WO2018059581A1 - Probe for hpv virus genotyping detection based on efirm technique, kit and use - Google Patents
Probe for hpv virus genotyping detection based on efirm technique, kit and use Download PDFInfo
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- WO2018059581A1 WO2018059581A1 PCT/CN2017/104829 CN2017104829W WO2018059581A1 WO 2018059581 A1 WO2018059581 A1 WO 2018059581A1 CN 2017104829 W CN2017104829 W CN 2017104829W WO 2018059581 A1 WO2018059581 A1 WO 2018059581A1
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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/708—Specific hybridization probes for papilloma
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Definitions
- the invention enjoys the invention entitled "probe combination, kit and method for genotyping detection of HPV high-risk strain” submitted on September 30, 2016, the application number is 201610877670.0; the invention name is “an HPV virus based on EFIRM technology” Detected probe combination, kit and method” application number is 201610881088.1 and the invention name is "a probe combination, kit and method for virus genotyping detection", and three invention patent applications with application number 201610877702.7 Priority of the Chinese Utility Model Patent Application No. 201620769829.2, entitled “Detection Electrode Structure and Detection Orifice", submitted on December 26, 2016, the entire contents of the above four Chinese patent applications This is hereby incorporated by reference in its entirety for all purposes.
- the invention relates to the field of gene detection technology, in particular to a probe kit, a kit and a method based on the EFIRM technology HPV virus detection.
- Cervical cancer is one of the most common malignant tumors in the female genital tract.
- HPV human papillomavirus
- multiple infections are one of the important causes of cervical cancer, a worldwide study.
- the results showed that the presence of high-risk HPV DNA was detected in 99.7% of cervical cancer patients.
- HPVs there are more than 200 types of HPVs, including low-risk and high-risk types. Different types have different correlations with different diseases.
- Low-risk HPV infection may cause genital warts and high-risk HPV infection is associated with cervical cancer and vaginal cancer.
- there is still a lack of effective treatments for HPV so early detection and early prevention of cervical HPV is the key to blocking cancer.
- HPV nucleic acid detection technology mainly includes hybridization capture method, PCR-fluorescence probe method, transcription-mediated nucleic acid amplification technology and PCR-hybridization method.
- the early screening method for cervical cancer is to use Pap smear and improved TCT technology. These methods can only evaluate the incidence of cervical cancer from the perspective of cytological lesions, that is, it needs to wait until the cells infected with the virus begin to appear. It can be detected when there is a significant change, and the specificity and sensitivity are not ideal.
- HPV gene detection has become more widely used as a screening method for cervical cancer.
- HPV typing detection technology is mainly based on polymerase chain reaction, such as fluorescence quantification, reverse dot blot hybridization and gene chip technology.
- HC2 is a nucleic acid hybridization detection method using microplate chemiluminescence for signal amplification, and is recognized as the gold standard for evaluating new HPV detection technology.
- the HC2 detection method is expensive, and the method is cumbersome to operate, and it is easy to cross-contamination during operation and false positives occur.
- a contribution of an aspect of the present invention is to provide a probe kit based on EFIRM technology HPV virus detection, which can detect genotype of virus in a sample to be tested, has strong specificity, high sensitivity and Low false positive rate, and the detection results are accurate and reliable.
- the second aspect of the present invention is to provide a kit and a detection plate for detecting HPV virus based on EFIRM technology, which is capable of genotyping detection of a virus in a sample to be tested, which has strong specificity and high sensitivity. Sensitivity, and the detection results are accurate and reliable, the false positive rate is low, and the operation is simple and convenient.
- the contribution of the three aspects of the present invention is to provide a method for genotyping detection of HPV virus based on EFIRM technology, which can detect genotype of virus in a sample to be tested, has strong specificity and high sensitivity, and is detected. The result is accurate and reliable, and the detection method is simple and easy to operate.
- a probe kit for HPV virus genotyping detection characterized in that
- each of the capture probes being used to bind a target sequence on a HPV viral genotype
- the plurality of capture probes include: a first capture probe and a second capture probe, at least one selected from the group consisting of the third to 11th capture probes, and at least one selected from the group consisting of the 12th to 16th capture probes; ,among them,
- a first capture probe for detecting a HPV 16 subtype the base sequence of which is set forth in SEQ ID NO.
- a second capture probe for detecting a HPV 18 subtype the base sequence of which is set forth in SEQ ID NO.
- a third capture probe for detecting a HPV 31 subtype the base sequence of which is set forth in SEQ ID NO.
- a fourth capture probe for detecting HPV 33 subtype or 52 subtype or 58 subtype the base sequence is shown in SEQ ID NO.
- a fifth capture probe for detecting the HPV 35 subtype the base sequence of which is set forth in SEQ ID NO.
- a sixth capture probe for detecting a subtype of HPV 39 the base sequence of which is set forth in SEQ ID NO.
- a 7th capture probe for detecting the HPV 45 subtype the base sequence of which is set forth in SEQ ID NO.
- An 8th capture probe for detecting a HPV 51 subtype the base sequence of which is set forth in SEQ ID NO.
- a ninth capture probe for detecting a HPV 56 subtype the base sequence of which is set forth in SEQ ID NO.
- a 10th capture probe for detecting a HPV 59 subtype the base sequence of which is set forth in SEQ ID NO.
- An 11th capture probe for detecting the HPV 68 subtype the base sequence is set forth in SEQ ID NO.
- the base sequence is set forth in SEQ ID NO.
- the base sequence is set forth in SEQ ID NO.
- the base sequence is set forth in SEQ ID NO.
- the base sequence is set forth in SEQ ID NO.
- the 16th capture probe was used to detect the HPV 82 subtype, and the base sequence is shown in SEQ ID NO.
- the method further comprises a plurality of detection probes that cooperate with the capture probe and can bind the corresponding target sequence, as follows:
- the base sequence of the detection probe complexed with the first capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the second capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the third capture probe or the fifth capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the fourth capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the sixth capture probe or the 11th capture probe capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the seventh capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the eighth capture probe or the ninth capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the 10th capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the 12th capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the 13th capture probe or the 14th capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the 15th capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe to which the 16th capture probe is ligated is shown in SEQ ID NO.
- a probe kit for genotyping detection of high-risk HPV viruses characterized in that
- each of the capture probes being used to bind a target sequence on an HPV viral genotype
- a third capture probe for detecting a HPV 31 subtype the base sequence of which is set forth in SEQ ID NO.
- a fourth capture probe for detecting HPV 33 subtype or 52 subtype or 58 subtype the base sequence is shown in SEQ ID NO.
- a fifth capture probe for detecting the HPV 35 subtype the base sequence of which is set forth in SEQ ID NO.
- a sixth capture probe for detecting a subtype of HPV 39 the base sequence of which is set forth in SEQ ID NO.
- a 7th capture probe for detecting the HPV 45 subtype the base sequence of which is set forth in SEQ ID NO.
- An 8th capture probe for detecting a HPV 51 subtype the base sequence of which is set forth in SEQ ID NO.
- a ninth capture probe for detecting a HPV 56 subtype the base sequence of which is set forth in SEQ ID NO.
- the 10th capture probe was used to detect the HPV 59 subtype, and the base sequence is shown in SEQ ID NO.
- it also includes
- a first capture probe for detecting the HPV 16 subtype the base sequence is shown in SEQ ID NO. 1; and a second capture probe for detecting the HPV 18 subtype, the base sequence is SEQ ID NO. Shown.
- the method further comprises a plurality of detection probes that cooperate with the capture probe and can bind the corresponding target sequence, as follows:
- the base sequence of the detection probe complexed with the first capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the second capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the third capture probe or the fifth capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the fourth capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the sixth capture probe or the 11th capture probe capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the seventh capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the eighth capture probe or the ninth capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the 10th capture probe is shown in SEQ ID NO.
- a probe kit for high-risk HPV virus genotyping detection comprising at least one selected from the group consisting of 12th to 16th capture probes, each of which is used to bind an HPV virus a target sequence on a genotype;
- the base sequence is set forth in SEQ ID NO.
- the base sequence is set forth in SEQ ID NO.
- the base sequence is set forth in SEQ ID NO.
- the base sequence is set forth in SEQ ID NO.
- the 16th capture probe was used to detect the HPV 82 subtype, and the base sequence is shown in SEQ ID NO.
- the base sequence is set forth in SEQ ID NO. 1; and a second capture probe for detecting the HPV 18 subtype, the base sequence is as SEQ ID NO. 3 is shown.
- the method further comprises a plurality of detection probes that cooperate with the capture probe and can bind the corresponding target sequence, as follows:
- the base sequence of the detection probe complexed with the first capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the second capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the 12th capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the 13th capture probe or the 14th capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the 15th capture probe is as shown in SEQ ID NO.
- the base sequence of the detection probe complexed with the 16th capture probe is shown in SEQ ID NO.
- a probe kit for HPV virus genotyping detection characterized in that
- a first capture probe for detecting a HPV 16 subtype the base sequence of which is set forth in SEQ ID NO.
- the detection probe for detecting type 16 HPV is further included, and the base sequence is as shown in SEQ ID NO.
- the detection probe of type 18 HPV was detected, and the base sequence is shown in SEQ ID NO.
- the 3' or 5' end of the detection probe is labeled with an affinity for binding to a catalytic enzyme for catalyzing the chemical reaction of the substrate to form a stream of electrons.
- a kit for detecting HPV virus based on EFIRM technology characterized in that it comprises a probe in any of the above probe kits.
- the kit further comprises a fixture for fixing the capture probe to a detection orifice plate, the fixture comprising a conductive polymer and an ionic compound;
- the conductive polymer is any one selected from the group consisting of pyrrole, aniline and thiophene;
- the ionic compound is any one selected from the group consisting of sodium chloride and potassium chloride.
- the kit further comprises the catalytic enzyme, the catalytic enzyme is a labeled horseradish peroxidase or alkaline phosphatase, and the label is used for binding to the affinity,
- the marker is any one of a digoxin antibody, a fluorescein isothiocyanate antibody, and streptavidin;
- the affinity labeled with the 3' or 5' end of the detection probe is one of digoxin, fluorescein isothiocyanate and biotin corresponding to the label.
- the kit further comprises the substrate of the catalytic enzyme
- the substrate is any one of TMB, ABTS and OPD;
- the substrate is a combination of BCIP and NBT, p-nitrophenyl phosphate, disodium 4-nitrobenzene phosphate, naphthol AS-BI phosphate, Any of naphthol-AS-MX-phosphate.
- the kit further includes a cleaning solution comprising a lotion A and a lotion B, the lotion A being an SDS-containing SSC buffer, and the lotion B is a Tween 20-containing PBS buffer.
- the kit further includes the detection well plate, wherein the capture probes are respectively fixed in different reaction wells of the detection well plate according to their corresponding HPV virus genotypes; There is a working electrode and is configured to apply a voltage to form an electric field.
- the bottom of the reaction hole is further provided with an opposite electrode, and the opposite electrode is disposed on the bottom plate of the reaction hole and configured to acquire a detection signal and output the detection signal;
- the working electrode includes at least one uniform width a first linear portion, the opposite electrode includes at least one second linear portion having a uniform width, the first linear portion and the second linear portion are alternately arranged at a bottom of the reaction hole; at least two The working electrodes in the adjacent reaction wells are electrically connected.
- a detection plate for HPV virus genotyping detection based on EFIRM technology is characterized in that a bottom of a reaction well of the detection plate is provided with a working electrode and is configured to apply a voltage to form electric field;
- a capture probe is dispensed and fixed in the reaction well of the detection well plate, and the dispensed and fixed capture probe is selected from any one of the following groups:
- Group 1 comprising a first capture probe and a second capture probe, at least one selected from the group consisting of the third to 11th capture probes, and at least one selected from the group consisting of the 12th to 16th capture probes;
- Group 2 comprising at least one of the third to eleven capture probes
- Group 3 comprising at least one of the 12th to 16th capture probes
- Group 4 comprising at least one of a first capture probe and a second capture probe, and a third to eleven capture probe;
- Group 5 comprising at least one of a first capture probe and a second capture probe, and a 12th to 16th capture probe;
- a first capture probe for detecting a HPV 16 subtype the base sequence of which is set forth in SEQ ID NO.
- a second capture probe for detecting a HPV 18 subtype the base sequence of which is set forth in SEQ ID NO.
- a third capture probe for detecting a HPV 31 subtype the base sequence of which is set forth in SEQ ID NO.
- a fourth capture probe for detecting HPV 33 subtype or 52 subtype or 58 subtype the base sequence is shown in SEQ ID NO.
- a fifth capture probe for detecting the HPV 35 subtype the base sequence of which is set forth in SEQ ID NO.
- a sixth capture probe for detecting a subtype of HPV 39 the base sequence of which is set forth in SEQ ID NO.
- a 7th capture probe for detecting the HPV 45 subtype the base sequence of which is set forth in SEQ ID NO.
- An 8th capture probe for detecting a HPV 51 subtype the base sequence of which is set forth in SEQ ID NO.
- a ninth capture probe for detecting a HPV 56 subtype the base sequence of which is set forth in SEQ ID NO.
- a 10th capture probe for detecting a HPV 59 subtype the base sequence of which is set forth in SEQ ID NO.
- An 11th capture probe for detecting the HPV 68 subtype the base sequence is set forth in SEQ ID NO.
- the base sequence is set forth in SEQ ID NO.
- the base sequence is set forth in SEQ ID NO.
- the base sequence is set forth in SEQ ID NO.
- the base sequence is set forth in SEQ ID NO.
- the base sequence is set forth in SEQ ID NO.
- the dispensing means that each of the capture probes is immobilized in a different one of the reaction wells.
- the bottom of the reaction well is further provided with an opposite electrode, and the opposite electrode is disposed on the reaction well bottom plate and configured to acquire a detection signal and output the detection signal;
- the working electrode includes at least one first linear portion having a uniform width, the opposite electrode including at least one second linear portion having a uniform width, and the first linear portion and the second linear portion are in the reaction The bottoms of the holes are alternately arranged;
- the working electrodes of at least two adjacent ones of the reaction wells are electrically connected.
- the capturing probe is mixed with a conductive polymer and an ionic compound to form a mixed solution, and then added to the reaction hole, and then the first square wave electric field is applied through the working electrode.
- a conductive polymer and an ionic compound to form a mixed solution, and then added to the reaction hole, and then the first square wave electric field is applied through the working electrode.
- the parameters of the first electric field are: voltage A: 350 mV, 1 s; voltage B: 950 mV, 1 s; 9 cycles are performed.
- a method for detecting HPV virus genotyping based on EFIRM technology characterized in that the kit according to any one of claims 13-19 is used, the steps are as follows:
- the detection orifice plate according to any one of claims 20-22, or the capture probe is added to the blank detection orifice plate, and the bottom of the reaction well is provided with an electrode for turning on the EFIRM detector. Applying an electric field to the solution in the reaction well to carry out polymerization reaction; after the EFIRM detector is turned on, a first electric field is applied to the solution in the reaction well to carry out polymerization reaction; after the electric field treatment is completed, the detection orifice plate is cleaned.
- the parameters of the first electric field processing are: voltage 200-500 mV, 1-5 s; voltage 800-1500 mV, 1-5 s; 3-10 cycles;
- the concentration of the detection probe in the detection probe solution is 0.5-1.5 ⁇ mol/L;
- the beneficial effects of the probes, kits and methods based on the EFIRM technology HPV virus detection provided by the present invention are that the probe combination based on the EFIRM technology HPV virus detection provided by the present invention comprises a plurality of probe pairs, each probe pair comprises There is a capture probe for binding to a target sequence and a detection probe corresponding to the capture probe and capable of binding the target sequence, and the capture probe binds to the target sequence through the principle of base complementation, and the target sequence is captured and fixed for the first time.
- the detection probe binds specifically to the target sequence through the principle of base complementation, and the detection probe is bound and immobilized; the labeled affinity of the 3' or 5' end of the detection probe binds the catalytic enzyme
- the catalytic enzyme catalyzes the release of the current signal from the substrate, detects the released current signal, and detects the viral genotype. Since the target sequence can be signaled only after two specific bindings simultaneously with the capture probe and the detection probe, which greatly increases the specificity of the detection, making the detection result accurate and reliable, false positive The rate is very low.
- the present invention provides a base sequence of a capture probe of each probe pair, which is capable of separately capturing, for example, a common type of HPV 16 subtype, 18 subtype, and a medium and high risk type of HPV 26, 53, 66, 73, 82.
- the target sequence in the subtype which in turn can detect whether the sample to be tested contains one of the common type viruses such as HPV 16 subtype, 18 subtype, and high risk type viruses such as HPV HPV26, 53, 66, 73, 82 subtypes. Or a variety of viral subtypes.
- Embodiment 1 is an exemplary detection result of Embodiment 1 of the present invention.
- Embodiment 2 is an exemplary detection result of Embodiment 2 of the present invention.
- Embodiment 3 is an exemplary detection result of Embodiment 3 of the present invention.
- Embodiment 4 is an exemplary detection result of Embodiment 4 of the present invention.
- FIG. 5 is an exemplary detection result according to Embodiment 5 of the present invention.
- Embodiment 6 is an exemplary detection result of Embodiment 6 of the present invention.
- FIG. 7 is a schematic plan view showing the structure of a detecting electrode in the detecting orifice plate of the present invention.
- FIG. 8 is a schematic plan view showing another structure of a detecting electrode in the detecting orifice plate of the present invention.
- FIG. 9 is a schematic plan view showing another structure of a detecting electrode in the detecting orifice plate of the present invention.
- FIG. 10 is a schematic plan view showing another structure of a detecting electrode in the detecting orifice plate of the present invention.
- Figure 11a is a perspective view of a detection orifice plate according to the present invention.
- Figure 11b is a schematic plan view of a detecting orifice plate according to the present invention.
- FIG. 12 is a partial perspective view of a detecting orifice plate according to the present invention.
- Figure 13 is a partial perspective view of a detecting orifice plate according to the present invention.
- Figure 14 is a partial perspective view of a detecting orifice plate according to the present invention.
- Figure 15 is a partial side elevational view of the detection orifice plate of the present invention.
- the invention is illustrated by the following examples, which are not indicated in the examples, and are carried out according to conventional conditions or conditions recommended by the manufacturer.
- the reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained by commercially available purchase.
- the EFIRM detector used in the present invention is produced by Guangzhou Yihuo Biotech Co., Ltd., and is described in "Felvi Chemical Sensor for Multiplex Biomarkers Detection, Clin Cancer Res. 2009 Jul 1; 15 (13) published by Fang Wei et al. In 4446–4452, the electrochemical detector used therein.
- a square wave (csw E-field) can be applied to the reaction well by a general square wave-generating instrument, and the living biotechnology limited can also be adopted.
- the company's pre-developed EFIRMY instruments and supporting software are implemented.
- HPV16, 18 subtypes are the most common clinical, and the two types of viruses have the highest probability of occurrence in cervical cancer patients; HPV 31, 33, 52, 58, 35, 39, 45, 51, 56, 59, 68 subtypes are high Risk type (collectively referred to as high risk group), HPV26, 53, 66, 73, 82 subtypes are medium risk type (collectively referred to as medium and high risk groups), and the present invention is based on the above virus subtypes.
- the probe sequence is designed and the resulting probe combination can be used to detect the above viral subtypes.
- the invention provides a probe combination based on EFIRM technology HPV virus detection, which comprises a plurality of probe pairs.
- Each probe pair includes a capture probe for binding to a target sequence and a detection probe corresponding to the capture probe and conjugateable to the target sequence.
- the 3' or 5' end of the detection probe of each probe pair is labeled with an affinity for binding to a catalytic enzyme for catalyzing the chemical reaction of its substrate to form a stream of electrons.
- the plurality of probe pairs include a first probe pair, a second probe pair, and at least one selected from the group consisting of the 12th to 16th probe pairs.
- the base sequence of the capture probe and the detection probe detected by each probe pair, and the HPV subtype thereof detected will be described in detail below.
- the first probe pair for detecting the HPV16 subtype has a base sequence of the capture probe as shown in SEQ ID NO. 1, and the base sequence of the detection probe is shown in SEQ ID NO.
- the second probe pair for detecting the HPV18 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 3, and the base sequence of the detection probe is shown in SEQ ID NO.
- the 12th probe pair for detecting the HPV26 subtype is shown in SEQ ID NO. 20, and the base sequence of the detection probe is shown in SEQ ID NO.
- the base sequence of the capture probe is shown in SEQ ID NO. 21
- the base sequence of the detection probe is shown in SEQ ID NO.
- the 14th probe pair for detecting the HPV66 subtype is shown in SEQ ID NO. 22, and the base sequence of the detection probe is shown in SEQ ID NO.
- the 15th probe pair for detecting the HPV73 subtype is shown in SEQ ID NO. 23, and the base sequence of the detection probe is shown in SEQ ID NO.
- the 16th probe pair for detecting the HPV82 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 24, and the base sequence of the detection probe is shown in SEQ ID NO.
- the plurality of probe pairs may further comprise probe pairs for detecting high risk HPV:
- a third probe pair for detecting the HPV31 subtype the base sequence of the capture probe is shown in SEQ ID NO. 5, and the base sequence of the detection probe is shown in SEQ ID NO.
- a fourth probe pair for detecting HPV33 subtype or 52 subtype or 58 subtype the base sequence of the capture probe is shown in SEQ ID NO. 6, and the base sequence of the detection probe is SEQ ID NO. As shown in .15, the three subtypes share the fourth probe pair.
- the 5th probe pair for detecting the HPV35 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 7, and the base sequence of the detection probe is shown in SEQ ID NO.
- the 6th probe pair for detecting the HPV39 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 8, and the base sequence of the detection probe is shown in SEQ ID NO.
- the 7th probe pair for detecting the HPV45 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 9, and the base sequence of the detection probe is shown in SEQ ID NO.
- the 8th probe pair for detecting the HPV51 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 10, and the base sequence of the detection probe is shown in SEQ ID NO.
- the ninth probe pair for detecting the HPV 56 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 11, and the base sequence of the detection probe is shown in SEQ ID NO.
- the 10th probe pair for detecting the HPV59 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 12, and the base sequence of the detection probe is shown in SEQ ID NO.
- the 11th probe pair for detecting the HPV68 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 13, and the base sequence of the detection probe is shown in SEQ ID NO.
- the detection probe corresponds to the capture probe, and one capture probe and one detection probe form a probe pair, which can be used for detecting a certain type of virus.
- Both the capture probe and the detection probe are based on the same type of virus of the same type. Designing a target sequence or a conserved DNA fragment, the capture probe and the detection probe can bind to different regions or binding sites of the same conserved gene or conserved DNA fragment of the virus type through the principle of base complementation, but capture The probe is not complementary to the detection probe, and the base sequence is also different, and the binding sites of the two and the target sequence do not overlap or overlap. Further, the binding region of the capture probe and the detection probe to the target sequence may be adjacent or may be separated by a plurality of bases. As long as the capture probe and the detection probe are capable of binding to the same conserved gene of the same type of virus or the same conserved DNA fragment by the principle of base complementation.
- the capture probe sequence of each probe pair and the base sequence of the detection probe sequence may not correspond to each other, that is, in other embodiments, the capture probe of each probe pair
- the sequence may be the base sequence described above, and the base sequence of the corresponding detection probe may be complementary to other sequences taken from the same DNA conserved region of the same detection subtype virus; or in other embodiments,
- the detection probe sequence of each probe pair may be the above-mentioned base sequence, and the base sequence of the corresponding capture probe may be another sequence selected from the same DNA conserved region of the same detection subtype virus.
- the base sequences of the capture probes and the detection probes of the probe pairs provided by the present invention have the advantages of stronger specificity, higher sensitivity, etc. when they are detected, and the detection results can be seen in the examples. .
- group detection can be performed, for example, only the virus containing a certain risk level (for example, a high-risk group or a medium-high-risk group) is detected, and it is not necessary to detect that it contains the group.
- a certain risk level for example, a high-risk group or a medium-high-risk group
- Specific type of virus for example, specific subtypes can also be detected, for example, by detecting a subtype of a particular high-risk group, such as the HPV53 subtype virus, in the sample.
- the specific detection method is used to detect the degree to which the detection result is obtained, and the tester can select according to the specific situation to provide a more reasonable guiding significance.
- the specific group detection is described in the embodiment.
- the probe combination for viral gene analysis detection may include only one or two or three or more probe pairs of the first to 16th probe pairs, as long as Any combination selected from the first to 16th probe pairs is within the scope of the present invention.
- the affinity of the 3' or 5' end of each of the detection probes is biotin, and the biotin acts to bind to the streptavidin-labeled catalytic enzyme and catalyze the catalytic end.
- the current generated by the object releases the detection signal.
- the detection probe may bind to the catalytic enzyme without the recognition system of biotin-streptavidin, and may be combined with other catalytic systems such as antibody/antigen, ligand/receptor, etc.
- the enzyme therefore, the affinity may be one of an antigen/antibody.
- the affinity may be digoxin or fluorescein isothiocyanate, and correspondingly, the catalytic enzyme may be labeled with a high-octane antibody or a fluorescein isothiocyanate antibody.
- the affinity may be labeled at the 3' end of the detection probe or at the 5' end, either.
- the kit for detecting HPV virus based on EFIRM technology comprises any one of the probe combinations described above.
- each probe is independently present in the form of a solution, for example, the capture probe is present in the form of a capture probe solution containing a capture probe, and the detection probe is present in the form of a test probe solution containing the detection probe.
- the concentration of the probe contained in each probe solution can be set according to actual conditions.
- each probe solution contains a probe having a final concentration of 0.5 to 1.5 ⁇ M.
- the kit for detecting HPV virus based on EFIRM technology may further comprise a fixture for fixing the capture probe of the probe pair to the detection well plate.
- the fixture includes a conductive polymer and an ionic compound.
- the conductive polymer is selected from one of pyrrole, aniline and thiophene.
- the conductive polymer may also be other conductive polymer materials.
- the ionic compound is selected from any one of sodium chloride and potassium chloride.
- the conductive polymer is positively charged, and forms a network cross-linked structure under the action of an electric field, and is deposited at the bottom of the reaction hole.
- the mesh cross-linked structure can stably fix the capture probe at the bottom, which helps to improve the capture probe. Needle stability and capture ability.
- the kit for detecting HPV virus based on EFIRM technology may further comprise a catalytic enzyme, the catalytic enzyme is a horseradish peroxidase with a label, preferably, the catalytic enzyme is labeled with streptavidin. Horseradish peroxidase.
- the catalytic enzyme may also be a labeled alkaline phosphatase, and the label is any one of a digoxin antibody, a fluorescein isothiocyanate antibody or streptavidin, a label and an affinity. Correspondingly, it can be selected based on the type of affinity on the detection probe.
- the affinity is biotin
- the marker is streptavidin
- the affinity is digoxin
- the marker is a digoxin antibody
- the affinity is fluorescein isothiocyanate
- the marker is a fluorescein isothiocyanate antibody.
- kit for detecting HPV virus based on EFIRM technology may further comprise a substrate, and the class of the substrate is selected according to the type of catalytic enzyme.
- the substrate is TMB (Tetramethylbenzidine, tetramethylbenzidine), ABTS (2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate, 2,2-diazepine) - bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) and OPD (o-Phenylenediamine, o-phenylenediamine).
- TMB Tetramethylbenzidine, tetramethylbenzidine
- ABTS 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate, 2,2-diazepine) - bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt
- OPD o-Phenylenediamine, o-phenylenediamine
- TMB, ABTS and OPD are both horseradish peroxidation
- the substrate of the enzyme under the catalysis of horseradish peroxidase, undergoes a color reaction and is accompanied by a current generation, which contributes to an increase in the release of the detection signal.
- the substrate is BCIP (5-Bromo-4-Chloro-3-Indolyl Phosphate, 5-bromo-4-chloro-3-indolyl-phosphate) and NBT (Nitrotetrazolium). Blue chloride, tetrazolium nitroblue) composition, nitrophenyl phosphate, disodium 4-nitrobenzene phosphate, naphthol AS-BI phosphate, naphthol-AS-MX-phosphate .
- kit for detecting HPV virus based on EFIRM technology may further include a cleaning solution including washing liquid A and washing liquid B, washing liquid A is SDS buffer containing SDS, and washing liquid B is containing Tween20. PBS buffer.
- kit for detecting HPV virus based on EFIRM technology may further comprise a diluent, which is a casein-containing PBS buffer.
- the invention provides a method for detecting HPV virus based on EFIRM technology, which comprises:
- Providing a plurality of probe pairs comprising: a first probe pair, a second probe pair, and at least one selected from the group consisting of 3rd to 11th probe pairs; each probe pair including for binding a capture probe of the target sequence and a detection probe corresponding to the capture probe and capable of binding to the target sequence, and the 3' or 5' end of the detection probe is labeled for binding to catalyze the formation of a chemical reaction of the corresponding substrate.
- base sequence of the capture probe of the first probe pair is as shown in SEQ ID NO. 1
- base sequence of the capture probe of the second probe pair is as shown in SEQ ID NO. 3, and 12 to 16
- the base sequences of the capture probes of the probe pair are shown in SEQ ID NO. 20 to 24, respectively.
- Capture probe immobilization step The capture probe for binding to the target sequence is first fixed to the reaction well of the detection well by an electric field. The capture probe moves to the bottom of the reaction well under the action of an electric field and is fixed at the bottom of the reaction well.
- Sample hybridization step The sample to be tested is added to the reaction well.
- the target sequence and the capture probe in the sample to be tested are captured and fixed at the bottom of the reaction well by the principle of base complementation.
- Detection probe binding step adding a detection probe corresponding to the capture probe and binding the target sequence to the reaction well, and the 3' end or the 5' end of the detection probe is labeled for binding to catalyze the corresponding substrate production
- the chemical reaction forms an affinity for the catalytic enzyme of the electron flow.
- the detection probe is immobilized in the reaction well by binding to the target sequence through the principle of base complementation.
- Enzyme catalytic reaction detection step a catalytic enzyme and a substrate are added to the reaction well, and the current in the reaction well is detected by a current detecting device.
- the detection probe binds to the catalytic enzyme through the affinity, and the catalytic enzyme re-catalyzes the substrate to generate a current release detection signal, which is recognized and amplified by the current detecting device, and the result is detected.
- the detection principle of the present invention is to rapidly detect viral genotypes by using Electric Field-Induced Release and Measurement (EFIRM) technology, especially for detecting 18 high-risk HPVs.
- EFIRM Electric Field-Induced Release and Measurement
- the capture probe is immobilized on the bottom of the reaction well on the detection well plate under the action of the electric field; the capture probe captures the target sequence in the sample to be tested, ie, viral DNA, by the principle of base complementary pairing under the electric field;
- the labeled detection probe is further bound to the target sequence; the catalytic enzyme is recognized by the labeled streptavidin and biotin, and the substrate of the catalytic enzyme is added to the reaction well to generate a redox reaction, current generation, and instrument detection.
- the current signal is further determined to have a corresponding target sequence in the sample to be tested. Since both the capture probe and the detection probe are designed according to a conserved region of a specific virus type, the type of the virus can be known.
- kits for detecting HPV virus based on EFIRM technology provided in the present embodiment is described by taking the HPV genotype as an example.
- the kit for detecting a virus genotyping includes a probe combination comprising 16 probe pairs for detecting HPV subtypes, and the capture probes and detection probes of each probe pair are The solution forms were independent and the final concentration of the probe was 1 ⁇ M.
- the 16 probe pairs are: a first probe pair for detecting the HPV 16 subtype, and the base sequence of the capture probe is as shown in SEQ ID NO. 1, and the base sequence of the detection probe is SEQ. ID NO. 2; a second probe pair for detecting the HPV 18 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 3, and the base sequence of the detection probe is SEQ ID NO.
- a probe combination comprising 16 probe pairs for detecting HPV subtypes, and the capture probes and detection probes of each probe pair are The solution forms were independent and the final concentration of the probe was 1 ⁇ M.
- the 16 probe pairs are: a first probe pair for detecting the HPV 16 subtype, and the base sequence of the capture probe is as shown in SEQ ID
- a third probe pair for detecting the HPV 31 subtype the base sequence of the capture probe is shown in SEQ ID NO. 5, and the base sequence of the detection probe is SEQ ID NO.
- the base sequence of the capture probe is shown in SEQ ID NO. 6, and the base sequence of the detection probe is shown.
- SEQ ID NO. 15 a 5th probe pair for detecting the HPV 35 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 7, and the base sequence of the detection probe is SEQ. ID NO. 14; a 6th probe pair for detecting the HPV 39 subtype, the base sequence of the capture probe is shown in SEQ ID NO.
- the base sequence of the detection probe is SEQ ID NO. .16; used to detect HPV 45 a 7th probe pair of the type, the base sequence of the capture probe is shown in SEQ ID NO. 9, and the base sequence of the detection probe is shown in SEQ ID NO. 17; for detecting HPV 51 subtype
- the eighth probe pair the base sequence of the capture probe is shown in SEQ ID NO. 10, the base sequence of the detection probe is shown in SEQ ID NO. 18, and the ninth for detecting the HPV 56 subtype.
- the probe pair has a base sequence of the capture probe as shown in SEQ ID NO. 11, a base sequence of the detection probe as shown in SEQ ID NO. 18, and a 10th probe for detecting the HPV 59 subtype.
- the base sequence of the capture probe is shown in SEQ ID NO. 12, the base sequence of the detection probe is shown in SEQ ID NO. 19, and the 11th probe pair for detecting the HPV 68 subtype,
- the base sequence of the capture probe is shown in SEQ ID NO. 13, the base sequence of the detection probe is shown in SEQ ID NO. 16, and the 12th probe pair for detecting the HPV 26 subtype is captured.
- the base sequence of the probe is shown in SEQ ID NO. 20, the base sequence of the detection probe is shown in SEQ ID NO. 26; the 13th probe pair for detecting the HPV 53 subtype, and the capture probe thereof.
- Base sequence such as SEQ As shown in ID NO. 21, the base sequence of the detection probe is shown in SEQ ID NO.
- the base sequence of the capture probe is SEQ ID NO. 25; the 14th probe pair for detecting the HPV 66 subtype, the base sequence of the capture probe is SEQ ID NO. As shown in Fig. 22, the base sequence of the detection probe is shown in SEQ ID NO. 25; the 15th probe pair for detecting the HPV 73 subtype, the base sequence of the capture probe is SEQ ID NO. As shown, the base sequence of the detection probe is shown in SEQ ID NO. 27; the 16th probe pair for detecting the HPV 82 subtype, the base sequence of the capture probe is shown in SEQ ID NO. The base sequence of the detection probe is shown in SEQ ID NO. The base sequences of the respective HPV subtypes and their corresponding capture probes and detection probes are shown in Table 1.
- the capture probes of the 33, 52, and 58 subtypes have the same base sequence
- the detection probes of the 33, 52, and 58 subtypes have the same base sequence, that is, the capture probe shown by SEQ ID NO.
- the probe and the detection probe shown in SEQ ID NO. 15 are capable of detecting HPV 33, 52 and 58 subtype viruses, and the detection results indicate that the sample has at least one of HPV types 33, 52 and 58, but specifically Which of the three subtypes of the virus was not detected.
- the detection probe sequences of the 39 and 68 subtypes are identical
- the detection probe sequences of the 51 and 56 subtypes are identical
- the detection probe sequences of the 53 and 66 subtypes are identical.
- the 5'-end labeled biotin was used to detect the 16 and 18 subtypes of the detection probe, and the remaining subtypes of the detection probe were labeled with biotin at the 3' end (as shown in Table 1).
- the specific detection steps for the sample 1 and the sample 2 using the virus genotyping detection kit provided in the present embodiment are as follows.
- E-plate On a 96-well detection plate (E-plate) (the structure and working principle can be seen in the priority document 201620769829.2), add 30 ⁇ l of the prepared mixture of pyrrole and CP to the reaction well according to the instructions.
- the tip of the gun is attached to the bottom of the hole, but does not touch the bottom electrode, after tilting or tapping the E-plate, the liquid is evenly covered on the surface of the electrode in the hole, and then immediately go to the EFIRM instrument and operate the electric field according to its operating instructions. .
- the corresponding column for the experiment was selected on the EFIRM software.
- the electric field parameters were set to: voltage A: 350 mV, 1 s; voltage B: 950 mV, 1 s; 9 cycles were performed. After the electric field treatment is completed, remove it immediately and clean the E-plate plate.
- the lotion A was a 2 ⁇ SSC buffer containing 0.05% by mass of SDS.
- the hybrid buffer (purchased from thermo fisher) was treated in a water bath at 90 ° C for 10 min in a water bath and then allowed to cool at room temperature for 20 min.
- the sample to be tested was taken out from the -20 ° C refrigerator and placed in a refrigerator at 4 ° C to be thawed. After complete dissolution, the sample to be tested is pretreated by boiling or 0.4M NaOH, and then the sample to be tested is mixed with the hybrid buffer by a volume ratio of 1:2, vortexed and centrifuged, and then the sample is loaded for detection.
- four detection groups are set, which are 16 subtype group, 18 subtype group, 11 high risk type groups, and 5 medium and high risk type groups.
- the 16 subtype group was used to detect the HPV16 subtype (the corresponding pair of probe pairs was added to each well in the group)
- the 18 subtype group was used to detect the HPV18 subtype (the corresponding probe was added to each well of the group)
- 11 high-risk groups were used to detect one of 11 high-risk HPVs (the group included 3 to 11 probe pairs for each well, and 5 medium- and high-risk types for 5 medium- and high-risk HPVs).
- One of the groups (the pair of holes corresponds to the 12th to 16th probe pairs). It should be noted that the number of detection groups can be designed according to the accuracy of the required test results.
- the common type HPV, the high-risk type virus, and the medium-high-risk type virus can be set according to the setting method of the detection group in this embodiment. If only the sample is required to detect whether the sample contains HPV, only one detection group can be set, and the specific detection method Refer to Embodiment 2. If it is necessary to detect that the sample specifically contains a certain type of virus, 18 detection groups can be set, and the specific detection method is referred to Embodiment 3.
- a corresponding 30 ⁇ l of a hybrid buffer containing a positive oligonucleotide having a final concentration of 1 pM (which is capable of complementary pairing with the corresponding capture probe and detection probe) was added as a positive control to the positive control well (remarks that The positive control of 11 high-risk groups only selected 52 subtype nucleotide sequences (line 9 in Table 2), and the positive control of 5 medium-high-risk groups only selected 26 subtype nucleotide sequences (Table 2) Line 13) can serve as a positive control.
- the base sequence of the positive oligonucleotide corresponding to each HPV subtype is shown in Table 2. 30 ⁇ l of hybridization buffer was added to the negative control well as a negative control.
- the electric field parameters are set to: voltage A: 300 mV, 1 s; voltage B: 500 mV, 1 s; 150 cycles. After the electric field treatment is completed, remove it immediately and clean the E-plate plate.
- the dilution solution was taken out from the refrigerator at 4 ° C, and a 1.5 mL centrifuge tube was taken. 990 ⁇ l of the dilution solution was added, and 10 ⁇ l of DP (100 ⁇ M) was added to the reaction wells of each test group, vortexed and mixed, centrifuged, and set aside. Among them, the diluent was PBS buffer (pH 7.4) containing 0.1% (mass by volume) casein. The role of casein is to block non-specific sites to increase the sensitivity and accuracy of the assay.
- the lotion B was a PBS buffer containing 0.1% by mass of Tween20.
- the substrate is a solution containing TMB (commercially available from Thermo Fisher, Cat. No. 34028 the product, the name 1-Step TM Ultra TMB-ELISA ).
- TMB commercially available from Thermo Fisher, Cat. No. 34028 the product, the name 1-Step TM Ultra TMB-ELISA ).
- the substrate of the enzyme is added, a redox reaction occurs, a current is generated, and the current value in each well is detected to complete the entire detection process.
- the corresponding column for the experiment was selected on the EFIRM software.
- the electric field parameters were set to: voltage A: -200 mV, 60 s; voltage B: 0 mV, 0 s; one cycle was performed. After the electric field treatment is completed, remove it immediately and clean the E-plate plate.
- the instrument will automatically complete the test and the test data will be automatically uploaded to the cloud computing platform.
- the histogram is drawn according to the detected data, the abscissa is the category of the detection group, and the ordinate is the current value (Current) of each detection hole in each detection group, and the unit is nanoamperes (-nA, negative sign indicates direction).
- the detection results of this embodiment are shown in FIG.
- the test result of the sample 1 was positive, and it contained the HPV16 subtype virus (the negative control current value was 28.67 nA, the standard deviation was 5.62, and the positive control current value was 130.22 nA, the sample The current value of 1 is 192.05 nA, and the current value of sample 2 is 30.53 nA).
- the test result of sample 2 is positive (the negative control current value is 24.94 nA, and the standard deviation is 5.91, positive control)
- the current value is 132.94nA
- the current value of sample 1 is 29.57nA
- the current value of sample 2 is 151.30nA), indicating that it contains high-risk HPV31, 33, 52, 58, 35, 39, 45, 51, 56, 59, At least one of the 68 subtypes.
- the kit for detecting HPV virus based on EFIRM technology includes a probe combination (same as in Example 1), a fixative, streptavidin-labeled horseradish peroxidase (present in solution), and a bottom thereof.
- the immobilizer is a conductive polymer and an ionic compound, wherein the conductive polymer is pyrrole and the ionic compound is potassium chloride.
- the substrate is a solution containing TMB. The rest are the same as in the first embodiment.
- the detection group is set to one, and the 18 subtypes are simultaneously detected, and the detection group includes one positive control hole, one negative control hole (repeated 4 times), one sample 1 detection hole and one sample. 2 detection holes.
- the 16 probe pairs described in Example 1 were added to each well and 18 HPV subtypes were simultaneously detected). The rest are the same as in the first embodiment. If there is a positive detection result, the detected result can indicate that the sample to be tested contains HPV 16, 18, 31, 33, 52, 58, 35, 39, 45, 51, 56, 59, 68, 26, 53, 66, At least one of the 73 and 82 subtypes (as shown in Figure 2).
- the detection result of this embodiment is as shown in FIG. 2.
- test result of sample 1 is positive (the negative control current value is 26.59 nA, the standard deviation is 7.91, the positive control current value is 110.46 nA, and the sample 1 current value is 189.66 nA), indicating that it contains HPV16, At least one of 18, 31, 33, 52, 58, 35, 39, 45, 51, 56, 59, 68, 26, 53, 66, 73, 82 subtypes; sample 2 is positive (negative)
- the control current value was 26.59 nA, the standard deviation was 7.91, the positive control current value was 110.46 nA, and the sample 2 current value was 80.60 nA), indicating that sample 2 contained HPV 16, 18, 31, 33, 52, 58, 35, 39. At least one of the 45, 51, 56, 59, 68, 26, 53, 66, 73, 82 subtypes.
- the kit for detecting HPV virus based on EFIRM technology includes a probe combination (same as in Example 1), a fixative, streptavidin-labeled horseradish peroxidase (present in solution), and a bottom thereof. Matter, cleaning solution.
- the anchor is a conductive polymer and an ionic compound, wherein the conductive polymer is pyrrole and the ionic compound is potassium chloride.
- the substrate is a solution containing TMB.
- the cleaning solution includes a lotion A and a lotion B, the lot A is an SDS buffer containing SDS, and the lotion B is a PBS buffer containing Tween 20. The rest are the same as in the first embodiment.
- the detection group is set to 16 groups, which are 16 subtype group, 18 subtype group, 26 subtype group, 31 subtype group, 35 subtype group, 39 subtype group, and 45 subtype group. , 51 subtype group, 52 subtype group, 53 subtype group, 56 subtype group, 59 subtype group, 66 subtype group, 68 subtype group, 73 subtype group and 82 subtype group.
- One positive control well was set for each test group (each test group was added with the corresponding positive oligonucleotide shown in Table 2), and one negative control well (heavy Repeat 4 times), one sample 1 detection hole and one sample 2 detection hole, and each corresponding detection hole is added to its corresponding probe pair. The rest are the same as in the first embodiment. If each test group has a positive detection result, the detected result can indicate the corresponding HPV subtype virus contained in the sample to be tested.
- the positive result detected by the 52 subtype indicates that the sample to be tested contains one or more of the HPV 33, 52, and 58 subtypes.
- the detection result of this embodiment is shown in FIG.
- the test result of the sample 1 was positive (the negative control current value was 28.67 nA, the standard deviation was 5.62, the positive control current value was 130.22 nA, and the sample 1 current value was 192.05 nA.
- the current value of sample 2 is 30.53nA), indicating that sample 1 contains HPV 16 subtype virus; in group 52, sample 2 is positive (negative control current value is 25.58nA, and its standard deviation is 5.33, The positive control current value was 75.70 nA, the sample 1 current value was 23.90 nA, and the sample 2 current value was 166.10 nA).
- sample 2 test result was positive (the negative control current value was 25.92).
- nA its standard deviation is 6.20
- positive control current value is 115.75nA
- sample 1 current value is 34.47nA
- sample 2 current value is 72.72nA)
- the detection group of 18 by setting the detection group of 18 to detect the detection result of the sample to be tested, the specific virus subtype contained therein can be detected, and the detection result is accurate and reliable.
- the kit for detecting HPV virus based on EFIRM technology includes a probe combination, a detection plate, a fluorescein isothiocyanate antibody-labeled horseradish peroxidase (present in solution) and a substrate thereof (ABTS) , in the form of a solution), cleaning solution.
- the probe combination comprises 9 probe pairs, respectively: a first probe pair for detecting the HPV 16 subtype, a second probe pair for detecting the HPV 18 subtype, and For detecting the third probe pair of the HPV 31 subtype, the fourth probe pair for detecting the HPV 33 subtype or the 52 subtype or the 58 subtype, and the eighth probe pair for detecting the HPV 51 subtype, 9th probe pair for detecting HPV 56 subtype, 12th probe pair for detecting HPV 26 subtype, 13th probe pair for detecting HPV53 subtype, 15th for detecting HPV 73 subtype Probe pair.
- Each test probe was provided with fluorescein isothiocyanate at the 3' end.
- the base sequences of the respective probe pairs and the corresponding capture probes and detection probes are shown in Table 1 in Example 1.
- the capture probe of the above probe pair is fixed in the reaction well of the detection well plate, and the method of fixing the capture probe to the detection well plate is the same as the "1 capture probe fixation" step in the first embodiment, of course, in other implementations.
- the detection of the capture probe of the present invention by using other methods to the detection well plate is also within the scope of the present invention.
- the cleaning solution includes a lotion A and a lotion B, the lotion A is an SSS buffer containing SDS, and the lotion B is a PBS buffer containing Tween20.
- the rest are the same as in the first embodiment.
- the sample 1 and the sample 2 are simultaneously detected by using the embodiment, and the detecting step is basically the same as that of the first embodiment, and the detection result is as shown in FIG. 4 .
- sample 1 contains HPV16 subtype virus
- sample 2 is positive (negative control current value is 24.25nA, standard deviation is 5.27, positive control current value is 115.92nA, sample)
- the current value of 2 is 170.42 nA), indicating that it contains at least one of the high-risk types HPV 31, 33, 51, 52, 58 and 56 subtypes.
- the kit for detecting HPV virus based on EFIRM technology includes a probe combination, a fixer, a digoxin antibody-labeled alkaline phosphatase enzyme (present in solution) and a substrate thereof (including BCIP and NBT) A solution of the composition is present), a cleaning solution.
- the probe combination includes four probe pairs, which are: a first probe pair for detecting the HPV 16 subtype, a second probe pair for detecting the HPV 18 subtype, and A 4th probe pair for detecting HPV 33 subtype or 52 subtype or 58 subtype, and a 12th probe pair for detecting HPV 26 subtype.
- Each detection probe has a digoxin at the 5' end.
- the base sequences of the respective probe pairs and the corresponding capture probes and detection probes are shown in Table 1 in Example 1.
- the anchor is a conductive polymer and an ionic compound, wherein the conductive polymer is thiophene, and of course, in other embodiments, aniline.
- the ionic compound is sodium chloride.
- the cleaning solution includes a lotion A and a lotion B, the lot A is an SDS buffer containing SDS, and the lotion B is a PBS buffer containing Tween 20. The rest are the same as in the first embodiment.
- the sample 1 and the sample 2 are simultaneously detected by using the embodiment, and the detection step is basically the same as that of the embodiment 1, and the detection result is shown in FIG. 5.
- the test result of the sample 1 was positive (the negative control current value was 26.31 nA, the standard deviation was 5.29, the positive control current value was 118.74 nA, and the sample 1 current value was 185.47 nA. ), indicating that sample 1 contains HPV16 subtype virus; in the three high-risk groups, sample 2 was positive (negative control current value was 27.45 nA, standard deviation was 5.15, positive control current value was 122.62 nA, sample) The current value of 2 is 150.07 nA), indicating that sample 2 contains at least one of the high-risk types HPV 33, 51, 52, and 58 subtypes.
- the kit for detecting HPV virus based on EFIRM technology includes a probe combination, a immobilizer, streptavidin-labeled horseradish peroxidase (present in solution) and a substrate thereof, and the immobilizer is conductive A polymer and an ionic compound, wherein the conductive polymer is pyrrole and the ionic compound is potassium chloride.
- the substrate is a solution containing TMB. The rest are the same as in the first embodiment.
- the probe combination includes only the first probe pair, the second probe pair, the twelfth probe pair, the thirteenth probe pair, the fourteen probe pair, the fifteenth probe pair, and the sixteenth probe pair (each The sequence of the capture probe and the detection probe of the probe pair is the same as in Example 1).
- the probe combination includes a first probe pair, a second probe pair, and at least one selected from the group consisting of 12th to 16th probe pairs, for example, two or three types. .
- the sample 2 was only tested by the kit of the present embodiment, and the detection step was basically the same as that of the first embodiment, and the detection result is shown in FIG. 6.
- the test result of sample 2 is positive (the negative control current value is 28.36nA, the standard deviation is 7.15, the positive control current value is 147.47nA, and the current value of sample 2 is 66.91nA), indicating that sample 2 contains one or more of five medium-high-risk viruses; in the 16-subtype group (negative control current value is 26.36nA, standard deviation is 5.10, positive control current value is 142.15nA) , sample 2 current value is 28.54nA) and 18 subtype group (negative control current value is 23.51nA, its standard deviation is 5.90, positive control current value is 116.36nA, sample 2 current value is 31.21nA), sample The test result of 2 was negative, indicating that sample 2 did not contain HPV16 subtype and HPV18 subtype.
- the capture probe is fixed at the bottom of the detection orifice plate.
- the capture solution contained the following components: 5% by weight of thiophene, 2 mol/L of NaCl, and 1.5 ⁇ mol/L of capture probe.
- the corresponding column for the experiment was selected on the EFIRM software.
- the electric field parameters were set to: voltage A: 500 mV, 1 s; voltage B: 1500 mV, 1 s; 10 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
- the hybrid buffer (using the hybrid buffer 3 in the Summary of the Invention) was treated in a water bath at 95 ° C for 5 min in a water bath and then allowed to cool at room temperature.
- the samples were taken out from the -20 ° C refrigerator and thawed in a 4 ° C refrigerator. After complete dissolution, the sample and the hybrid buffer are mixed at a volume ratio of 1:2.5, vortexed and centrifuged, and the sample can be tested.
- a blank control buffer, a corresponding concentration of the negative control (WT) and a positive control (MT) were added to the corresponding wells, and the sample volume was 80 ⁇ l.
- WT negative control
- MT positive control
- the tip of the gun is attached to the bottom of the hole, but the bottom electrode is not touched.
- the tilting or tapping of the detecting plate allows the liquid to uniformly cover the surface of the electrode in the hole, and then immediately goes to the EFIMR for electric field operation.
- the corresponding column for the experiment was selected on the EFIRM software.
- the electric field parameters were set to: voltage A: 500 mV, 1 s; voltage B: 800 mV, 1 s; 10 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
- the detection solution was PBS as a solvent, wherein the weight percentage of casein was 5%, the concentration of the detection probe was 1.5 ⁇ mol/L, vortexed and mixed, centrifuged, and set aside.
- the corresponding column for the experiment was selected on the EFIRM software.
- the electric field parameters were set to: voltage A: 500 mV, 1 s; voltage B: 800 mV, 1 s; 8 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
- TMB/H2O2 solution (purchased from thermo fisher, product no. 34022, named as
- the electric field parameter is set to: voltage A: -300mV, 100s, and the current reading is obtained.
- test results were in agreement with the results of Examples 1-6 obtained based on the procedure described in Example 1.
- test sample the specific steps are as follows:
- the capture probe is fixed at the bottom of the detection orifice plate.
- the capture solution contained the following components: the weight percentage of aniline was 0.1%, the concentration of NaCl was 0.01 mol/L, and the concentration of the capture probe was 0.5 ⁇ mol/L.
- the corresponding column for the experiment was selected on the EFIRM software.
- the electric field parameters were set to: voltage A: 200 mV, 5 s; voltage B: 800 mV, 5 s; 3 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
- the hybrid buffer (using the hybrid buffer 6 in the Summary of the Invention) was treated in a water bath at 85 ° C for 15 min in a water bath and then left to cool at room temperature.
- the samples were taken out from the -20 ° C refrigerator and thawed in a 4 ° C refrigerator. After complete dissolution, the sample and the hybrid buffer are mixed at a volume ratio of 1:1.5, vortexed and centrifuged, and the sample can be tested.
- a blank control buffer, a corresponding concentration of the negative control (WT) and a positive control (MT) were added to the corresponding wells, and the amount of the sample was 20 ⁇ l.
- WT negative control
- MT positive control
- the tip of the gun is attached to the bottom of the hole, but the bottom electrode is not touched.
- the tilting or tapping of the detecting plate allows the liquid to uniformly cover the surface of the electrode in the hole, and then immediately goes to the EFIMR for electric field operation.
- the corresponding column for the experiment was selected on the EFIRM software.
- the electric field parameters were set to: voltage A: 200 mV, 5 s; voltage B: 300 mV, 5 s; 3 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
- the detection solution was PBS as a solvent, wherein the weight percentage of casein was 0.1%, the concentration of the detection probe was 0.5 ⁇ mol/L, vortexed and mixed, centrifuged, and set aside.
- the corresponding column for the experiment was selected on the EFIRM software.
- the electric field parameters were set to: voltage A: 200 mV, 5 s; voltage B: 300 mV, 5 s; 3 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
- the electric field parameter is set to: voltage A: -100mV, 40s, and the current reading is obtained.
- the detecting orifice plate used in the method of the present invention has the common feature that the inner bottom of the reaction hole is provided with an electrode for applying an electric field to the solution in the reaction hole after the EFIRM detector is turned on, and the commercially available product can be used.
- the capture probe is immobilized in the reaction well during use, or prefabricated for use or for sale.
- the detection orifice plate is shown in FIG. 7 and includes: a reaction well bottom plate 101, the reaction well bottom plate 101 includes at least one detection region 102, a working electrode 103, and the working electrode 103 is disposed on the reaction well bottom plate 101. And configured to apply a voltage to form an electric field; and the opposite electrode 104, the opposite electrode 104 is disposed on the reaction aperture substrate 101 and configured to acquire a detection signal and output the detection signal.
- the working electrode 103 and the opposite electrode 104 are both disposed on the same surface of the reaction cell bottom plate, and therefore, the working electrode 103 and the opposite electrode 104 may be in the same plane. As shown in FIG.
- the working electrode 103 includes at least one first linear portion 1031 having a uniform width; the opposite electrode 104 includes at least one second linear portion 1041 of uniform width; the first linear portion 1031 and the second linear portion 1041 They are disposed within the detection zone 102 and are alternately spaced apart from one another.
- the electrode 104 located on the right side of FIG. 1 can be configured as a working electrode to apply a voltage to form an electric field; the electrode 103 located on the left side of FIG.
- the counter electrode is configured to acquire a detection signal and output the detection signal, and the disclosure is not limited herein.
- the working electrode 103 can apply a voltage to generate an electric field to move and concentrate the target substance.
- the working electrode 103 can apply a square wave alternating voltage to first include the target substance in the liquid to be detected.
- the charged substance moves to the working electrode 103 to be enriched, so that the target substance can be combined with the probe on the working electrode 103, and then the polarity of the voltage is changed, so that other substances in the charged substance that are not combined with the probe are away from the working electrode 103 (
- the force of the electric field on the target substance is set to be smaller than the binding force of the target substance and the probe; then, the opposite electrode 104 can acquire a detection signal about the target substance and output the detection signal, for example, a target substance bound to the probe.
- the current reacts with a specific reagent to generate a current, so the opposite electrode 104 can acquire a detection signal about the target substance by detecting the current and output the detection signal; and then, by analyzing the output detection signal, the target substance can be obtained.
- Information (such as the concentration of the target substance) so that it can be detected quickly and accurately. Since the first linear portion 1031 and the second linear portion 1041 are linear structures having a uniform width, and within the detection region 102, the first linear portion 1031 and the second linear portion 1041 are alternately equidistantly spaced within the detection region 102.
- the first linear portion 1031 of the working electrode 103 can generate a uniform electric field in the detection region 102, and the second linear portion 1041 of the opposite electrode 104 can detect a minute current in the detection region 102, thus Can improve the accuracy of detection.
- the density and uniformity of the probe formed on the working electrode 103 can be controlled, and the probe is not overly dense, thereby giving The binding of the target substance to the probe provides space to increase the efficiency of binding of the target substance to the probe, thereby improving the reaction speed of the liquid biopsy and further improving the accuracy of the detection.
- the detecting electrode structure in the detecting orifice plate has a circular shape
- the working electrode 103 may include an arc-shaped first body portion 1030 and a plurality of extending from the first body portion 1030.
- the first linear portion 1031 that is parallel to each other.
- the counter electrode 104 includes an arc-shaped second body portion 1040 and a plurality of second linear portions 1041 that are parallel to each other and extend from the second body portion 1040.
- the first body portion 1030 is disposed opposite to the second body portion 1040, and the plurality of first linear portions 1031 and the plurality of second linear portions 1041 are disposed in the detection region 102 and are alternately spaced and equidistantly disposed.
- the working electrode 103 and the opposite electrode 104 have a comb-like structure, and the working electrode 103 and the opposite electrode 104 cross each other to form an interdigitated structure.
- the range of the detection area may include a plurality of first linear portions and a plurality of second linear portions, and may further include a first main body portion and a second main body portion, which are not limited herein.
- the working electrode may not be provided with a fixing portion that is in direct contact with the probe, so the first linear portion may be formed into a line having a uniform width, thereby providing a more uniform electric field, so that the arrangement of the probe is more regular, thereby improving the efficiency and accuracy of detection. .
- the detecting area 102 has a circular shape
- the working electrode 103 includes a first linear portion 1031 which is spirally arranged
- the opposite electrode 104 includes a second line which is spirally arranged.
- the portion 1041, the first linear portion 1031 and the second linear portion 1041 are disposed in the detection region 102 and are alternately spaced and equidistantly disposed.
- the width of the first linear portion is the same as the width of the second linear portion, so that the accuracy of the detection can be improved; in addition, the width of the first linear portion and the second linear portion
- the range can be 3-20 mils (thousandths of an inch).
- the pitch of the first linear portion and the second linear portion may range from 3 to 20 mils (thousandths of an inch).
- the width of the first linear portion and the width of the second linear portion are equal to the spacing between the first linear portion and the second linear portion.
- FIG. 9 is a plan view showing another structure of the detecting electrode
- FIG. 10 is a plan view showing another structure of the detecting electrode.
- the detecting electrode structure provided in an example of the embodiment further includes setting.
- the reference electrode At the reference electrode at the edge of the detection zone, since the reference electrode is disposed at the edge of the detection zone, the outer side of the first linear portion and the second linear portion, the reference electrode can provide a contrast in the process of acquiring the detection signal about the target substance. The polarity error of the working electrode is eliminated, thereby further improving the accuracy of the detection.
- the material of the working electrode and the opposing electrode includes gold. Since the chemical nature of the gold element is stable and does not react with the liquid to be detected and has a lower impedance, the accuracy of the detection can be further improved.
- other conductive materials such as platinum or indium tin oxide may also be used.
- Figures 11a and 11b illustrate a detection orifice plate used in the method of the present invention, the detection orifice plate comprising: a cartridge body 200 and a detection electrode structure 100, the cartridge body 200 comprising a plurality of reaction wells 211, the size of the reaction wells Reference may be made to the design of a conventional 96-well plate. Of course, the present disclosure includes but is not limited thereto, and the size of the reaction well may be designed according to the concentration and kind of the liquid to be detected. As shown in FIGS.
- the detecting electrode structure 100 is disposed at the bottom of the casing 200
- the detecting electrode structure 100 may be the detecting electrode structure of any of the above-described first embodiment
- the detecting portion 102 is disposed at the bottom of the reaction hole 211.
- the bottom of the reaction well 211 is sealed.
- the reaction cell bottom plate 101 of the detecting electrode structure 100 and the casing 200 can be made of the same material. Thereby, the liquid to be detected can be contained in the accommodating space composed of the detection zone 102 and the reaction well 211, thereby detecting the liquid to be detected.
- the working electrode 103 may apply a square wave alternating voltage to form a vertical electric field perpendicular to the bottom surface of the through hole 211, first causing the charged substance including the target substance in the liquid to be detected from the respective positions of the reaction hole 211 to the reaction hole 211.
- the bottom moves and moves to the working electrode 103 to enrich, so that the target substance can be combined with the probe on the working electrode 103 (a substance that can bind to the target substance, such as a DNA polymer molecule), and then the polarity of the voltage is converted to make it perpendicular to the pass.
- the direction of the vertical electric field on the bottom surface of the hole 211 is reversed, so that other substances not charged with the probe in the charged substance in the liquid to be detected move from the bottom of the reaction hole 211 to the upper portion of the reaction hole 211, thereby causing other substances in the charged substance.
- the substance not bound to the probe is away from the working electrode 103 (the force of the electric field on the target substance is set to be smaller than the binding force of the target substance and the probe); then, the opposite electrode 104 can acquire the detection signal about the target substance and detect
- the signal output for example, the target substance bound to the probe reacts with a specific reagent to generate a current, so the opposite electrode 104 can pass the inspection.
- the current is measured to obtain a detection signal about the target substance and the detection signal is output; then, the information about the target substance (for example, the concentration of the target substance) can be obtained by analyzing the output detection signal.
- a plurality of reaction wells 211 are arranged in a matrix in the cartridge body 200.
- the plurality of reaction holes 211 are cylindrical through holes.
- the shape of the reaction well includes, but is not limited to, the shape of the plurality of reaction holes 211 may also be a square cylinder, a triangular cylinder or other cylinders.
- the number of the plurality of reaction holes 211 is a multiple of four, as shown in FIG. 8, four adjacent reaction holes 211 correspond to the four working electrodes 103 in the detection electrode structure. Electrically connected.
- the reaction hole bottom plate 101 is formed with a wire 111 and a wire 112.
- the wire 111 electrically connects the four working electrodes 103.
- the wires 112 are electrically connected to the four opposite electrodes 104 to extract the electrical signals of the opposite electrode 104.
- the four adjacent through holes can be used as a detection group.
- the voltages applied to the four working electrodes are uniform, and four adjacent through holes are The control experiment can be performed better, so the detection accuracy can be further improved.
- any number of working electrodes in the detection electrode structure corresponding to any number of through holes may be electrically connected to provide a uniform voltage.
- the detection aperture board used in the present invention further includes: a circuit board 110 electrically connected to the detection pole structure.
- An amplification circuit may be disposed on the circuit board 110 to amplify the electrical signal outputted by the opposite electrode or the reference electrode to improve detection accuracy;
- a voltage stabilization circuit may also be disposed on the circuit board 110 to provide a stable voltage to the working electrode to improve detection accuracy.
- the present disclosure includes but is not limited thereto, and an overcurrent, overvoltage protection circuit, or the like may be disposed on the circuit board 110.
- the circuit board 110 can be disposed under the reaction hole bottom plate 101, so that the space can be utilized more reasonably.
- the circuit board 110 can also be disposed at other positions, and the disclosure is not limited herein.
- virus genotyping detection kit provided by the present invention has the following advantages:
- the conventional probe fixing method is to fix one end of the probe on the planar support. This method reduces the hybridization efficiency between the probe and the target DNA to be detected due to the hydrophobicity of the surface of the support, etc.
- the invention fixes the capture probe in the polypyrrole hole by charge adsorption to ensure the ultra-high activity of the capture probe; the traditional nucleic acid hybridization process improves the hybridization efficiency by controlling the hybridization temperature, the salt ion, the reaction time, etc., and the invention increases the electric field.
- the capture efficiency of the capture probe to the DNA of the target sequence is improved by the electric field; in this method, the electronic signal generated by the HRP-catalyzed oxidation of TMB is determined as a detection result, since the catalytic efficiency of the enzyme is very high.
- High, indirect amplification of the results of the hybridization reaction increases the sensitivity of the assay.
- the three core technologies of instantaneous target molecular capture, ultra-high activity molecular probe immobilization, and capture molecular signal specific amplification ensure that the method has ultra-high sensitivity, far higher than Pap smear, TCT technology and HC2 technology, and Polymerase chain reaction and gene chip and other technologies are equivalent.
- each subtype of HPV detection process comprises a capture probe and a detection probe, and the probe length is between 25-40 bp, and the hybridization efficiency is affected by the mismatched base. Obviously, only the target sequence DNA and the two probes can be accurately paired at the same time to have a detection signal, which greatly improves the specificity of the detection.
- the sample does not need to be pre-processed such as purification and PCR amplification, thereby effectively avoiding false positives caused by environmental pollution, and the test operation does not need to be specialized.
- the operator does not need to obtain the permission for clinical gene amplification, and can be operated by general technicians, and the requirements for the experimental environment and the quality of the operators are low.
- EFIRM technology is based on the principle of nucleic acid hybridization, using a uniquely designed electrochemical technique.
- the nucleic acid probe used in the present invention has a length of 25-40 bp, and is selected from the E7 region or the L1 region with a large difference between the HPV subtypes, and artificial synthetic oligonucleotide probes are used, wherein the CP does not need to be modified, DP
- Biotin modification method the preparation of the probe is completed by a commercial DNA chemical synthesis company, which has low technical difficulty and good stability, and some subtypes share CP or DP, which reduces the total number of probes and reduces the cost. .
- PCR-based fluorescence quantification requires both ends of the probe to be modified, and one end is a fluorescent group, and the synthesis cost is high; reverse dot blot hybridization and gene chip technology mostly need to immobilize the hybridization probe to the solid phase carrier, and the treatment process Complex, increasing the cost of testing, and the probe passes The immobilization activity is reduced to a certain extent; the probe used for HC2 is a full-length RNA probe with a length of 7000-8000 bases.
- the preparation process is complicated, time-consuming, costly, and because the probe is very long, hybridization Efficiency is less affected by mismatched bases, and subtypes may cross.
- the EFIRM technology saves the steps of DNA extraction and purification during the detection process, so the cost of the detection reagent is greatly reduced compared to other techniques.
- the EFIRM-based HPV probe detection method has the characteristics of high sensitivity, high specificity, short detection time, low experimental site requirement, low cost, and the like, and is suitable for a large number of clinical tests and large-scale epidemics. Pathological screening.
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Abstract
Disclosed are a probe combination for HPV virus genotyping detection based on an EFIRM technique, a kit and a method, relating to the field of gene detection technology. The probe combination comprises a plurality of probe pairs, and each probe pair is directed towards one HPV virus genotype, can detect the genotype of the HPV virus in a sample to be detected, and has a relatively strong specificity, a relatively high sensitivity and a relatively low false positive rate. The detection results are accurate and reliable.
Description
交叉引用cross reference
本发明享有2016年9月30日提交的发明名称为“HPV高危毒株基因分型检测的探针组合、试剂盒以及方法”,申请号为201610877670.0;发明名称为“一种基于EFIRM技术HPV病毒检测的探针组合、试剂盒以及方法”申请号为201610881088.1以及发明名称为“一种用于病毒基因分型检测的探针组合、试剂盒以及方法”,申请号为201610877702.7的三项发明专利申请的优先权,以及2016年12月26日提交的,实用新型名称为“检测电极结构以及检测孔板”申请号为201620769829.2的中国实用新型专利申请的优先权,上述四个中国专利申请的全部内容,在此基于所有的目的,通过引用的方式并入本发明中。The invention enjoys the invention entitled "probe combination, kit and method for genotyping detection of HPV high-risk strain" submitted on September 30, 2016, the application number is 201610877670.0; the invention name is "an HPV virus based on EFIRM technology" Detected probe combination, kit and method" application number is 201610881088.1 and the invention name is "a probe combination, kit and method for virus genotyping detection", and three invention patent applications with application number 201610877702.7 Priority of the Chinese Utility Model Patent Application No. 201620769829.2, entitled "Detection Electrode Structure and Detection Orifice", submitted on December 26, 2016, the entire contents of the above four Chinese patent applications This is hereby incorporated by reference in its entirety for all purposes.
本发明涉及基因检测技术领域,具体而言,涉及一种基于EFIRM技术HPV病毒检测的探针试剂盒、试剂盒以及方法。The invention relates to the field of gene detection technology, in particular to a probe kit, a kit and a method based on the EFIRM technology HPV virus detection.
宫颈癌是女性生殖道最常见的恶性肿瘤之一,已有研究表明,高危型人乳头瘤病毒(Human Papillomavirus,HPV)持续感染及多重感染是导致宫颈癌变的重要原因之一,全球范围的研究结果显示,在99.7%的宫颈癌患者体内检测到高危型HPV DNA的存在。目前已经发现的HPV有200多种型别,主要包括低危型和高危型两大类。不同型别与不同疾病的相关性也不一样,低危型HPV感染可能会引起生殖道湿疣病变,高危型HPV感染则与宫颈癌、阴道癌相关。目前还缺乏针对HPV公认的有效治疗手段,因此宫颈HPV早发现、早预防是阻断癌变的关键。Cervical cancer is one of the most common malignant tumors in the female genital tract. Studies have shown that high-risk human papillomavirus (HPV) persistent infection and multiple infections are one of the important causes of cervical cancer, a worldwide study. The results showed that the presence of high-risk HPV DNA was detected in 99.7% of cervical cancer patients. At present, there are more than 200 types of HPVs, including low-risk and high-risk types. Different types have different correlations with different diseases. Low-risk HPV infection may cause genital warts and high-risk HPV infection is associated with cervical cancer and vaginal cancer. At present, there is still a lack of effective treatments for HPV, so early detection and early prevention of cervical HPV is the key to blocking cancer.
多年来,国际上通用的宫颈上皮内瘤变及宫颈癌的诊断主要遵循“三阶梯式”诊断程序,即宫颈细胞学、阴道镜及组织病理学检查。由于HPV不能体外培养,HPV检测方法主要基于HPV DNA的分子分型。HPV核酸检测技术主要包括杂交捕获法,PCR-荧光探针法、转录介导的核酸扩增技术以及PCR-杂交法等。For many years, the internationally accepted diagnosis of cervical intraepithelial neoplasia and cervical cancer mainly follows the "three-step" diagnostic procedure, namely cervical cytology, colposcopy and histopathology. Since HPV cannot be cultured in vitro, the HPV assay is based primarily on molecular typing of HPV DNA. HPV nucleic acid detection technology mainly includes hybridization capture method, PCR-fluorescence probe method, transcription-mediated nucleic acid amplification technology and PCR-hybridization method.
宫颈癌的早期筛查方式是采用巴氏涂片以及改进后的TCT技术,这些方法只能从细胞学病变的角度来评估宫颈癌的发病情况,也就是说需要等到遭受病毒感染的细胞开始出现明显变化时才可以检测出来,特异性和灵敏度均不够理想。随着对HPV感染导致宫颈癌的认识加深以及分子诊断技术的发展,HPV基因检测作为宫颈癌的一种筛查手段得到了更广泛的应用。目前HPV分型检测技术主要基于聚合酶链反应,如荧光定量、反向斑点杂交和基因芯片技术等,这些技术表现出高灵敏度、高特异性,可以进行多重感染检测等优点,但这些技术对实验条件和检验人员素质要求高,检测费用也较高,不太适用于大量的临床检测和大规模的流行病学筛查。第二代基因杂交捕获技术检测采用美国Digene公司的HC2技术试剂盒,HC2是一种应用微孔板化学发光进行信号放大的核酸杂交检测方法,被公认为评价HPV检测新技术的金标准。然而,HC2检测方法价格昂贵,且其方法操作繁琐,操作过程中容易交叉污染而出现假阳性。The early screening method for cervical cancer is to use Pap smear and improved TCT technology. These methods can only evaluate the incidence of cervical cancer from the perspective of cytological lesions, that is, it needs to wait until the cells infected with the virus begin to appear. It can be detected when there is a significant change, and the specificity and sensitivity are not ideal. With the deepening of the understanding of cervical cancer caused by HPV infection and the development of molecular diagnostic techniques, HPV gene detection has become more widely used as a screening method for cervical cancer. At present, HPV typing detection technology is mainly based on polymerase chain reaction, such as fluorescence quantification, reverse dot blot hybridization and gene chip technology. These technologies show high sensitivity, high specificity, and can carry out multiple infection detection, but these technologies are The experimental conditions and the quality of the inspectors are high, and the testing costs are also high, which is not suitable for a large number of clinical tests and large-scale epidemiological screening. The second generation of gene hybridization capture technology is detected by the HC2 technology kit of Digene, USA. HC2 is a nucleic acid hybridization detection method using microplate chemiluminescence for signal amplification, and is recognized as the gold standard for evaluating new HPV detection technology. However, the HC2 detection method is expensive, and the method is cumbersome to operate, and it is easy to cross-contamination during operation and false positives occur.
发明内容Summary of the invention
本发明的一方面的贡献在于提供基于EFIRM技术HPV病毒检测的探针试剂盒,该探针组合能够对待测样本中的病毒进行基因型检测,具有较强的特异性、较高的灵敏度以及较低的假阳性率,且检出结果准确可靠。A contribution of an aspect of the present invention is to provide a probe kit based on EFIRM technology HPV virus detection, which can detect genotype of virus in a sample to be tested, has strong specificity, high sensitivity and Low false positive rate, and the detection results are accurate and reliable.
本发明的第二方面的贡献在于在于提供基于EFIRM技术HPV病毒检测的试剂盒和检测孔板,该试剂盒能够对待测样本中的病毒进行基因型检测,其具有较强的特异性和较高的灵敏度,且检出结果准确可靠、假阳性率低、操作简单方便。The second aspect of the present invention is to provide a kit and a detection plate for detecting HPV virus based on EFIRM technology, which is capable of genotyping detection of a virus in a sample to be tested, which has strong specificity and high sensitivity. Sensitivity, and the detection results are accurate and reliable, the false positive rate is low, and the operation is simple and convenient.
本发明的三方面的贡献在于提供基于EFIRM技术HPV病毒基因分型检测的方法,该方法能够对待测样本中的病毒进行基因型检测,具有较强的特异性和较高的灵敏度,且检出结果准确可靠,检测方法简单易操作。The contribution of the three aspects of the present invention is to provide a method for genotyping detection of HPV virus based on EFIRM technology, which can detect genotype of virus in a sample to be tested, has strong specificity and high sensitivity, and is detected. The result is accurate and reliable, and the detection method is simple and easy to operate.
本发明解决其技术问题是采用以下技术方案来实现的。The technical problem solved by the present invention is achieved by the following technical solutions.
一种用于HPV病毒基因分型检测的探针试剂盒,其特征在于,A probe kit for HPV virus genotyping detection, characterized in that
包括多条捕获探针,每条所述捕获探针用于结合一种HPV病毒基因型上的目标序列;
Including a plurality of capture probes, each of the capture probes being used to bind a target sequence on a HPV viral genotype;
所述多个捕获探针包括:第1捕获探针和第2捕获探针、选自第3~11捕获探针的至少一个、以及选自第12~16捕获探针中的至少一个;其中,其中,The plurality of capture probes include: a first capture probe and a second capture probe, at least one selected from the group consisting of the third to 11th capture probes, and at least one selected from the group consisting of the 12th to 16th capture probes; ,among them,
第1捕获探针,用于检测HPV 16亚型,碱基序列如SEQ ID NO.1所示;a first capture probe for detecting a HPV 16 subtype, the base sequence of which is set forth in SEQ ID NO.
第2捕获探针,用于检测HPV 18亚型,碱基序列如SEQ ID NO.3所示;a second capture probe for detecting a HPV 18 subtype, the base sequence of which is set forth in SEQ ID NO.
第3捕获探针,用于检测HPV 31亚型,碱基序列如SEQ ID NO.5所示;a third capture probe for detecting a HPV 31 subtype, the base sequence of which is set forth in SEQ ID NO.
第4捕获探针,用于检测HPV 33亚型或52亚型或58亚型的,碱基序列如SEQ ID NO.6所示;a fourth capture probe for detecting HPV 33 subtype or 52 subtype or 58 subtype, the base sequence is shown in SEQ ID NO.
第5捕获探针,用于检测HPV 35亚型,碱基序列如SEQ ID NO.7所示;a fifth capture probe for detecting the HPV 35 subtype, the base sequence of which is set forth in SEQ ID NO.
第6捕获探针,用于检测HPV 39亚型,碱基序列如SEQ ID NO.8所示;a sixth capture probe for detecting a subtype of HPV 39, the base sequence of which is set forth in SEQ ID NO.
第7捕获探针,用于检测HPV 45亚型,碱基序列如SEQ ID NO.9所示;a 7th capture probe for detecting the HPV 45 subtype, the base sequence of which is set forth in SEQ ID NO.
第8捕获探针,用于检测HPV 51亚型,碱基序列如SEQ ID NO.10所示;An 8th capture probe for detecting a HPV 51 subtype, the base sequence of which is set forth in SEQ ID NO.
第9捕获探针,用于检测HPV 56亚型,碱基序列如SEQ ID NO.11所示;a ninth capture probe for detecting a HPV 56 subtype, the base sequence of which is set forth in SEQ ID NO.
第10捕获探针,用于检测HPV 59亚型,碱基序列如SEQ ID NO.12所示;a 10th capture probe for detecting a HPV 59 subtype, the base sequence of which is set forth in SEQ ID NO.
第11捕获探针,用于检测HPV 68亚型,碱基序列如SEQ ID NO.13所示,An 11th capture probe for detecting the HPV 68 subtype, the base sequence is set forth in SEQ ID NO.
第12捕获探针,用于检测HPV 26亚型,碱基序列如SEQ ID NO.20所示,a 12th capture probe for detecting the HPV 26 subtype, the base sequence is set forth in SEQ ID NO.
第13捕获探针,用于检测HPV 53亚型,碱基序列如SEQ ID NO.21所示,a 13th capture probe for detecting the HPV 53 subtype, the base sequence is set forth in SEQ ID NO.
第14捕获探针,用于检测HPV 66亚型,碱基序列如SEQ ID NO.22所示a 14th capture probe for detecting the HPV 66 subtype, the base sequence is set forth in SEQ ID NO.
第15捕获探针,用于检测HPV 73亚型,碱基序列如SEQ ID NO.23所示,a 15th capture probe for detecting the HPV 73 subtype, the base sequence is set forth in SEQ ID NO.
第16捕获探针,用于检测HPV 82亚型,碱基序列如SEQ ID NO.24所示。The 16th capture probe was used to detect the HPV 82 subtype, and the base sequence is shown in SEQ ID NO.
优选地,还包括多条与所述捕获探针相配合且可结合相应的所述目标序列的检测探针,具体如下:Preferably, the method further comprises a plurality of detection probes that cooperate with the capture probe and can bind the corresponding target sequence, as follows:
与所述第1捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.2所示,The base sequence of the detection probe complexed with the first capture probe is as shown in SEQ ID NO.
与所述第2捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.4所示,The base sequence of the detection probe complexed with the second capture probe is as shown in SEQ ID NO.
与所述第3捕获探针或所述第5捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.14所示,The base sequence of the detection probe complexed with the third capture probe or the fifth capture probe is as shown in SEQ ID NO.
与所述第4捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.15所示,The base sequence of the detection probe complexed with the fourth capture probe is as shown in SEQ ID NO.
与所述第6捕获探针或所述第11捕获探针捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.16所示,The base sequence of the detection probe complexed with the sixth capture probe or the 11th capture probe capture probe is as shown in SEQ ID NO.
与所述第7捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.17所示,The base sequence of the detection probe complexed with the seventh capture probe is as shown in SEQ ID NO.
与所述第8捕获探针或所述第9捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.18所示,The base sequence of the detection probe complexed with the eighth capture probe or the ninth capture probe is as shown in SEQ ID NO.
与所述第10捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.19所示,The base sequence of the detection probe complexed with the 10th capture probe is as shown in SEQ ID NO.
与所述第12捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.26所示,The base sequence of the detection probe complexed with the 12th capture probe is as shown in SEQ ID NO.
与所述第13捕获探针或所述第14捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.25所示,The base sequence of the detection probe complexed with the 13th capture probe or the 14th capture probe is as shown in SEQ ID NO.
与所述第15捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.27所示,The base sequence of the detection probe complexed with the 15th capture probe is as shown in SEQ ID NO.
所述第16捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.28所示。The base sequence of the detection probe to which the 16th capture probe is ligated is shown in SEQ ID NO.
一种用于中高危HPV病毒基因分型检测的探针试剂盒,其特征在于,A probe kit for genotyping detection of high-risk HPV viruses, characterized in that
包括第3~11捕获探针中的至少一个,每条所述捕获探针用于结合一种HPV病毒基因型上的目标序列;Including at least one of the 3rd to 11th capture probes, each of the capture probes being used to bind a target sequence on an HPV viral genotype;
其中,among them,
第3捕获探针,用于检测HPV 31亚型,碱基序列如SEQ ID NO.5所示;a third capture probe for detecting a HPV 31 subtype, the base sequence of which is set forth in SEQ ID NO.
第4捕获探针,用于检测HPV 33亚型或52亚型或58亚型的,碱基序列如SEQ ID NO.6所示;a fourth capture probe for detecting HPV 33 subtype or 52 subtype or 58 subtype, the base sequence is shown in SEQ ID NO.
第5捕获探针,用于检测HPV 35亚型,碱基序列如SEQ ID NO.7所示;a fifth capture probe for detecting the HPV 35 subtype, the base sequence of which is set forth in SEQ ID NO.
第6捕获探针,用于检测HPV 39亚型,碱基序列如SEQ ID NO.8所示;a sixth capture probe for detecting a subtype of HPV 39, the base sequence of which is set forth in SEQ ID NO.
第7捕获探针,用于检测HPV 45亚型,碱基序列如SEQ ID NO.9所示;a 7th capture probe for detecting the HPV 45 subtype, the base sequence of which is set forth in SEQ ID NO.
第8捕获探针,用于检测HPV 51亚型,碱基序列如SEQ ID NO.10所示;An 8th capture probe for detecting a HPV 51 subtype, the base sequence of which is set forth in SEQ ID NO.
第9捕获探针,用于检测HPV 56亚型,碱基序列如SEQ ID NO.11所示;a ninth capture probe for detecting a HPV 56 subtype, the base sequence of which is set forth in SEQ ID NO.
第10捕获探针,用于检测HPV 59亚型,碱基序列如SEQ ID NO.12所示。
The 10th capture probe was used to detect the HPV 59 subtype, and the base sequence is shown in SEQ ID NO.
优选地,还包括Preferably, it also includes
第1捕获探针,用于检测HPV 16亚型,碱基序列如SEQ ID NO.1所示;和第2捕获探针,用于检测HPV 18亚型,碱基序列如SEQ ID NO.3所示。a first capture probe for detecting the HPV 16 subtype, the base sequence is shown in SEQ ID NO. 1; and a second capture probe for detecting the HPV 18 subtype, the base sequence is SEQ ID NO. Shown.
优选地,还包括多条与所述捕获探针相配合且可结合相应的所述目标序列的检测探针,具体如下:Preferably, the method further comprises a plurality of detection probes that cooperate with the capture probe and can bind the corresponding target sequence, as follows:
与所述第1捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.2所示,The base sequence of the detection probe complexed with the first capture probe is as shown in SEQ ID NO.
与所述第2捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.4所示,The base sequence of the detection probe complexed with the second capture probe is as shown in SEQ ID NO.
与所述第3捕获探针或所述第5捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.14所示,The base sequence of the detection probe complexed with the third capture probe or the fifth capture probe is as shown in SEQ ID NO.
与所述第4捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.15所示,The base sequence of the detection probe complexed with the fourth capture probe is as shown in SEQ ID NO.
与所述第6捕获探针或所述第11捕获探针捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.16所示,The base sequence of the detection probe complexed with the sixth capture probe or the 11th capture probe capture probe is as shown in SEQ ID NO.
与所述第7捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.17所示,The base sequence of the detection probe complexed with the seventh capture probe is as shown in SEQ ID NO.
与所述第8捕获探针或所述第9捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.18所示,The base sequence of the detection probe complexed with the eighth capture probe or the ninth capture probe is as shown in SEQ ID NO.
与所述第10捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.19所示。The base sequence of the detection probe complexed with the 10th capture probe is shown in SEQ ID NO.
一种用于高危HPV病毒基因分型检测的探针试剂盒,其特征在于,包括选自第12~16捕获探针中的至少一个,每条所述捕获探针用于结合一种HPV病毒基因型上的目标序列;其中,A probe kit for high-risk HPV virus genotyping detection, comprising at least one selected from the group consisting of 12th to 16th capture probes, each of which is used to bind an HPV virus a target sequence on a genotype;
第12捕获探针,用于检测HPV 26亚型,碱基序列如SEQ ID NO.20所示,a 12th capture probe for detecting the HPV 26 subtype, the base sequence is set forth in SEQ ID NO.
第13捕获探针,用于检测HPV 53亚型,碱基序列如SEQ ID NO.21所示,a 13th capture probe for detecting the HPV 53 subtype, the base sequence is set forth in SEQ ID NO.
第14捕获探针,用于检测HPV 66亚型,碱基序列如SEQ ID NO.22所示a 14th capture probe for detecting the HPV 66 subtype, the base sequence is set forth in SEQ ID NO.
第15捕获探针,用于检测HPV 73亚型,碱基序列如SEQ ID NO.23所示,a 15th capture probe for detecting the HPV 73 subtype, the base sequence is set forth in SEQ ID NO.
第16捕获探针,用于检测HPV 82亚型,碱基序列如SEQ ID NO.24所示。The 16th capture probe was used to detect the HPV 82 subtype, and the base sequence is shown in SEQ ID NO.
优选地,还包括第1捕获探针,用于检测HPV 16亚型,碱基序列如SEQ ID NO.1所示;和第2捕获探针,用于检测HPV 18亚型,碱基序列如SEQ ID NO.3所示。Preferably, further comprising a first capture probe for detecting the HPV 16 subtype, the base sequence is set forth in SEQ ID NO. 1; and a second capture probe for detecting the HPV 18 subtype, the base sequence is as SEQ ID NO. 3 is shown.
优选地,还包括多条与所述捕获探针相配合且可结合相应的所述目标序列的检测探针,具体如下:Preferably, the method further comprises a plurality of detection probes that cooperate with the capture probe and can bind the corresponding target sequence, as follows:
与所述第1捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.2所示,The base sequence of the detection probe complexed with the first capture probe is as shown in SEQ ID NO.
与所述第2捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.4所示,The base sequence of the detection probe complexed with the second capture probe is as shown in SEQ ID NO.
与所述第12捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.26所示,The base sequence of the detection probe complexed with the 12th capture probe is as shown in SEQ ID NO.
与所述第13捕获探针或所述第14捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.25所示,The base sequence of the detection probe complexed with the 13th capture probe or the 14th capture probe is as shown in SEQ ID NO.
与所述第15捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.27所示,The base sequence of the detection probe complexed with the 15th capture probe is as shown in SEQ ID NO.
与所述第16捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.28所示。The base sequence of the detection probe complexed with the 16th capture probe is shown in SEQ ID NO.
一种用于HPV病毒基因分型检测的探针试剂盒,其特征在于,包括A probe kit for HPV virus genotyping detection, characterized in that
第1捕获探针,用于检测HPV 16亚型,碱基序列如SEQ ID NO.1所示;a first capture probe for detecting a HPV 16 subtype, the base sequence of which is set forth in SEQ ID NO.
和第2捕获探针,用于检测HPV 18亚型,碱基序列如SEQ ID NO.3所示。And a second capture probe for detecting the HPV 18 subtype, the base sequence is shown in SEQ ID NO.
优选地,还包括检测16型HPV的检测探针,碱基序列如SEQ ID NO.2所示,Preferably, the detection probe for detecting type 16 HPV is further included, and the base sequence is as shown in SEQ ID NO.
检测18型HPV的检测探针,碱基序列如SEQ ID NO.4所示。The detection probe of type 18 HPV was detected, and the base sequence is shown in SEQ ID NO.
优选地,所述检测探针的3’端或5’端标记有用于结合催化酶的亲和物,所述催化酶用于催化底物产生化学反应形成电子流。
Preferably, the 3' or 5' end of the detection probe is labeled with an affinity for binding to a catalytic enzyme for catalyzing the chemical reaction of the substrate to form a stream of electrons.
一种基于EFIRM技术的HPV病毒检测的试剂盒,其特征在于,其包括上述任一探针试剂盒中的探针。A kit for detecting HPV virus based on EFIRM technology, characterized in that it comprises a probe in any of the above probe kits.
优选地,所述试剂盒还包括用于将所述捕获探针固定至检测孔板的固定物,所述固定物包括导电聚合物和离子化合物;Preferably, the kit further comprises a fixture for fixing the capture probe to a detection orifice plate, the fixture comprising a conductive polymer and an ionic compound;
所述导电聚合物为选自吡咯、苯胺和噻吩中的任一种;The conductive polymer is any one selected from the group consisting of pyrrole, aniline and thiophene;
所述离子化合物为选自氯化钠和氯化钾中的任一种。The ionic compound is any one selected from the group consisting of sodium chloride and potassium chloride.
优选地,所述试剂盒还包括所述催化酶,所述催化酶是带有标记物的辣根过氧化物酶或碱性磷酸酶,所述标记物用于与所述亲和物结合,所述标记物为地高辛抗体、异硫氰酸荧光素抗体和链霉亲和素中的任一种;Preferably, the kit further comprises the catalytic enzyme, the catalytic enzyme is a labeled horseradish peroxidase or alkaline phosphatase, and the label is used for binding to the affinity, The marker is any one of a digoxin antibody, a fluorescein isothiocyanate antibody, and streptavidin;
所述检测探针的3’端或5’端标记的所述亲和物是与所述标记物相对应的地高辛、异硫氰酸荧光素和生物素中一种。The affinity labeled with the 3' or 5' end of the detection probe is one of digoxin, fluorescein isothiocyanate and biotin corresponding to the label.
优选地,所述试剂盒还包括所述催化酶的所述底物;Preferably, the kit further comprises the substrate of the catalytic enzyme;
当所述催化酶为所述辣根过氧化物酶时,所述底物是TMB、ABTS和OPD中的任一种;When the catalytic enzyme is the horseradish peroxidase, the substrate is any one of TMB, ABTS and OPD;
当所述催化酶为所述碱性磷酸酶时,所述底物是BCIP和NBT的组合物、对硝基苯磷酸盐、4-硝基苯磷酸二钠、萘酚AS-BI磷酸盐、萘酚-AS-MX-磷酸盐中的任一种。When the catalytic enzyme is the alkaline phosphatase, the substrate is a combination of BCIP and NBT, p-nitrophenyl phosphate, disodium 4-nitrobenzene phosphate, naphthol AS-BI phosphate, Any of naphthol-AS-MX-phosphate.
所述试剂盒还包括清洗液,所述清洗液包括洗液A和洗液B,所述洗液A是含SDS的SSC缓冲液,所述洗液B是含Tween20的PBS缓冲液。The kit further includes a cleaning solution comprising a lotion A and a lotion B, the lotion A being an SDS-containing SSC buffer, and the lotion B is a Tween 20-containing PBS buffer.
所述试剂盒还包括所述检测孔板,所述捕获探针按其对应的HPV病毒基因型种类分别固定在所述检测孔板的不同反应孔内;所述检测孔板的反应孔底部设置有工作电极并配置为可施加电压以形成电场。The kit further includes the detection well plate, wherein the capture probes are respectively fixed in different reaction wells of the detection well plate according to their corresponding HPV virus genotypes; There is a working electrode and is configured to apply a voltage to form an electric field.
优选地,所述反应孔底部还设置有对置电极,所述对置电极设置在所述反应孔底板上并配置为获取检测信号并输出所述检测信号;所述工作电极包括至少一个宽度均匀的第一线状部,所述对置电极包括至少一个宽度均匀的第二线状部,所述第一线状部和所述第二线状部在所述反应孔底部相互交替设置;至少两个相邻的所述反应孔中的所述工作电极电性相连。Preferably, the bottom of the reaction hole is further provided with an opposite electrode, and the opposite electrode is disposed on the bottom plate of the reaction hole and configured to acquire a detection signal and output the detection signal; the working electrode includes at least one uniform width a first linear portion, the opposite electrode includes at least one second linear portion having a uniform width, the first linear portion and the second linear portion are alternately arranged at a bottom of the reaction hole; at least two The working electrodes in the adjacent reaction wells are electrically connected.
如图7-15所示,一种基于EFIRM技术的HPV病毒基因分型检测的检测孔板,其特征在于,所述检测孔板的反应孔底部设置有工作电极并配置为可施加电压以形成电场;As shown in FIG. 7-15, a detection plate for HPV virus genotyping detection based on EFIRM technology is characterized in that a bottom of a reaction well of the detection plate is provided with a working electrode and is configured to apply a voltage to form electric field;
所述检测孔板的反应孔内分配并固定有捕获探针,所述分配并固定的捕获探针选自以下任一组:A capture probe is dispensed and fixed in the reaction well of the detection well plate, and the dispensed and fixed capture probe is selected from any one of the following groups:
第1组:包括第1捕获探针和第2捕获探针、选自第3~11捕获探针的至少一个和选自第12~16捕获探针中的至少一个;Group 1 : comprising a first capture probe and a second capture probe, at least one selected from the group consisting of the third to 11th capture probes, and at least one selected from the group consisting of the 12th to 16th capture probes;
第2组:包括第3~11捕获探针中的至少一个;Group 2: comprising at least one of the third to eleven capture probes;
第3组:包括第12~16捕获探针中的至少一个;Group 3: comprising at least one of the 12th to 16th capture probes;
第4组:包括第1捕获探针和第2捕获探针,以及第3~11捕获探针中的至少一个;Group 4: comprising at least one of a first capture probe and a second capture probe, and a third to eleven capture probe;
第5组:包括第1捕获探针和第2捕获探针,以及第12~16捕获探针中的至少一个;其中Group 5: comprising at least one of a first capture probe and a second capture probe, and a 12th to 16th capture probe;
第1捕获探针,用于检测HPV 16亚型,碱基序列如SEQ ID NO.1所示;a first capture probe for detecting a HPV 16 subtype, the base sequence of which is set forth in SEQ ID NO.
第2捕获探针,用于检测HPV 18亚型,碱基序列如SEQ ID NO.3所示;a second capture probe for detecting a HPV 18 subtype, the base sequence of which is set forth in SEQ ID NO.
第3捕获探针,用于检测HPV 31亚型,碱基序列如SEQ ID NO.5所示;a third capture probe for detecting a HPV 31 subtype, the base sequence of which is set forth in SEQ ID NO.
第4捕获探针,用于检测HPV 33亚型或52亚型或58亚型的,碱基序列如SEQ ID NO.6所示;a fourth capture probe for detecting HPV 33 subtype or 52 subtype or 58 subtype, the base sequence is shown in SEQ ID NO.
第5捕获探针,用于检测HPV 35亚型,碱基序列如SEQ ID NO.7所示;a fifth capture probe for detecting the HPV 35 subtype, the base sequence of which is set forth in SEQ ID NO.
第6捕获探针,用于检测HPV 39亚型,碱基序列如SEQ ID NO.8所示;a sixth capture probe for detecting a subtype of HPV 39, the base sequence of which is set forth in SEQ ID NO.
第7捕获探针,用于检测HPV 45亚型,碱基序列如SEQ ID NO.9所示;a 7th capture probe for detecting the HPV 45 subtype, the base sequence of which is set forth in SEQ ID NO.
第8捕获探针,用于检测HPV 51亚型,碱基序列如SEQ ID NO.10所示;An 8th capture probe for detecting a HPV 51 subtype, the base sequence of which is set forth in SEQ ID NO.
第9捕获探针,用于检测HPV 56亚型,碱基序列如SEQ ID NO.11所示;a ninth capture probe for detecting a HPV 56 subtype, the base sequence of which is set forth in SEQ ID NO.
第10捕获探针,用于检测HPV 59亚型,碱基序列如SEQ ID NO.12所示;a 10th capture probe for detecting a HPV 59 subtype, the base sequence of which is set forth in SEQ ID NO.
第11捕获探针,用于检测HPV 68亚型,碱基序列如SEQ ID NO.13所示,An 11th capture probe for detecting the HPV 68 subtype, the base sequence is set forth in SEQ ID NO.
第12捕获探针,用于检测HPV 26亚型,碱基序列如SEQ ID NO.20所示,a 12th capture probe for detecting the HPV 26 subtype, the base sequence is set forth in SEQ ID NO.
第13捕获探针,用于检测HPV 53亚型,碱基序列如SEQ ID NO.21所示,
a 13th capture probe for detecting the HPV 53 subtype, the base sequence is set forth in SEQ ID NO.
第14捕获探针,用于检测HPV 66亚型,碱基序列如SEQ ID NO.22所示a 14th capture probe for detecting the HPV 66 subtype, the base sequence is set forth in SEQ ID NO.
第15捕获探针,用于检测HPV 73亚型,碱基序列如SEQ ID NO.23所示,a 15th capture probe for detecting the HPV 73 subtype, the base sequence is set forth in SEQ ID NO.
第16捕获探针,用于检测HPV 82亚型,碱基序列如SEQ ID NO.24所示,a 16th capture probe for detecting the HPV 82 subtype, the base sequence is set forth in SEQ ID NO.
所述分配是指每一种所述捕获探针固定在不同的所述反应孔中。The dispensing means that each of the capture probes is immobilized in a different one of the reaction wells.
优选地,所述的检测孔板中,所述反应孔底部还设置有对置电极,所述对置电极设置在所述反应孔底板上并配置为获取检测信号并输出所述检测信号;Preferably, in the detecting orifice plate, the bottom of the reaction well is further provided with an opposite electrode, and the opposite electrode is disposed on the reaction well bottom plate and configured to acquire a detection signal and output the detection signal;
所述工作电极包括至少一个宽度均匀的第一线状部,所述对置电极包括至少一个宽度均匀的第二线状部,所述第一线状部和所述第二线状部在所述反应孔底部相互交替设置;The working electrode includes at least one first linear portion having a uniform width, the opposite electrode including at least one second linear portion having a uniform width, and the first linear portion and the second linear portion are in the reaction The bottoms of the holes are alternately arranged;
至少两个相邻的所述反应孔中的所述工作电极电性相连。The working electrodes of at least two adjacent ones of the reaction wells are electrically connected.
优选地,所述的检测孔板中,所述捕获探针是与导电聚合物和离子化合物混合成混合液后加到所述反应孔中,然后通过所述工作电极施加第一方波电场后固定在所述反应孔内底部表面制成;Preferably, in the detecting orifice plate, the capturing probe is mixed with a conductive polymer and an ionic compound to form a mixed solution, and then added to the reaction hole, and then the first square wave electric field is applied through the working electrode. Fixed on the bottom surface of the reaction well;
所述第一电场的参数为:电压A:350mV,1s;电压B:950mV,1s;进行9个循环。The parameters of the first electric field are: voltage A: 350 mV, 1 s; voltage B: 950 mV, 1 s; 9 cycles are performed.
一种基于EFIRM技术的HPV病毒基因分型检测方法,其特征在于,采用权利要求13-19任一所述的试剂盒,步骤如下:A method for detecting HPV virus genotyping based on EFIRM technology, characterized in that the kit according to any one of claims 13-19 is used, the steps are as follows:
(1)采用权利要求20-22任一所述的检测孔板,或所述捕获探针加入到空白检测孔板中,所述反应孔内底部设置有电极,用于接通EFIRM检测仪后对反应孔内溶液施加电场进行聚合反应;接通EFIRM检测仪后对反应孔内溶液施加第一电场进行聚合反应;电场处理完毕,清洗检测孔板(1) The detection orifice plate according to any one of claims 20-22, or the capture probe is added to the blank detection orifice plate, and the bottom of the reaction well is provided with an electrode for turning on the EFIRM detector. Applying an electric field to the solution in the reaction well to carry out polymerization reaction; after the EFIRM detector is turned on, a first electric field is applied to the solution in the reaction well to carry out polymerization reaction; after the electric field treatment is completed, the detection orifice plate is cleaned.
所述第一电场处理的参数为:电压200-500mV,1-5s;电压800-1500mV,1-5s;3-10个循环;The parameters of the first electric field processing are: voltage 200-500 mV, 1-5 s; voltage 800-1500 mV, 1-5 s; 3-10 cycles;
(2)加入待测样本与杂交buffer的混合溶液,对反应孔施加第二电场:电压200-500mV,1-5s;电压300-800mV,1-5s;3-10个循环;清洗检测孔板;(2) adding a mixed solution of the sample to be tested and the hybrid buffer, applying a second electric field to the reaction well: voltage 200-500 mV, 1-5 s; voltage 300-800 mV, 1-5 s; 3-10 cycles; cleaning detection orifice ;
(3)加入所述检测探针溶液,在EFIRM上进行电场操作;(3) adding the detection probe solution and performing an electric field operation on the EFIRM;
所述检测探针溶液中检测探针的浓度为0.5-1.5μmol/L;The concentration of the detection probe in the detection probe solution is 0.5-1.5 μmol/L;
(4)加入酶液,孵育后清洗;(4) adding the enzyme solution, washing after incubation;
(5)加入底物,在电场电压A:-200mV,60s;电压B:0mV,0s;进行1个循环处理后读数,得到电流值。
(5) Adding a substrate, the electric field voltage A: -200 mV, 60 s; voltage B: 0 mV, 0 s; after 1 cycle of processing, reading, to obtain a current value.
本发明提供的基于EFIRM技术HPV病毒检测的探针、试剂盒以及方法的有益效果是:本发明提供的基于EFIRM技术HPV病毒检测的探针组合包括多个探针对,每个探针对包括有用于结合目标序列的捕获探针和与捕获探针相对应的且可结合目标序列的检测探针,捕获探针通过碱基互补原则与目标序列结合,将目标序列捕获固定,进行第一次特异性结合固定;检测探针通过碱基互补原则与目标序列第二特异性结合,检测探针被结合固定;检测探针的3’端或5’端的通过标记的亲和物将催化酶结合,催化酶催化底物释放电流信号,检测释放的电流信号,进而检测出病毒基因型。由于,目标序列只有与捕获探针和检测探针同时准确配对后即经过两次特异性结合,才能有信号的产生,这大大地提高了检测的特异性,使得检出结果准确可靠,假阳性率非常低。且本发明的提供了各探针对的捕获探针的碱基序列,其能够分别捕获固定例如常见型的HPV 16亚型、18亚型以及中高危型的HPV26、53、66、73、82亚型中的目标序列,进而可检测出待测样本中是否含有常见型病毒例如HPV 16亚型、18亚型以及高危型病毒例如HPV HPV26、53、66、73、82亚型中的一种或多种病毒亚型。The beneficial effects of the probes, kits and methods based on the EFIRM technology HPV virus detection provided by the present invention are that the probe combination based on the EFIRM technology HPV virus detection provided by the present invention comprises a plurality of probe pairs, each probe pair comprises There is a capture probe for binding to a target sequence and a detection probe corresponding to the capture probe and capable of binding the target sequence, and the capture probe binds to the target sequence through the principle of base complementation, and the target sequence is captured and fixed for the first time. Specific binding is fixed; the detection probe binds specifically to the target sequence through the principle of base complementation, and the detection probe is bound and immobilized; the labeled affinity of the 3' or 5' end of the detection probe binds the catalytic enzyme The catalytic enzyme catalyzes the release of the current signal from the substrate, detects the released current signal, and detects the viral genotype. Since the target sequence can be signaled only after two specific bindings simultaneously with the capture probe and the detection probe, which greatly increases the specificity of the detection, making the detection result accurate and reliable, false positive The rate is very low. And the present invention provides a base sequence of a capture probe of each probe pair, which is capable of separately capturing, for example, a common type of HPV 16 subtype, 18 subtype, and a medium and high risk type of HPV 26, 53, 66, 73, 82. The target sequence in the subtype, which in turn can detect whether the sample to be tested contains one of the common type viruses such as HPV 16 subtype, 18 subtype, and high risk type viruses such as HPV HPV26, 53, 66, 73, 82 subtypes. Or a variety of viral subtypes.
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly described below. It should be understood that the following drawings show only certain embodiments of the present invention, and therefore It should be seen as a limitation on the scope, and those skilled in the art can obtain other related drawings according to these drawings without any creative work.
图1为本发明实施例1的示例性检测结果;1 is an exemplary detection result of Embodiment 1 of the present invention;
图2为本发明实施例2的示例性检测结果;2 is an exemplary detection result of Embodiment 2 of the present invention;
图3为本发明实施例3的示例性检测结果;3 is an exemplary detection result of Embodiment 3 of the present invention;
图4为本发明实施例4的示例性检测结果;4 is an exemplary detection result of Embodiment 4 of the present invention;
图5为本发明实施例5的示例性检测结果;FIG. 5 is an exemplary detection result according to Embodiment 5 of the present invention; FIG.
图6为本发明实施例6的示例性检测结果;6 is an exemplary detection result of Embodiment 6 of the present invention;
图7为本发明公开的检测孔板中的检测电极结构的平面示意图;7 is a schematic plan view showing the structure of a detecting electrode in the detecting orifice plate of the present invention;
图8为本发明公开的检测孔板中另一种检测电极结构的平面示意图;8 is a schematic plan view showing another structure of a detecting electrode in the detecting orifice plate of the present invention;
图9为本发明公开的检测孔板中另一种检测电极结构的平面示意图;9 is a schematic plan view showing another structure of a detecting electrode in the detecting orifice plate of the present invention;
图10为本发明公开的检测孔板中另一种检测电极结构的平面示意图;10 is a schematic plan view showing another structure of a detecting electrode in the detecting orifice plate of the present invention;
图11a为本发明公开的一种检测孔板的立体示意图;Figure 11a is a perspective view of a detection orifice plate according to the present invention;
图11b为本发明公开的一种检测孔板的平面示意图;Figure 11b is a schematic plan view of a detecting orifice plate according to the present invention;
图12为本发明公开的一种检测孔板局部立体示意图;12 is a partial perspective view of a detecting orifice plate according to the present invention;
图13为本发明公开的一种检测孔板的局部立体示意图;Figure 13 is a partial perspective view of a detecting orifice plate according to the present invention;
图14为本发明公开的一种检测孔板的局部立体示意图;Figure 14 is a partial perspective view of a detecting orifice plate according to the present invention;
图15为本发明公开的检测孔板的局部侧视示意图。Figure 15 is a partial side elevational view of the detection orifice plate of the present invention.
以下通过示例性实施例说明本发明,实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。The invention is illustrated by the following examples, which are not indicated in the examples, and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained by commercially available purchase.
本发明采用的EFIRM检测仪,为广州易活生物公司出品,记载于Fang Wei等2009年发表于Clinic Cancer Research上的“Electrochemical Sensor for Multiplex Biomarkers Detection,Clin Cancer Res.2009Jul 1;15(13):4446–4452中,即其中采用的电化学检测仪。The EFIRM detector used in the present invention is produced by Guangzhou Yihuo Biotech Co., Ltd., and is described in "Felvi Chemical Sensor for Multiplex Biomarkers Detection, Clin Cancer Res. 2009 Jul 1; 15 (13) published by Fang Wei et al. In 4446–4452, the electrochemical detector used therein.
本领域技术人员根据本发明的描述,以及上述提到的现有技术,可以采用一般的可发生方波的仪器对反应孔施加方波(csw E-field),也可以采用易活生物科技有限公司前期研发的EFIRMY仪器及配套的软件来实现。According to the description of the present invention and the above-mentioned prior art, a square wave (csw E-field) can be applied to the reaction well by a general square wave-generating instrument, and the living biotechnology limited can also be adopted. The company's pre-developed EFIRMY instruments and supporting software are implemented.
下面对本发明实施例的基于EFIRM技术HPV病毒检测的探针、试剂盒以及方法进行具体说明。
The probes, kits and methods based on the EFIRM technology HPV virus detection according to the examples of the present invention are specifically described below.
通常来说,根据各种HPV亚型在宫颈癌患者出现的概率大小,一般情况下将上述人乳头瘤病毒(Human Papillomavirus,HPV)作如下分类。HPV16、18亚型是临床最常见的,也是该两类型病毒在宫颈癌患者出现的概率最大;HPV31、33、52、58、35、39、45、51、56、59、68亚型是高等风险型型别的(可统称为高危型组),HPV26、53、66、73、82亚型是中等风险型别的(可统称为中高危型组),本发明根据上述病毒亚型的进行探针序列设计,得到的探针组合可用于检测上述病毒亚型。Generally, according to the probability of occurrence of various HPV subtypes in cervical cancer patients, the above-mentioned human papillomavirus (HPV) is generally classified as follows. HPV16, 18 subtypes are the most common clinical, and the two types of viruses have the highest probability of occurrence in cervical cancer patients; HPV 31, 33, 52, 58, 35, 39, 45, 51, 56, 59, 68 subtypes are high Risk type (collectively referred to as high risk group), HPV26, 53, 66, 73, 82 subtypes are medium risk type (collectively referred to as medium and high risk groups), and the present invention is based on the above virus subtypes. The probe sequence is designed and the resulting probe combination can be used to detect the above viral subtypes.
本发明提供的基于EFIRM技术HPV病毒检测的探针组合,其包括多个探针对。每个探针对包括用于结合目标序列的捕获探针和与捕获探针相对应的且可结合目标序列的检测探针。每个探针对的检测探针的3’端或5’端标记有用于结合催化酶的亲和物,该催化酶用于催化其底物产生化学反应形成电子流。The invention provides a probe combination based on EFIRM technology HPV virus detection, which comprises a plurality of probe pairs. Each probe pair includes a capture probe for binding to a target sequence and a detection probe corresponding to the capture probe and conjugateable to the target sequence. The 3' or 5' end of the detection probe of each probe pair is labeled with an affinity for binding to a catalytic enzyme for catalyzing the chemical reaction of its substrate to form a stream of electrons.
多个探针对包括:第1探针对、第2探针对、选自第12~16探针对中的至少一种。以下对各探针对检测的捕获探针和检测探针的碱基序列,以及其检测的HPV亚型进行详细说明。The plurality of probe pairs include a first probe pair, a second probe pair, and at least one selected from the group consisting of the 12th to 16th probe pairs. The base sequence of the capture probe and the detection probe detected by each probe pair, and the HPV subtype thereof detected will be described in detail below.
具体地,用于检测HPV16亚型的第1探针对,其捕获探针的碱基序列如SEQ ID NO.1所示,其检测探针的碱基序列如SEQ ID NO.2所示。Specifically, the first probe pair for detecting the HPV16 subtype has a base sequence of the capture probe as shown in SEQ ID NO. 1, and the base sequence of the detection probe is shown in SEQ ID NO.
用于检测HPV18亚型的第2探针对,其捕获探针的碱基序列如SEQ ID NO.3所示,其检测探针的碱基序列如SEQ ID NO.4所示。The second probe pair for detecting the HPV18 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 3, and the base sequence of the detection probe is shown in SEQ ID NO.
用于检测HPV26亚型的第12探针对,其捕获探针的碱基序列如SEQ ID NO.20所示,其检测探针的碱基序列如SEQ ID NO.26所示;The 12th probe pair for detecting the HPV26 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 20, and the base sequence of the detection probe is shown in SEQ ID NO.
用于检测HPV53亚型的第13探针对,其捕获探针的碱基序列如SEQ ID NO.21所示,其检测探针的碱基序列如SEQ ID NO.25所示;a 13th probe pair for detecting a subtype of HPV53, the base sequence of the capture probe is shown in SEQ ID NO. 21, and the base sequence of the detection probe is shown in SEQ ID NO.
用于检测HPV66亚型的第14探针对,其捕获探针的碱基序列如SEQ ID NO.22所示,其检测探针的碱基序列如SEQ ID NO.25所示;The 14th probe pair for detecting the HPV66 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 22, and the base sequence of the detection probe is shown in SEQ ID NO.
用于检测HPV73亚型的第15探针对,其捕获探针的碱基序列如SEQ ID NO.23所示,其检测探针的碱基序列如SEQ ID NO.27所示;The 15th probe pair for detecting the HPV73 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 23, and the base sequence of the detection probe is shown in SEQ ID NO.
用于检测HPV82亚型的第16探针对,其捕获探针的碱基序列如SEQ ID NO.24所示,其检测探针的碱基序列如SEQ ID NO.28所示。The 16th probe pair for detecting the HPV82 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 24, and the base sequence of the detection probe is shown in SEQ ID NO.
优选地,多个探针对还可包括用于检测高危型HPV的探针对:Preferably, the plurality of probe pairs may further comprise probe pairs for detecting high risk HPV:
用于检测HPV31亚型的第3探针对,其捕获探针的碱基序列如SEQ ID NO.5所示,其检测探针的碱基序列如SEQ ID NO.14所示。A third probe pair for detecting the HPV31 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 5, and the base sequence of the detection probe is shown in SEQ ID NO.
用于检测HPV33亚型或52亚型或58亚型的第4探针对,其捕获探针的碱基序列如SEQ ID NO.6所示,其检测探针的碱基序列如SEQ ID NO.15所示,该三种亚型共用第4探针对。A fourth probe pair for detecting HPV33 subtype or 52 subtype or 58 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 6, and the base sequence of the detection probe is SEQ ID NO. As shown in .15, the three subtypes share the fourth probe pair.
用于检测HPV35亚型的第5探针对,其捕获探针的碱基序列如SEQ ID NO.7所示,其检测探针的碱基序列如SEQ ID NO.14所示。The 5th probe pair for detecting the HPV35 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 7, and the base sequence of the detection probe is shown in SEQ ID NO.
用于检测HPV39亚型的第6探针对,其捕获探针的碱基序列如SEQ ID NO.8所示,其检测探针的碱基序列如SEQ ID NO.16所示。The 6th probe pair for detecting the HPV39 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 8, and the base sequence of the detection probe is shown in SEQ ID NO.
用于检测HPV45亚型的第7探针对,其捕获探针的碱基序列如SEQ ID NO.9所示,其检测探针的碱基序列如SEQ ID NO.17所示。The 7th probe pair for detecting the HPV45 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 9, and the base sequence of the detection probe is shown in SEQ ID NO.
用于检测HPV51亚型的第8探针对,其捕获探针的碱基序列如SEQ ID NO.10所示,其检测探针的碱基序列如SEQ ID NO.18所示。The 8th probe pair for detecting the HPV51 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 10, and the base sequence of the detection probe is shown in SEQ ID NO.
用于检测HPV 56亚型的第9探针对,其捕获探针的碱基序列如SEQ ID NO.11所示,其检测探针的碱基序列如SEQ ID NO.18所示。The ninth probe pair for detecting the HPV 56 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 11, and the base sequence of the detection probe is shown in SEQ ID NO.
用于检测HPV59亚型的第10探针对,其捕获探针的碱基序列如SEQ ID NO.12所示,其检测探针的碱基序列如SEQ ID NO.19所示。The 10th probe pair for detecting the HPV59 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 12, and the base sequence of the detection probe is shown in SEQ ID NO.
用于检测HPV68亚型的第11探针对,其捕获探针的碱基序列如SEQ ID NO.13所示,其检测探针的碱基序列如SEQ ID NO.16所示。The 11th probe pair for detecting the HPV68 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 13, and the base sequence of the detection probe is shown in SEQ ID NO.
其中,检测探针与捕获探针相对应是指,一个捕获探针和一个检测探针组成一个探针对,即可用于检测某类型的病毒。捕获探针和检测探针均根据同一类型的病毒的同一个保
守基因或保守DNA片段即目标序列进行设计的,捕获探针和检测探针能够与该病毒类型的同一个保守基因或保守DNA片段的不同区域或结合位点通过碱基互补原则结合,但捕获探针与检测探针并不互补,碱基序列也不相同,二者与目标序列的结合位点不重合、也不重叠。另外,捕获探针和检测探针与目标序列的结合区域可以是相邻,也可以是间隔多个碱基。只要捕获探针和检测探针能够与同一类型的病毒的同一个保守基因或同一段保守DNA片段通过碱基互补原则结合即可。Wherein, the detection probe corresponds to the capture probe, and one capture probe and one detection probe form a probe pair, which can be used for detecting a certain type of virus. Both the capture probe and the detection probe are based on the same type of virus of the same type.
Designing a target sequence or a conserved DNA fragment, the capture probe and the detection probe can bind to different regions or binding sites of the same conserved gene or conserved DNA fragment of the virus type through the principle of base complementation, but capture The probe is not complementary to the detection probe, and the base sequence is also different, and the binding sites of the two and the target sequence do not overlap or overlap. Further, the binding region of the capture probe and the detection probe to the target sequence may be adjacent or may be separated by a plurality of bases. As long as the capture probe and the detection probe are capable of binding to the same conserved gene of the same type of virus or the same conserved DNA fragment by the principle of base complementation.
需要说明的是,上述各探针对的捕获探针序列和检测探针序列的碱基序列可以是不相互对应的,也就是说,在其他的实施例中,各探针对的捕获探针序列可以是上述所述的碱基序列,而其相应的检测探针的碱基序列可以是取自同一检测亚型病毒的同一DNA保守区域其他的序列的互补;或者在其他的实施例中,各探针对的检测探针序列可以是上述所述的碱基序列,而其相应的捕获探针的碱基序列可以是其他的序列选择自同一检测亚型病毒的同一DNA保守区域其他的序列的互补序列。但本发明给出的各探针对的捕获探针和检测探针的碱基序列,在其进行检测时,其具有更强的特异性、更高的灵敏度等优点,具有检测结果可见实施例。It should be noted that the capture probe sequence of each probe pair and the base sequence of the detection probe sequence may not correspond to each other, that is, in other embodiments, the capture probe of each probe pair The sequence may be the base sequence described above, and the base sequence of the corresponding detection probe may be complementary to other sequences taken from the same DNA conserved region of the same detection subtype virus; or in other embodiments, The detection probe sequence of each probe pair may be the above-mentioned base sequence, and the base sequence of the corresponding capture probe may be another sequence selected from the same DNA conserved region of the same detection subtype virus. Complementary sequence. However, the base sequences of the capture probes and the detection probes of the probe pairs provided by the present invention have the advantages of stronger specificity, higher sensitivity, etc. when they are detected, and the detection results can be seen in the examples. .
另外,在对这些亚型病毒检测中,可以进行分组检测例如仅检测出样本含有某个危险级别(例如高危型组或中高危型组)的病毒,而不必检测出其含有那个组别中的具体某类型病毒。当然,也可以进行具体亚型的检测,例如检测出样本中具体含有高危型组中的某类亚型例如HPV53亚型病毒。具体采用何种检测方法检测至何种程度,得到何种程度的检测结果,检测人员可以根据具体情况来选择,以提供较合理的指导意义,具体分组检测的情况见实施例。In addition, in the detection of these subtype viruses, group detection can be performed, for example, only the virus containing a certain risk level (for example, a high-risk group or a medium-high-risk group) is detected, and it is not necessary to detect that it contains the group. Specific type of virus. Of course, specific subtypes can also be detected, for example, by detecting a subtype of a particular high-risk group, such as the HPV53 subtype virus, in the sample. The specific detection method is used to detect the degree to which the detection result is obtained, and the tester can select according to the specific situation to provide a more reasonable guiding significance. The specific group detection is described in the embodiment.
当然,在其他的实施例中,用于病毒基因分析检测的探针组合可以仅包括第1~16探针对中的一种或二种或三种或多种探针对的情况,只要是从第1~16探针对中选出的任意组合,均属于本发明的保护范围。Of course, in other embodiments, the probe combination for viral gene analysis detection may include only one or two or three or more probe pairs of the first to 16th probe pairs, as long as Any combination selected from the first to 16th probe pairs is within the scope of the present invention.
另外,优选地,上述每个检测探针的3’端或5’端标记的亲和物是生物素,生物素的作用在于与链霉亲和素标记的催化酶结合,通过催化酶催化底物产生的电流释放检测信号。当然,在其他的实施例中,检测探针可以不通过生物素-链霉亲和素的识别系统结合催化酶,可以通过其他的例如抗体/抗原、配体/受体等结合系统来结合催化酶,因此,亲和物可以抗原/抗体中的一种。例如亲和物可以是地高辛或者异硫氰酸荧光素,对应地,催化酶上标记有高辛抗体或异硫氰酸荧光素抗体即可。且亲和物可以标记在检测探针的3’端记,也可以标记在其5’端,二者均可以。In addition, preferably, the affinity of the 3' or 5' end of each of the detection probes is biotin, and the biotin acts to bind to the streptavidin-labeled catalytic enzyme and catalyze the catalytic end. The current generated by the object releases the detection signal. Of course, in other embodiments, the detection probe may bind to the catalytic enzyme without the recognition system of biotin-streptavidin, and may be combined with other catalytic systems such as antibody/antigen, ligand/receptor, etc. The enzyme, therefore, the affinity may be one of an antigen/antibody. For example, the affinity may be digoxin or fluorescein isothiocyanate, and correspondingly, the catalytic enzyme may be labeled with a high-octane antibody or a fluorescein isothiocyanate antibody. The affinity may be labeled at the 3' end of the detection probe or at the 5' end, either.
此外,本发明提供的基于EFIRM技术HPV病毒检测的试剂盒,其包括上述所述的任意一项探针组合。优选地,各探针以溶液的形式独立存在,例如捕获探针以含有捕获探针的捕获探针溶液的形式存在,检测探针以含有检测探针的检测探针溶液的形式存在。各探针溶液含有的探针浓度为可以根据实际情况设置。优选地,各探针溶液含有的探针终浓度为0.5~1.5μM。Furthermore, the kit for detecting HPV virus based on EFIRM technology provided by the present invention comprises any one of the probe combinations described above. Preferably, each probe is independently present in the form of a solution, for example, the capture probe is present in the form of a capture probe solution containing a capture probe, and the detection probe is present in the form of a test probe solution containing the detection probe. The concentration of the probe contained in each probe solution can be set according to actual conditions. Preferably, each probe solution contains a probe having a final concentration of 0.5 to 1.5 μM.
另外,本发明提供的基于EFIRM技术HPV病毒检测的试剂盒还可包括将探针对的捕获探针固定至检测孔板的固定物。固定物包括导电聚合物和离子化合物。导电聚合物选自吡咯、苯胺和噻吩中的一种,当然,导电聚合物也可以是其他的导电聚合物材料。离子化合物选自氯化钠和氯化钾中的任一种。导电聚合物带正电,其在电场的作用下形成网状交联结构,被沉积在反应孔的底部,网状交联结构能够稳定地将捕获探针固定在底部,有助于提高捕获探针的稳定性和捕获能力。In addition, the kit for detecting HPV virus based on EFIRM technology provided by the present invention may further comprise a fixture for fixing the capture probe of the probe pair to the detection well plate. The fixture includes a conductive polymer and an ionic compound. The conductive polymer is selected from one of pyrrole, aniline and thiophene. Of course, the conductive polymer may also be other conductive polymer materials. The ionic compound is selected from any one of sodium chloride and potassium chloride. The conductive polymer is positively charged, and forms a network cross-linked structure under the action of an electric field, and is deposited at the bottom of the reaction hole. The mesh cross-linked structure can stably fix the capture probe at the bottom, which helps to improve the capture probe. Needle stability and capture ability.
另外,本发明提供的基于EFIRM技术HPV病毒检测的试剂盒还可包括催化酶,催化酶是带有标记物的辣根过氧化物酶,优选地,催化酶是带有链霉亲和素标记的辣根过氧化物酶。当然,催化酶也可以是带有标记物的碱性磷酸酶,标记物为地高辛抗体、异硫氰酸荧光素抗体或链霉亲和素中的任一种,标记物与亲和物相对应,其可根据检测探针上的亲和物的类别进行选择。当亲和物是生物素时,标记物为链霉亲和素;当亲和物是地高辛时,
标记物为地高辛抗体;当亲和物是异硫氰酸荧光素时,标记物为异硫氰酸荧光素抗体。只要亲和物与标记物相对应,可相互结合即可。In addition, the kit for detecting HPV virus based on EFIRM technology may further comprise a catalytic enzyme, the catalytic enzyme is a horseradish peroxidase with a label, preferably, the catalytic enzyme is labeled with streptavidin. Horseradish peroxidase. Of course, the catalytic enzyme may also be a labeled alkaline phosphatase, and the label is any one of a digoxin antibody, a fluorescein isothiocyanate antibody or streptavidin, a label and an affinity. Correspondingly, it can be selected based on the type of affinity on the detection probe. When the affinity is biotin, the marker is streptavidin; when the affinity is digoxin,
The marker is a digoxin antibody; when the affinity is fluorescein isothiocyanate, the marker is a fluorescein isothiocyanate antibody. As long as the affinity corresponds to the label, it can be combined with each other.
另外,本发明提供的基于EFIRM技术HPV病毒检测的试剂盒还可包括底物,底物的类别根据催化酶的类别选择。In addition, the kit for detecting HPV virus based on EFIRM technology provided by the present invention may further comprise a substrate, and the class of the substrate is selected according to the type of catalytic enzyme.
当催化酶为辣根过氧化物酶时,底物是TMB(Tetramethylbenzidine、四甲基联苯胺)、ABTS(2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐)和OPD(o-Phenylenediamine、邻苯二胺)中的任一种。TMB、ABTS和OPD均是辣根过氧化物酶的底物,在辣根过氧化物酶的催化作用下发生显色反应并伴随电流产生,有助于提高检测信号的释放。When the catalytic enzyme is horseradish peroxidase, the substrate is TMB (Tetramethylbenzidine, tetramethylbenzidine), ABTS (2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate, 2,2-diazepine) - bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) and OPD (o-Phenylenediamine, o-phenylenediamine). TMB, ABTS and OPD are both horseradish peroxidation The substrate of the enzyme, under the catalysis of horseradish peroxidase, undergoes a color reaction and is accompanied by a current generation, which contributes to an increase in the release of the detection signal.
当催化酶为碱性磷酸酶时,底物是对BCIP(5-Bromo-4-Chloro-3-Indolyl Phosphate、5-溴-4-氯-3-吲哚基-磷酸盐)和NBT(Nitrotetrazolium Blue chloride、四唑硝基蓝)的组合物、硝基苯磷酸盐、4-硝基苯磷酸二钠、萘酚AS-BI磷酸盐、萘酚-AS-MX-磷酸盐中的任一种。When the catalytic enzyme is alkaline phosphatase, the substrate is BCIP (5-Bromo-4-Chloro-3-Indolyl Phosphate, 5-bromo-4-chloro-3-indolyl-phosphate) and NBT (Nitrotetrazolium). Blue chloride, tetrazolium nitroblue) composition, nitrophenyl phosphate, disodium 4-nitrobenzene phosphate, naphthol AS-BI phosphate, naphthol-AS-MX-phosphate .
另外,本发明提供的基于EFIRM技术HPV病毒检测的试剂盒还可包括清洗液,清洗液包括洗液A和洗液B,洗液A是含SDS的SSC缓冲液,洗液B是含Tween20的PBS缓冲液。In addition, the kit for detecting HPV virus based on EFIRM technology may further include a cleaning solution including washing liquid A and washing liquid B, washing liquid A is SDS buffer containing SDS, and washing liquid B is containing Tween20. PBS buffer.
另外,本发明提供的基于EFIRM技术HPV病毒检测的试剂盒还可包括稀释液,稀释液是含酪蛋白的PBS缓冲液。In addition, the kit for detecting HPV virus based on EFIRM technology provided by the present invention may further comprise a diluent, which is a casein-containing PBS buffer.
本发明提供的基于EFIRM技术HPV病毒检测的方法,其包括:The invention provides a method for detecting HPV virus based on EFIRM technology, which comprises:
提供多个探针对,多个探针对包括:第1探针对、第2探针对以及选自第3~11探针对中的至少一种;每个探针对包括用于结合目标序列的捕获探针和与捕获探针相对应的且可结合目标序列的检测探针,检测探针的3’端或5’端标记有可用于结合用于催化相应底物产生化学反应形成电子流的催化酶的亲和物;Providing a plurality of probe pairs, the plurality of probe pairs comprising: a first probe pair, a second probe pair, and at least one selected from the group consisting of 3rd to 11th probe pairs; each probe pair including for binding a capture probe of the target sequence and a detection probe corresponding to the capture probe and capable of binding to the target sequence, and the 3' or 5' end of the detection probe is labeled for binding to catalyze the formation of a chemical reaction of the corresponding substrate. An affinity for a catalytic enzyme of electron flow;
其中,第1探针对的捕获探针的碱基序列如SEQ ID NO.1所示,第2探针对的捕获探针的碱基序列如SEQ ID NO.3所示,第12~16探针对的捕获探针的碱基序列分别如SEQ ID NO.20~24所示。Wherein the base sequence of the capture probe of the first probe pair is as shown in SEQ ID NO. 1, and the base sequence of the capture probe of the second probe pair is as shown in SEQ ID NO. 3, and 12 to 16 The base sequences of the capture probes of the probe pair are shown in SEQ ID NO. 20 to 24, respectively.
捕获探针固定步骤:先将用于结合目标序列的捕获探针通过电场作用固定至检测孔板的反应孔内。捕获探针在电场作用下,往反应孔底部移动沉积,并被固定在反应孔底部。Capture probe immobilization step: The capture probe for binding to the target sequence is first fixed to the reaction well of the detection well by an electric field. The capture probe moves to the bottom of the reaction well under the action of an electric field and is fixed at the bottom of the reaction well.
样本杂交步骤:将待测样本加入至反应孔内。待测样本中的目标序列与捕获探针通过碱基互补原则被捕获固定在反应孔底部。Sample hybridization step: The sample to be tested is added to the reaction well. The target sequence and the capture probe in the sample to be tested are captured and fixed at the bottom of the reaction well by the principle of base complementation.
检测探针结合步骤:加入与捕获探针对应的且可结合目标序列的检测探针至反应孔内,检测探针的3’端或5’端标记有可用于结合用于催化相应底物产生化学反应形成电子流的催化酶的亲和物。检测探针通过碱基互补原则与目标序列结合固定在反应孔内。Detection probe binding step: adding a detection probe corresponding to the capture probe and binding the target sequence to the reaction well, and the 3' end or the 5' end of the detection probe is labeled for binding to catalyze the corresponding substrate production The chemical reaction forms an affinity for the catalytic enzyme of the electron flow. The detection probe is immobilized in the reaction well by binding to the target sequence through the principle of base complementation.
酶催化反应检测步骤:加入催化酶和底物至反应孔内,通过电流检测装置检测反应孔内的电流。检测探针通过亲和物与催化酶结合,催化酶再催化底物产生电流释放检测信号,该信号被电流检测装置识别放大,进而检出结果。Enzyme catalytic reaction detection step: a catalytic enzyme and a substrate are added to the reaction well, and the current in the reaction well is detected by a current detecting device. The detection probe binds to the catalytic enzyme through the affinity, and the catalytic enzyme re-catalyzes the substrate to generate a current release detection signal, which is recognized and amplified by the current detecting device, and the result is detected.
需要说明的是,上述方法中所采用的检测孔板和电流检测装置均可通过市面上购买得到。It should be noted that the detection orifice plate and the current detecting device used in the above method are commercially available.
本发明的检测原理是利用电场诱导释放和测量(Electric Field–Induced Release and Measurement,EFIRM)技术快速检测病毒基因型,尤其是对18种高危型HPV进行检测。EFIRM的基本原理是:The detection principle of the present invention is to rapidly detect viral genotypes by using Electric Field-Induced Release and Measurement (EFIRM) technology, especially for detecting 18 high-risk HPVs. The basic principles of EFIRM are:
在电场作用下捕获探针被固定在检测孔板上的反应孔的孔底;捕获探针在电场作用下通过碱基互补配对原则捕获待测样本中的目标序列即病毒DNA;带有生物素标记的检测探针再与目标序列结合;催化酶通过其标记的链霉亲合素与生物素识别,结合;往反应孔加入催化酶的底物,产生氧化还原反应,有电流产生,仪器检测电流信号,进而判断出待测样本中有相应的目标序列,由于捕获探针和检测探针均是根据特定病毒类型的保守区域进行设计的,进而可知该病毒的类型。The capture probe is immobilized on the bottom of the reaction well on the detection well plate under the action of the electric field; the capture probe captures the target sequence in the sample to be tested, ie, viral DNA, by the principle of base complementary pairing under the electric field; The labeled detection probe is further bound to the target sequence; the catalytic enzyme is recognized by the labeled streptavidin and biotin, and the substrate of the catalytic enzyme is added to the reaction well to generate a redox reaction, current generation, and instrument detection. The current signal is further determined to have a corresponding target sequence in the sample to be tested. Since both the capture probe and the detection probe are designed according to a conserved region of a specific virus type, the type of the virus can be known.
以下结合实施例对本发明的特征和性能作进一步的详细描述。
The features and performance of the present invention are further described in detail below in conjunction with the embodiments.
以下本实施例1-6,采用不同的hpv探针组合对对取自两个不同的宫颈癌患者的宫颈口处的细胞(即待测样本)检测其HPV亚型,取自两个不同患者的待测样本分别命名为样本1和样本2,并对样本1和样本2同时检测,取好的样本1和样本2于-20℃存储。In the following Examples 1-6, different HPV probe combinations were used to detect HPV subtypes from cells at the cervix of two different cervical cancer patients (ie, samples to be tested), taken from two different patients. The samples to be tested are named Sample 1 and Sample 2, respectively, and Sample 1 and Sample 2 are simultaneously detected, and Sample 1 and Sample 2 are stored at -20 °C.
其中设置的阳性对照如表2所示。The positive controls set therein are shown in Table 2.
实施例1Example 1
本实施例以检测HPV基因型为例,对本实施例提供的基于EFIRM技术HPV病毒检测的试剂盒进行说明。In this embodiment, a kit for detecting HPV virus based on EFIRM technology provided in the present embodiment is described by taking the HPV genotype as an example.
本实施例提供的病毒基因分型检测的试剂盒包括探针组合,该探针组合包括16个用于对HPV亚型检测的探针对,各探针对的捕获探针和检测探针以溶液形式独立存在,探针的终浓度均为1μM。该16个探针对分别是:用于检测HPV 16亚型的第1探针对,其捕获探针的碱基序列如SEQ ID NO.1所示,其检测探针的碱基序列如SEQ ID NO.2所示;用于检测HPV 18亚型的第2探针对,其捕获探针的碱基序列如SEQ ID NO.3所示,其检测探针的碱基序列如SEQ ID NO.4所示;用于检测HPV 31亚型的第3探针对,其捕获探针的碱基序列如SEQ ID NO.5所示,其检测探针的碱基序列如SEQ ID NO.14所示;用于检测HPV 33亚型或52亚型或58亚型的第4探针对,其捕获探针的碱基序列如SEQ ID NO.6所示,其检测探针的碱基序列如SEQ ID NO.15所示;用于检测HPV 35亚型的第5探针对,其捕获探针的碱基序列如SEQ ID NO.7所示,其检测探针的碱基序列如SEQ ID NO.14所示;用于检测HPV 39亚型的第6探针对,其捕获探针的碱基序列如SEQ ID NO.8所示,其检测探针的碱基序列如SEQ ID NO.16所示;用于检测HPV 45亚型的第7探针对,其捕获探针的碱基序列如SEQ ID NO.9所示,其检测探针的碱基序列如SEQ ID NO.17所示;用于检测HPV 51亚型的第8探针对,其捕获探针的碱基序列如SEQ ID NO.10所示,其检测探针的碱基序列如SEQ ID NO.18所示;用于检测HPV 56亚型的第9探针对,其捕获探针的碱基序列如SEQ ID NO.11所示,其检测探针的碱基序列如SEQ ID NO.18所示;用于检测HPV 59亚型的第10探针对,其捕获探针的碱基序列如SEQ ID NO.12所示,其检测探针的碱基序列如SEQ ID NO.19所示;用于检测HPV 68亚型的第11探针对,其捕获探针的碱基序列如SEQ ID NO.13所示,其检测探针的碱基序列如SEQ ID NO.16所示;用于检测HPV 26亚型的第12探针对,其捕获探针的碱基序列如SEQ ID NO.20所示,其检测探针的碱基序列如SEQ ID NO.26所示;用于检测HPV 53亚型的第13探针对,其捕获探针的碱基序列如SEQ ID NO.21所示,其检测探针的碱基序列如SEQ ID NO.25所示;用于检测HPV 66亚型的第14探针对,其捕获探针的碱基序列如SEQ ID NO.22所示,其检测探针的碱基序列如SEQ ID NO.25所示;用于检测HPV 73亚型的第15探针对,其捕获探针的碱基序列如SEQ ID NO.23所示,其检测探针的碱基序列如SEQ ID NO.27所示;用于检测HPV 82亚型的第16探针对,其捕获探针的碱基序列如SEQ ID NO.24所示,其检测探针的碱基序列如SEQ ID NO.28所示。各HPV亚型类别及其检测对应的捕获探针和检测探针的碱基序列见表1。The kit for detecting a virus genotyping provided in this embodiment includes a probe combination comprising 16 probe pairs for detecting HPV subtypes, and the capture probes and detection probes of each probe pair are The solution forms were independent and the final concentration of the probe was 1 μM. The 16 probe pairs are: a first probe pair for detecting the HPV 16 subtype, and the base sequence of the capture probe is as shown in SEQ ID NO. 1, and the base sequence of the detection probe is SEQ. ID NO. 2; a second probe pair for detecting the HPV 18 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 3, and the base sequence of the detection probe is SEQ ID NO. As shown in Fig. 4; a third probe pair for detecting the HPV 31 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 5, and the base sequence of the detection probe is SEQ ID NO. As shown in the fourth probe pair for detecting HPV 33 subtype or 52 subtype or 58 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 6, and the base sequence of the detection probe is shown. As shown in SEQ ID NO. 15, a 5th probe pair for detecting the HPV 35 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 7, and the base sequence of the detection probe is SEQ. ID NO. 14; a 6th probe pair for detecting the HPV 39 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 8, and the base sequence of the detection probe is SEQ ID NO. .16; used to detect HPV 45 a 7th probe pair of the type, the base sequence of the capture probe is shown in SEQ ID NO. 9, and the base sequence of the detection probe is shown in SEQ ID NO. 17; for detecting HPV 51 subtype The eighth probe pair, the base sequence of the capture probe is shown in SEQ ID NO. 10, the base sequence of the detection probe is shown in SEQ ID NO. 18, and the ninth for detecting the HPV 56 subtype. The probe pair has a base sequence of the capture probe as shown in SEQ ID NO. 11, a base sequence of the detection probe as shown in SEQ ID NO. 18, and a 10th probe for detecting the HPV 59 subtype. The base sequence of the capture probe is shown in SEQ ID NO. 12, the base sequence of the detection probe is shown in SEQ ID NO. 19, and the 11th probe pair for detecting the HPV 68 subtype, The base sequence of the capture probe is shown in SEQ ID NO. 13, the base sequence of the detection probe is shown in SEQ ID NO. 16, and the 12th probe pair for detecting the HPV 26 subtype is captured. The base sequence of the probe is shown in SEQ ID NO. 20, the base sequence of the detection probe is shown in SEQ ID NO. 26; the 13th probe pair for detecting the HPV 53 subtype, and the capture probe thereof. Base sequence such as SEQ As shown in ID NO. 21, the base sequence of the detection probe is shown in SEQ ID NO. 25; the 14th probe pair for detecting the HPV 66 subtype, the base sequence of the capture probe is SEQ ID NO. As shown in Fig. 22, the base sequence of the detection probe is shown in SEQ ID NO. 25; the 15th probe pair for detecting the HPV 73 subtype, the base sequence of the capture probe is SEQ ID NO. As shown, the base sequence of the detection probe is shown in SEQ ID NO. 27; the 16th probe pair for detecting the HPV 82 subtype, the base sequence of the capture probe is shown in SEQ ID NO. The base sequence of the detection probe is shown in SEQ ID NO. The base sequences of the respective HPV subtypes and their corresponding capture probes and detection probes are shown in Table 1.
表1.检测HPV亚型的捕获探针和检测探针的碱基序列Table 1. Base sequences of detection probes and detection probes for detecting HPV subtypes
其中,检测33、52和58亚型的捕获探针碱基序列相同,检测33、52和58亚型的检测探针碱基序列相同,也就是说用SEQ ID NO.6所示的捕获探针和SEQ ID NO.15所示的检测探针能够检测出HPV33、52和58亚型病毒,检出结果说明的是样品具有HPV33、52和58亚型中的至少一种,但具体是该三种亚型病毒中的哪一种亚型病毒并不能检测出。39和68亚型的检测探针序列相同,51和56亚型的检测探针序列相同,53和66亚型的检测探针序列相同。此外,用于检测16和18亚型的检测探针的5’端标记生物素,其余亚型的检测探针的3’端标记生物素(如表1所示)。Among them, the capture probes of the 33, 52, and 58 subtypes have the same base sequence, and the detection probes of the 33, 52, and 58 subtypes have the same base sequence, that is, the capture probe shown by SEQ ID NO. The probe and the detection probe shown in SEQ ID NO. 15 are capable of detecting HPV 33, 52 and 58 subtype viruses, and the detection results indicate that the sample has at least one of HPV types 33, 52 and 58, but specifically Which of the three subtypes of the virus was not detected. The detection probe sequences of the 39 and 68 subtypes are identical, the detection probe sequences of the 51 and 56 subtypes are identical, and the detection probe sequences of the 53 and 66 subtypes are identical. In addition, the 5'-end labeled biotin was used to detect the 16 and 18 subtypes of the detection probe, and the remaining subtypes of the detection probe were labeled with biotin at the 3' end (as shown in Table 1).
采用本实施例提供的病毒基因分型检测试剂盒对样本1和样本2的具体检测步骤如下。The specific detection steps for the sample 1 and the sample 2 using the virus genotyping detection kit provided in the present embodiment are as follows.
1捕获探针固定1 capture probe fixed
1.1配制吡咯(pyrrole)与CP的混合液1.1 Preparation of a mixture of pyrrole and CP
取1个1.5mL离心管,依次加入超纯水885μl,离子化合物100μl 3M KCl,涡旋震荡混匀,离心;加入导电聚合物5μl pyrrole(≥98.0%,购自Sigma,货号W338605),涡旋震荡混匀,离心;加入10μl CP(100μM)(在分组检测的情况下,将每种CP各加入10μl至检测孔中);涡旋震荡混匀后离心,备用。Take a 1.5mL centrifuge tube, add 885μl of ultrapure water, 100μl of ionic compound 3M KCl, vortex and mix, centrifuge; add 5μl pyrrole (≥98.0%, purchased from Sigma, catalog number W338605), vortex Shake well and centrifuge; add 10 μl CP (100 μM) (in the case of group detection, add 10 μl of each CP to the detection well); vortex and mix, centrifuge, and set aside.
1.2固定捕获探针1.2 fixed capture probe
在96孔的检测孔板(E-plate)(其结构和工作原理可见优先权文件201620769829.2)上,按其操作说明书,往反应孔加入30μl的已配制好的pyrrole与CP的混合液,加样时枪头贴近孔的底部,但是不接触到底部电极,加完后倾斜或拍打E-plate使液体在孔里的电极表面均匀覆盖,然后立刻到EFIRM仪器上,按其操作说明书,进行电场操作。On a 96-well detection plate (E-plate) (the structure and working principle can be seen in the priority document 201620769829.2), add 30 μl of the prepared mixture of pyrrole and CP to the reaction well according to the instructions. When the tip of the gun is attached to the bottom of the hole, but does not touch the bottom electrode, after tilting or tapping the E-plate, the liquid is evenly covered on the surface of the electrode in the hole, and then immediately go to the EFIRM instrument and operate the electric field according to its operating instructions. .
1.3EFIRM电场处理1.3EFIRM electric field treatment
在EFIRM软件上选择进行实验的对应列,电场参数设置为:电压A:350mV,1s;电压B:950mV,1s;进行9个循环。电场处理完毕,立刻取出,清洗E-plate板。The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: 350 mV, 1 s; voltage B: 950 mV, 1 s; 9 cycles were performed. After the electric field treatment is completed, remove it immediately and clean the E-plate plate.
1.4E-plate板清洗:1.4E-plate board cleaning:
在洗板机程序上选择对应的实验列,清洗程序选择(2bottom,2top),清洗液选择洗液A。清洗完毕,立刻进行下一步,样本上样操作。其中,洗液A为含0.05%(质量百分比)SDS的2×SSC缓冲液。Select the corresponding experiment column on the washing machine program, select the cleaning program (2bottom, 2top), and select the washing solution A for the cleaning solution. After the cleaning is completed, proceed to the next step and sample loading. Among them, the lotion A was a 2×SSC buffer containing 0.05% by mass of SDS.
2样本杂交
2 sample hybridization
2.1杂交buffer预处理2.1 hybrid buffer pretreatment
杂交buffer(购自thermo fisher)在水浴锅中90℃水浴处理10min,然后室温放置冷却20min。The hybrid buffer (purchased from thermo fisher) was treated in a water bath at 90 ° C for 10 min in a water bath and then allowed to cool at room temperature for 20 min.
2.2配制待测样本2.2 Preparation of samples to be tested
待测样本从-20℃冰箱取出,放进4℃冰箱解冻。完全溶解后,使用煮沸法或0.4M NaOH预处理待测样本,然后将待测样本与杂交buffer按体积比1:2混合,涡旋振荡后离心,即可上样进行检测。The sample to be tested was taken out from the -20 ° C refrigerator and placed in a refrigerator at 4 ° C to be thawed. After complete dissolution, the sample to be tested is pretreated by boiling or 0.4M NaOH, and then the sample to be tested is mixed with the hybrid buffer by a volume ratio of 1:2, vortexed and centrifuged, and then the sample is loaded for detection.
2.3加待测样本2.3 plus sample to be tested
在E-plate上,在对应的孔里加入上述待测样本与杂交buffer混合后的混合液30μl。(需要注意的是:加样时枪头贴近孔的底部,但是不接触到底部电极,加完后倾斜或拍打E-plate使液体在孔里的电极表面均匀覆盖,然后立刻到EFIRM上进行电场操作。)On the E-plate, 30 μl of the mixture of the above-mentioned sample to be tested and the hybrid buffer was added to the corresponding well. (It should be noted that the tip of the gun is attached to the bottom of the hole when loading, but does not touch the bottom electrode. After the addition, tilt or tap the E-plate to evenly cover the surface of the electrode in the hole, then immediately go to the EFIRM for the electric field. operating.)
在本实施例中,设置4个检测组,分别是16亚型组、18亚型组、11种高危型组以及5种中高危型组。每个检测组设置一个阳性对照孔,一个阴性对照孔(重复4次),一个样本1的检测孔以及一个样本2的检测孔。其中,16亚型组用于检测HPV16亚型(该组每个孔中对应加入第1探针对),18亚型组用于检测HPV18亚型(该组每个孔对应加入第2探针对),11种高危型组用于检测11种高危型HPV中的一种(该组每个孔对应加入第3~11探针对,5种中高危型用于检测5种中高危型HPV中的一种(该组每个孔对应加入第12~16探针对)。需要说明的是,检测组的数量可根据需要的检测结果准确程度进行设计。如果仅需检测出待测样本是否含有常见型HPV、高危型病毒以及中高危型病毒,则可按本实施例的检测组的设置方法进行设置。如果仅需检测出样本是否含有HPV,则可仅设置一个检测组,具体检测方法参考实施例2。如果需要检测出样本具体含有某类型的病毒,则可设置18个检测组,具体检测方法参考实施例3。In the present embodiment, four detection groups are set, which are 16 subtype group, 18 subtype group, 11 high risk type groups, and 5 medium and high risk type groups. One positive control well, one negative control well (repeated 4 times), one sample 1 test well and one sample 2 test well were set for each test group. Among them, the 16 subtype group was used to detect the HPV16 subtype (the corresponding pair of probe pairs was added to each well in the group), and the 18 subtype group was used to detect the HPV18 subtype (the corresponding probe was added to each well of the group) Yes, 11 high-risk groups were used to detect one of 11 high-risk HPVs (the group included 3 to 11 probe pairs for each well, and 5 medium- and high-risk types for 5 medium- and high-risk HPVs). One of the groups (the pair of holes corresponds to the 12th to 16th probe pairs). It should be noted that the number of detection groups can be designed according to the accuracy of the required test results. The common type HPV, the high-risk type virus, and the medium-high-risk type virus can be set according to the setting method of the detection group in this embodiment. If only the sample is required to detect whether the sample contains HPV, only one detection group can be set, and the specific detection method Refer to Embodiment 2. If it is necessary to detect that the sample specifically contains a certain type of virus, 18 detection groups can be set, and the specific detection method is referred to Embodiment 3.
其中,阳性对照孔中加入对应的30μl含终浓度为1pM的阳性寡核苷酸(其能够与对应的捕获探针和检测探针互补配对结合)的杂交buffer作为阳性对照(需要说明的是,11种高危型组的阳性对照仅选择52亚型核苷酸序列(表2中的第9行),5种中高危型组的阳性对照仅选择26亚型核苷酸序列(表2中的第13行),即可起到阳性对照的作用),各HPV亚型对应的阳性寡核苷酸的碱基序列见表2,阴性对照孔中加入30μl杂交buffer作为阴性对照。Wherein, a corresponding 30 μl of a hybrid buffer containing a positive oligonucleotide having a final concentration of 1 pM (which is capable of complementary pairing with the corresponding capture probe and detection probe) was added as a positive control to the positive control well (remarks that The positive control of 11 high-risk groups only selected 52 subtype nucleotide sequences (line 9 in Table 2), and the positive control of 5 medium-high-risk groups only selected 26 subtype nucleotide sequences (Table 2) Line 13) can serve as a positive control. The base sequence of the positive oligonucleotide corresponding to each HPV subtype is shown in Table 2. 30 μl of hybridization buffer was added to the negative control well as a negative control.
表2.用于检测HPV基因型的阳性对照的阳性寡核苷酸的碱基序列Table 2. Base sequence of positive oligonucleotides used to detect positive controls for HPV genotypes
2.4EFIRM电场处理2.4EFIRM electric field treatment
在EFIRM软件上选择进行实验的对应列,电场参数设置为:电压A:300mV,1s;电压B:500mV,1s;进行150个循环。电场处理完毕,立刻取出,清洗E-plate板。Select the corresponding column for the experiment on the EFIRM software. The electric field parameters are set to: voltage A: 300 mV, 1 s; voltage B: 500 mV, 1 s; 150 cycles. After the electric field treatment is completed, remove it immediately and clean the E-plate plate.
2.5室温孵育2.5 incubation at room temperature
盖上E-plate盖子,实验台上室温孵育30min。Cover the E-plate and incubate for 30 min at room temperature on the bench.
2.6E-plate板清洗2.6E-plate plate cleaning
在洗板机程序上选择对应的实验列,清洗程序选择(2bottom,2top),清洗液选择洗液A。清洗完毕,立刻进行DP加样操作。Select the corresponding experiment column on the washing machine program, select the cleaning program (2bottom, 2top), and select the washing solution A for the cleaning solution. After the cleaning is completed, the DP loading operation is performed immediately.
3DP结合3DP combination
3.1DP溶液配制3.1DP solution preparation
从4℃冰箱取出稀释液,取1个1.5mL离心管,加入990μl的稀释液,往各检测组的反应孔中加入对应的10μl DP(100μM),涡旋震荡混匀,离心,备用。其中,稀释液为含0.1%(质量体积比)酪蛋白的PBS缓冲液(pH7.4)。酪蛋白的作用是封闭非特异性位点,以提高检测的灵敏性和准确度。The dilution solution was taken out from the refrigerator at 4 ° C, and a 1.5 mL centrifuge tube was taken. 990 μl of the dilution solution was added, and 10 μl of DP (100 μM) was added to the reaction wells of each test group, vortexed and mixed, centrifuged, and set aside. Among them, the diluent was PBS buffer (pH 7.4) containing 0.1% (mass by volume) casein. The role of casein is to block non-specific sites to increase the sensitivity and accuracy of the assay.
3.2加样3.2 loading
根据实验设计在对应的孔加入对应DP溶液30μl,加样时枪头贴近孔的底部,但是不接触到底部电极,加完后倾斜或拍打E-plate使液体在孔里的电极表面均匀覆盖,然后立刻到EFIRM上进行电场操作,电场参数设置为:电压A:500mV,1s;电压B:800mV,1s;进行8个循环。电场处理完毕,立刻取出,清洗检测孔板。According to the experimental design, 30 μl of the corresponding DP solution was added to the corresponding hole. When the sample was applied, the tip of the gun was attached to the bottom of the hole, but the bottom electrode was not touched. After the addition, the E-plate was tilted or tapped to evenly cover the surface of the electrode in the hole. Then immediately go to EFIRM for electric field operation, the electric field parameters are set to: voltage A: 500mV, 1s; voltage B: 800mV, 1s; 8 cycles. After the electric field is processed, remove it immediately and clean the detection orifice.
3.3室温孵育3.3 incubation at room temperature
盖上E-plate盖子,实验台上室温孵育30min。Cover the E-plate and incubate for 30 min at room temperature on the bench.
3.4E-plate板清洗3.4E-plate plate cleaning
在洗板机程序上选择对应的实验列,清洗程序选择(2bottom,2top),清洗液选择洗液A。清洗完毕,立刻进行加样操作。Select the corresponding experiment column on the washing machine program, select the cleaning program (2bottom, 2top), and select the washing solution A for the cleaning solution. After the cleaning is completed, the sample loading operation is performed immediately.
4链霉亲和素标记的辣根过氧化物酶(Poly-HRP)与生物素结合4 streptavidin-labeled horseradish peroxidase (Poly-HRP) combined with biotin
4.1Poly-HRP溶液配制4.1Poly-HRP solution preparation
从4℃冰箱取出稀释液,取1个1.5mL离心管,加入999μl的稀释液,加入1μl的酶液(含Poly-HRP,浓度为0.5mg/ml,购自thermo fisher,产品名称为PierceTM Streptavidin Poly-HRP,货号为21140,单位规格为0.5mL),涡旋震荡混匀,离心,备用。Remove the dilution from the 4 ° C refrigerator, take a 1.5 mL centrifuge tube, add 999 μl of the dilution, add 1 μl of enzyme solution (containing Poly-HRP, concentration 0.5 mg / ml, purchased from thermo fisher, product name is Pierce TM Streptavidin Poly-HRP, item number 21140, unit size 0.5 mL), vortex and mix, centrifuge, and set aside.
4.2加酶液4.2 Add enzyme solution
在对应的各孔加入上述稀释液和酶液混合后的混合液30μl,Poly-HRP通过其标记的链霉亲和素与检测探针上的生物素识别并结合。30 μl of the mixture of the above diluted solution and the enzyme solution was added to each well, and Poly-HRP was recognized and bound by the labeled streptavidin and the biotin on the detection probe.
4.3室温孵育4.3 incubation at room temperature
盖上E-plate盖子,实验台上室温孵育30min。Cover the E-plate and incubate for 30 min at room temperature on the bench.
4.4E-plate板清洗4.4E-plate plate cleaning
在洗板机程序上选择对应的实验列,清洗程序选择(3bottom,3top),清洗液选择洗液B。清洗完毕,立刻进行TMB加样操作。其中,洗液B为含0.1%(质量百分比)Tween20的PBS缓冲液。Select the corresponding experiment column on the washing machine program, select the cleaning program (3bottom, 3top), and select the washing solution B for the cleaning solution. After the cleaning is completed, the TMB loading operation is performed immediately. Among them, the lotion B was a PBS buffer containing 0.1% by mass of Tween20.
5数据读取
5 data reading
5.1加底物5.1 plus substrate
在对应的各孔加入底物60μl,加样时枪头贴近孔的底部,但是不接触到底部电极。加完立刻到EFIRM上进行电场操作。其中,底物为含TMB的溶液(购自thermo fisher,产品货号为34028,名称为1-StepTM Ultra TMB-ELISA)。加入酶的底物,发生氧化还原反应,产生电流,检测各孔内电流值即完成整个检测过程。以阴性对照孔重复4次的电流平均值+3倍标准差的和作为阳性判定值,阳性判定值=AVG+3×SD,其中AVG为阴性对照孔重复4次的电流平均值,SD为阴性对照孔重复4次的标准差。若加入有待测样本的检测孔的电流值大于或等于该阳性判定值,则判定位阳性结果,说明待测样本中含有相应的HPV亚型病毒。60 μl of the substrate was added to the corresponding wells, and the tip of the gun was attached to the bottom of the well while the sample was applied, but did not touch the bottom electrode. Immediately after the addition, the electric field operation was performed on the EFIRM. Wherein the substrate is a solution containing TMB (commercially available from Thermo Fisher, Cat. No. 34028 the product, the name 1-Step TM Ultra TMB-ELISA ). The substrate of the enzyme is added, a redox reaction occurs, a current is generated, and the current value in each well is detected to complete the entire detection process. The sum of the current average + 3 standard deviations repeated 4 times in the negative control well was used as the positive judgment value, and the positive judgment value = AVG + 3 × SD, wherein AVG was the average value of the current of the negative control well repeated 4 times, and the SD was negative. Control wells were repeated 4 times standard deviation. If the current value of the detection hole to which the sample to be tested is added is greater than or equal to the positive determination value, the positive result is determined, indicating that the sample to be tested contains the corresponding HPV subtype virus.
5.2EFIRM电场读数5.2EFIRM electric field reading
在EFIRM软件上选择进行实验的对应列,电场参数设置为:电压A:-200mV,60s;电压B:0mV,0s;进行1个循环。电场处理完毕,立刻取出,清洗E-plate板。The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: -200 mV, 60 s; voltage B: 0 mV, 0 s; one cycle was performed. After the electric field treatment is completed, remove it immediately and clean the E-plate plate.
仪器将自动完成检测工作,检测数据自动上传到云计算平台。根据检测数据绘制柱状图,横坐标为检测组的类别,纵坐标为各检测组中各检测孔的电流值(Current),单位为纳安(-nA、负号表示方向)。本实施例的检测结果如图1所示。The instrument will automatically complete the test and the test data will be automatically uploaded to the cloud computing platform. The histogram is drawn according to the detected data, the abscissa is the category of the detection group, and the ordinate is the current value (Current) of each detection hole in each detection group, and the unit is nanoamperes (-nA, negative sign indicates direction). The detection results of this embodiment are shown in FIG.
由图1可知,在16亚型组中,样本1的检测结果呈阳性,其含有HPV16亚型病毒(阴性对照电流值为28.67nA、其标准差为5.62,阳性对照电流值为130.22nA,样本1的电流值为192.05nA,样本2的电流值为30.53nA);在11种高危型组中,样本2的检测结果呈阳性(阴性对照电流值为24.94nA、其标准差为5.91,阳性对照电流值为132.94nA,样本1的电流值为29.57nA,样本2的电流值为151.30nA),说明其含有高危型HPV31、33、52、58、35、39、45、51、56、59、68亚型中的至少一种。As can be seen from Fig. 1, in the 16 subtype group, the test result of the sample 1 was positive, and it contained the HPV16 subtype virus (the negative control current value was 28.67 nA, the standard deviation was 5.62, and the positive control current value was 130.22 nA, the sample The current value of 1 is 192.05 nA, and the current value of sample 2 is 30.53 nA). Among the 11 high-risk groups, the test result of sample 2 is positive (the negative control current value is 24.94 nA, and the standard deviation is 5.91, positive control) The current value is 132.94nA, the current value of sample 1 is 29.57nA, and the current value of sample 2 is 151.30nA), indicating that it contains high-risk HPV31, 33, 52, 58, 35, 39, 45, 51, 56, 59, At least one of the 68 subtypes.
实施例2Example 2
本实施例提供的基于EFIRM技术HPV病毒检测的试剂盒包括探针组合(同实施例1)、固定物、链霉亲和素标记的辣根过氧化物酶(以溶液形式存在)及其底物,固定物是导电聚合物和离子化合物,其中,导电聚合物是吡咯,离子化合物是氯化钾。底物是含TMB的溶液。其余同实施例1。The kit for detecting HPV virus based on EFIRM technology provided in this embodiment includes a probe combination (same as in Example 1), a fixative, streptavidin-labeled horseradish peroxidase (present in solution), and a bottom thereof. The immobilizer is a conductive polymer and an ionic compound, wherein the conductive polymer is pyrrole and the ionic compound is potassium chloride. The substrate is a solution containing TMB. The rest are the same as in the first embodiment.
在本实施例中,检测组设置为1个,命名为18种亚型同时检测组,检测组包括一个阳性对照孔,一个阴性对照孔(重复4次),一个样本1的检测孔以及一个样本2的检测孔。每个孔中都加入实施例1所述的16种探针对,18种HPV亚型同时检测)。其余同实施例1。如果有阳性检出结果,则检出的结果能说明待测样本含有HPV16、18、31、33、52、58、35、39、45、51、56、59、68、26、53、66、73、82亚型中的至少一种(如图2所示)。本实施例的检测结果如图2所示。In the present embodiment, the detection group is set to one, and the 18 subtypes are simultaneously detected, and the detection group includes one positive control hole, one negative control hole (repeated 4 times), one sample 1 detection hole and one sample. 2 detection holes. The 16 probe pairs described in Example 1 were added to each well and 18 HPV subtypes were simultaneously detected). The rest are the same as in the first embodiment. If there is a positive detection result, the detected result can indicate that the sample to be tested contains HPV 16, 18, 31, 33, 52, 58, 35, 39, 45, 51, 56, 59, 68, 26, 53, 66, At least one of the 73 and 82 subtypes (as shown in Figure 2). The detection result of this embodiment is as shown in FIG. 2.
由图2可知,样本1的检测结果呈阳性(阴性对照电流值为26.59nA、其标准差为7.91,阳性对照电流值为110.46nA,样本1的电流值为189.66nA),说明其含有HPV16、18、31、33、52、58、35、39、45、51、56、59、68、26、53、66、73、82亚型中的至少一种;样本2的检测结果呈阳性(阴性对照电流值为26.59nA、其标准差为7.91,阳性对照电流值为110.46nA,样本2的电流值为80.60nA),说明样本2含有HPV16、18、31、33、52、58、35、39、45、51、56、59、68、26、53、66、73、82亚型中的至少一种。It can be seen from Fig. 2 that the test result of sample 1 is positive (the negative control current value is 26.59 nA, the standard deviation is 7.91, the positive control current value is 110.46 nA, and the sample 1 current value is 189.66 nA), indicating that it contains HPV16, At least one of 18, 31, 33, 52, 58, 35, 39, 45, 51, 56, 59, 68, 26, 53, 66, 73, 82 subtypes; sample 2 is positive (negative) The control current value was 26.59 nA, the standard deviation was 7.91, the positive control current value was 110.46 nA, and the sample 2 current value was 80.60 nA), indicating that sample 2 contained HPV 16, 18, 31, 33, 52, 58, 35, 39. At least one of the 45, 51, 56, 59, 68, 26, 53, 66, 73, 82 subtypes.
实施例3Example 3
本实施例提供的基于EFIRM技术HPV病毒检测的试剂盒包括探针组合(同实施例1)、固定物、链霉亲和素标记的辣根过氧化物酶(以溶液形式存在)及其底物、清洗液。The kit for detecting HPV virus based on EFIRM technology provided in this embodiment includes a probe combination (same as in Example 1), a fixative, streptavidin-labeled horseradish peroxidase (present in solution), and a bottom thereof. Matter, cleaning solution.
固定物是导电聚合物和离子化合物,其中,导电聚合物是吡咯,离子化合物是氯化钾。底物是含TMB的溶液。清洗液包括包括洗液A和洗液B,洗液A是含SDS的SSC缓冲液,洗液B是含Tween20的PBS缓冲液。其余同实施例1。The anchor is a conductive polymer and an ionic compound, wherein the conductive polymer is pyrrole and the ionic compound is potassium chloride. The substrate is a solution containing TMB. The cleaning solution includes a lotion A and a lotion B, the lot A is an SDS buffer containing SDS, and the lotion B is a PBS buffer containing Tween 20. The rest are the same as in the first embodiment.
在本实施例中,检测组设置为16个组,分别是16亚型组、18亚型组、26亚型组、31亚型组、35亚型组、39亚型组、45亚型组、51亚型组、52亚型组、53亚型组、56亚型组、59亚型组、66亚型组、68亚型组、73亚型组以及82亚型组。每个检测组设置一个阳性对照孔(每个检测组分别加入表2中所示的对应的阳性寡核苷酸),一个阴性对照孔(重
复4次),一个样本1的检测孔以及一个样本2的检测孔,每个检测孔内加入其相应的探针对。其余同实施例1。如果各检测组有阳性检出结果,则检出的结果能够说明待测样本含有的相应的HPV亚型病毒。In this embodiment, the detection group is set to 16 groups, which are 16 subtype group, 18 subtype group, 26 subtype group, 31 subtype group, 35 subtype group, 39 subtype group, and 45 subtype group. , 51 subtype group, 52 subtype group, 53 subtype group, 56 subtype group, 59 subtype group, 66 subtype group, 68 subtype group, 73 subtype group and 82 subtype group. One positive control well was set for each test group (each test group was added with the corresponding positive oligonucleotide shown in Table 2), and one negative control well (heavy
Repeat 4 times), one sample 1 detection hole and one sample 2 detection hole, and each corresponding detection hole is added to its corresponding probe pair. The rest are the same as in the first embodiment. If each test group has a positive detection result, the detected result can indicate the corresponding HPV subtype virus contained in the sample to be tested.
需要说明的是,由于,HPV 33、52和58亚型的探针对通用,因此52亚型组检测出的阳性结果说明待测样本中含有HPV33、52和58亚型中的一种或几种。本实施例的检测结果如图3所示。It should be noted that since the probe pairs of the HPV types 33, 52, and 58 are common, the positive result detected by the 52 subtype indicates that the sample to be tested contains one or more of the HPV 33, 52, and 58 subtypes. Kind. The detection result of this embodiment is shown in FIG.
由图3可知,在16亚型组中,样本1的检测结果呈阳性(阴性对照电流值为28.67nA、其标准差为5.62,阳性对照电流值为130.22nA,样本1的电流值为192.05nA,样本2的电流值为30.53nA),说明样本1含有HPV 16亚型病毒;在52亚型组中,样本2的检测结果呈阳性(阴性对照电流值为25.58nA、其标准差为5.33,阳性对照电流值为75.70nA,样本1的电流值为23.90nA,样本2的电流值为166.10nA),另外,在66亚型组中,样本2的检测结果呈阳性(阴性对照电流值为25.92nA、其标准差为6.20,阳性对照电流值为115.75nA,样本1的电流值为34.47nA,样本2的电流值为72.72nA),说明样本2含有HPV 33、52、58亚型中的一种和HPV 66亚型。As can be seen from Fig. 3, in the 16 subtype group, the test result of the sample 1 was positive (the negative control current value was 28.67 nA, the standard deviation was 5.62, the positive control current value was 130.22 nA, and the sample 1 current value was 192.05 nA. The current value of sample 2 is 30.53nA), indicating that sample 1 contains HPV 16 subtype virus; in group 52, sample 2 is positive (negative control current value is 25.58nA, and its standard deviation is 5.33, The positive control current value was 75.70 nA, the sample 1 current value was 23.90 nA, and the sample 2 current value was 166.10 nA). In addition, in the 66 subtype group, the sample 2 test result was positive (the negative control current value was 25.92). nA, its standard deviation is 6.20, positive control current value is 115.75nA, sample 1 current value is 34.47nA, sample 2 current value is 72.72nA), indicating that sample 2 contains one of HPV 33, 52, 58 subtypes. And HPV 66 subtypes.
本实施例通过设置18检测组来检测待测样本的检测结果能够将其含有的具体病毒亚型检测出,其检测结果准确可靠。In this embodiment, by setting the detection group of 18 to detect the detection result of the sample to be tested, the specific virus subtype contained therein can be detected, and the detection result is accurate and reliable.
实施例4Example 4
本实施例提供的基于EFIRM技术HPV病毒检测的试剂盒包括探针组合、检测孔板、异硫氰酸荧光素抗体标记的辣根过氧化物酶(以溶液形式存在)及其底物(ABTS、以溶液形式存在)、清洗液。The kit for detecting HPV virus based on EFIRM technology provided by the present embodiment includes a probe combination, a detection plate, a fluorescein isothiocyanate antibody-labeled horseradish peroxidase (present in solution) and a substrate thereof (ABTS) , in the form of a solution), cleaning solution.
其中,探针组合包括9个探针对,该9个探针对分别是:用于检测HPV 16亚型的第1探针对、用于检测HPV 18亚型的第2探针对、用于检测HPV 31亚型的第3探针对、用于检测HPV 33亚型或52亚型或58亚型的第4探针对、用于检测HPV 51亚型的第8探针对、用于检测HPV 56亚型的第9探针对、用于检测HPV 26亚型的第12探针对、用于检测HPV53亚型的第13探针对、用于检测HPV 73亚型的第15探针对。各检测探针的3’端带有异硫氰酸荧光素。各探针对相应的捕获探针和检测探针的碱基序列见实施例1中的表1。Wherein, the probe combination comprises 9 probe pairs, respectively: a first probe pair for detecting the HPV 16 subtype, a second probe pair for detecting the HPV 18 subtype, and For detecting the third probe pair of the HPV 31 subtype, the fourth probe pair for detecting the HPV 33 subtype or the 52 subtype or the 58 subtype, and the eighth probe pair for detecting the HPV 51 subtype, 9th probe pair for detecting HPV 56 subtype, 12th probe pair for detecting HPV 26 subtype, 13th probe pair for detecting HPV53 subtype, 15th for detecting HPV 73 subtype Probe pair. Each test probe was provided with fluorescein isothiocyanate at the 3' end. The base sequences of the respective probe pairs and the corresponding capture probes and detection probes are shown in Table 1 in Example 1.
检测孔板的反应孔内固定有上述探针对的捕获探针,捕获探针固定至检测孔板的的方法同实施例1中的“1捕获探针固定”步骤,当然,在其他的实施例中,采用其他的方法将本发明的捕获探针固定至检测孔板上得到的检测孔板也属于本发明的保护范围。The capture probe of the above probe pair is fixed in the reaction well of the detection well plate, and the method of fixing the capture probe to the detection well plate is the same as the "1 capture probe fixation" step in the first embodiment, of course, in other implementations. In the examples, the detection of the capture probe of the present invention by using other methods to the detection well plate is also within the scope of the present invention.
清洗液包括洗液A和洗液B,洗液A是含SDS的SSC缓冲液,洗液B是含Tween20的PBS缓冲液。其余同实施例1。The cleaning solution includes a lotion A and a lotion B, the lotion A is an SSS buffer containing SDS, and the lotion B is a PBS buffer containing Tween20. The rest are the same as in the first embodiment.
采用本实施例对样本1和样本2同时检测,检测步骤与实施例1基本一致,检测结果如图4所示。The sample 1 and the sample 2 are simultaneously detected by using the embodiment, and the detecting step is basically the same as that of the first embodiment, and the detection result is as shown in FIG. 4 .
由图4可知,在16亚型组中,样本1的检测结果呈阳性(阴性对照电流值为25.22nA、其标准差为4.43,阳性对照电流值为108.10nA,样本1的电流值为166.03nA),说明样本1含有HPV16亚型病毒;在6种高危型组中,样本2的检测结果呈阳性(阴性对照电流值为24.25nA、其标准差为5.27,阳性对照电流值为115.92nA,样本2的电流值为170.42nA),说明其含有高危型HPV31、33、51、52、58以及56亚型中的至少一种。As can be seen from Fig. 4, in the 16 subtype group, the test result of the sample 1 was positive (the negative control current value was 25.22 nA, the standard deviation was 4.43, the positive control current value was 108.10 nA, and the sample 1 current value was 166.03 nA. ), indicating that sample 1 contains HPV16 subtype virus; in the six high-risk groups, sample 2 is positive (negative control current value is 24.25nA, standard deviation is 5.27, positive control current value is 115.92nA, sample) The current value of 2 is 170.42 nA), indicating that it contains at least one of the high-risk types HPV 31, 33, 51, 52, 58 and 56 subtypes.
实施例5Example 5
本实施例提供的基于EFIRM技术HPV病毒检测的试剂盒包括探针组合、固定物、地高辛抗体标记的碱性磷酸酶酶(以溶液形式存在)及其底物(以含BCIP和NBT的组合物的溶液存在)、清洗液。The kit for detecting HPV virus based on EFIRM technology provided by the present embodiment includes a probe combination, a fixer, a digoxin antibody-labeled alkaline phosphatase enzyme (present in solution) and a substrate thereof (including BCIP and NBT) A solution of the composition is present), a cleaning solution.
其中,探针组合包括4个探针对,该4个探针对分别是:用于检测HPV 16亚型的第1探针对、用于检测HPV 18亚型的第2探针对、用于检测HPV 33亚型或52亚型或58亚型的第4探针对、用于检测HPV 26亚型的第12探针对。各检测探针的5’端带有地高辛。各探针对相应的捕获探针和检测探针的碱基序列见实施例1中的表1。The probe combination includes four probe pairs, which are: a first probe pair for detecting the HPV 16 subtype, a second probe pair for detecting the HPV 18 subtype, and A 4th probe pair for detecting HPV 33 subtype or 52 subtype or 58 subtype, and a 12th probe pair for detecting HPV 26 subtype. Each detection probe has a digoxin at the 5' end. The base sequences of the respective probe pairs and the corresponding capture probes and detection probes are shown in Table 1 in Example 1.
固定物是导电聚合物和离子化合物,其中,导电聚合物是噻吩,当然,在其他的实施例中可以是苯胺。离子化合物是氯化钠。清洗液包括包括洗液A和洗液B,洗液A是含SDS的SSC缓冲液,洗液B是含Tween20的PBS缓冲液。其余同实施例1。
The anchor is a conductive polymer and an ionic compound, wherein the conductive polymer is thiophene, and of course, in other embodiments, aniline. The ionic compound is sodium chloride. The cleaning solution includes a lotion A and a lotion B, the lot A is an SDS buffer containing SDS, and the lotion B is a PBS buffer containing Tween 20. The rest are the same as in the first embodiment.
采用本实施例对样本1和样本2同时检测,检测步骤与实施例1基本一致,检测结果如图5所示。The sample 1 and the sample 2 are simultaneously detected by using the embodiment, and the detection step is basically the same as that of the embodiment 1, and the detection result is shown in FIG. 5.
由图5可知,在16亚型组中,样本1的检测结果呈阳性(阴性对照电流值为26.31nA、其标准差为5.29,阳性对照电流值为118.74nA,样本1的电流值为185.47nA),说明样本1含有HPV16亚型病毒;在3种高危型组中,样本2的检测结果呈阳性(阴性对照电流值为27.45nA、其标准差为5.15,阳性对照电流值为122.62nA,样本2的电流值为150.07nA),说明样本2含有高危型HPV33、51、52以及58亚型中的至少一种。As can be seen from Fig. 5, in the 16 subtype group, the test result of the sample 1 was positive (the negative control current value was 26.31 nA, the standard deviation was 5.29, the positive control current value was 118.74 nA, and the sample 1 current value was 185.47 nA. ), indicating that sample 1 contains HPV16 subtype virus; in the three high-risk groups, sample 2 was positive (negative control current value was 27.45 nA, standard deviation was 5.15, positive control current value was 122.62 nA, sample) The current value of 2 is 150.07 nA), indicating that sample 2 contains at least one of the high-risk types HPV 33, 51, 52, and 58 subtypes.
实施例6Example 6
本实施例提供的基于EFIRM技术HPV病毒检测的试剂盒包括探针组合、固定物、链霉亲和素标记的辣根过氧化物酶(以溶液形式存在)及其底物,固定物是导电聚合物和离子化合物,其中,导电聚合物是吡咯,离子化合物是氯化钾。底物是含TMB的溶液。其余同实施例1。The kit for detecting HPV virus based on EFIRM technology provided by the embodiment includes a probe combination, a immobilizer, streptavidin-labeled horseradish peroxidase (present in solution) and a substrate thereof, and the immobilizer is conductive A polymer and an ionic compound, wherein the conductive polymer is pyrrole and the ionic compound is potassium chloride. The substrate is a solution containing TMB. The rest are the same as in the first embodiment.
其中,探针组合仅包括第1探针对、第2探针对、第12探针对、第13探针对、第14探针对、第15探针对、第16探针对(各探针对的捕获探针和检测探针的序列同实施例1)。当然,在其他的实施例中,探针组合包括第1探针对、第2探针对、选自第12~16探针对中的至少一种例如两种或三种等多种的情形。The probe combination includes only the first probe pair, the second probe pair, the twelfth probe pair, the thirteenth probe pair, the fourteen probe pair, the fifteenth probe pair, and the sixteenth probe pair (each The sequence of the capture probe and the detection probe of the probe pair is the same as in Example 1). Of course, in other embodiments, the probe combination includes a first probe pair, a second probe pair, and at least one selected from the group consisting of 12th to 16th probe pairs, for example, two or three types. .
采用本实施例的试剂盒仅对样本2进行检测,检测步骤与实施例1基本一致,检测结果如图6所示。The sample 2 was only tested by the kit of the present embodiment, and the detection step was basically the same as that of the first embodiment, and the detection result is shown in FIG. 6.
由图6可知,在5种中高危型组中,样本2的检测结果呈阳性(阴性对照电流值为28.36nA、其标准差为7.15,阳性对照电流值为147.47nA,样本2的电流值为66.91nA),说明样本2含有5种中高危型病毒中的一种或多种;而在16亚型组(阴性对照电流值为26.36nA、其标准差为5.10,阳性对照电流值为142.15nA,样本2的电流值为28.54nA)和18亚型组(阴性对照电流值为23.51nA、其标准差为5.90,阳性对照电流值为116.36nA,样本2的电流值为31.21nA)中,样本2的检测结果呈阴性,说明样本2中不含HPV16亚型和HPV18亚型。It can be seen from Fig. 6 that in the five medium-high-risk groups, the test result of sample 2 is positive (the negative control current value is 28.36nA, the standard deviation is 7.15, the positive control current value is 147.47nA, and the current value of sample 2 is 66.91nA), indicating that sample 2 contains one or more of five medium-high-risk viruses; in the 16-subtype group (negative control current value is 26.36nA, standard deviation is 5.10, positive control current value is 142.15nA) , sample 2 current value is 28.54nA) and 18 subtype group (negative control current value is 23.51nA, its standard deviation is 5.90, positive control current value is 116.36nA, sample 2 current value is 31.21nA), sample The test result of 2 was negative, indicating that sample 2 did not contain HPV16 subtype and HPV18 subtype.
检测过程替代性方案:Alternative to the testing process:
对以上实施例1-6的检测,可以采用以下检测步骤替代:For the detection of the above Examples 1-6, the following detection steps can be used instead:
替代方法一.Alternative method one.
1、捕获探针固定在检测孔板的板底1. The capture probe is fixed at the bottom of the detection orifice plate.
1.1噻吩与捕获探针(简称CP)的混合液配制:1.1 Mixture of thiophene and capture probe (CP for short):
捕获溶液含有以下成分:噻吩的重量百分数为5%,NaCl的浓度为2mol/L,捕获探针的浓度为1.5μmol/L。The capture solution contained the following components: 5% by weight of thiophene, 2 mol/L of NaCl, and 1.5 μmol/L of capture probe.
1.2加样:1.2 loading:
在96孔的检测孔板上,每个孔加入20μl的已配制好的噻吩与CP混合液,加样时枪头贴近孔的底部,但是不接触到底部电极,加完后倾斜或拍打检测孔板使液体在孔里的电极表面均匀覆盖,然后立刻到EFIRM检测仪上进行电场操作。On a 96-well assay plate, add 20 μl of the prepared mixture of thiophene and CP to each well. When the sample is applied, the tip of the gun is attached to the bottom of the well, but the bottom electrode is not touched. After the addition, the tilt or tapping detection hole is applied. The plate evenly covers the surface of the electrode in the hole and immediately goes to the EFIRM detector for electric field operation.
1.3EFIRM电场处理:1.3EFIRM electric field treatment:
在EFIRM软件上选择进行实验的对应列,电场参数设置为:电压A:500mV,1s;电压B:1500mV,1s;进行10个循环。电场处理完毕,立刻取出,清洗检测孔板。The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: 500 mV, 1 s; voltage B: 1500 mV, 1 s; 10 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
1.4检测孔板清洗:1.4 detection orifice cleaning:
在洗板机程序上选择对应的实验列,清洗程序选择(2bottom,2top),洗液选择2XSSC(0.06%SDS)。清洗完毕,立刻进行下一步样本上样操作。Select the corresponding experiment column on the washer program, select the cleaning program (2bottom, 2top), and choose 2XSSC (0.06% SDS) for the lotion. After the cleaning is completed, proceed to the next sample loading operation.
2、样本杂交:2. Sample hybridization:
2.1杂交buffer预处理2.1 hybrid buffer pretreatment
杂交buffer(使用发明内容中的杂交buffer 3)在水浴锅中95℃水浴处理5min,然后室温放置冷却。
The hybrid buffer (using the hybrid buffer 3 in the Summary of the Invention) was treated in a water bath at 95 ° C for 5 min in a water bath and then allowed to cool at room temperature.
2.2样本的配制2.2 Preparation of the sample
样本从-20℃冰箱取出,放进4℃冰箱解冻。完全溶解后,样本与杂交buffer按体积比1:2.5混合,涡旋振荡后离心,即可上样进行检测。The samples were taken out from the -20 ° C refrigerator and thawed in a 4 ° C refrigerator. After complete dissolution, the sample and the hybrid buffer are mixed at a volume ratio of 1:2.5, vortexed and centrifuged, and the sample can be tested.
2.3加样:2.3 loading:
在检测孔板上,在对应的孔里加入空白对照buffer、相应浓度的阴性对照(WT)和阳性对照(MT),上样量80μl。加样时枪头贴近孔的底部,但是不接触到底部电极,加完后倾斜或拍打检测孔板使液体在孔里的电极表面均匀覆盖,然后立刻到EFIRM上进行电场操作。On the detection well plate, a blank control buffer, a corresponding concentration of the negative control (WT) and a positive control (MT) were added to the corresponding wells, and the sample volume was 80 μl. When the sample is applied, the tip of the gun is attached to the bottom of the hole, but the bottom electrode is not touched. After the addition, the tilting or tapping of the detecting plate allows the liquid to uniformly cover the surface of the electrode in the hole, and then immediately goes to the EFIMR for electric field operation.
2.4EFIRM电场处理:2.4EFIRM electric field treatment:
在EFIRM软件上选择进行实验的对应列,电场参数设置为:电压A:500mV,1s;电压B:800mV,1s;进行10个循环。电场处理完毕,立刻取出,清洗检测孔板。The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: 500 mV, 1 s; voltage B: 800 mV, 1 s; 10 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
2.5检测孔板清洗:2.5 detection orifice cleaning:
在洗板机程序上选择对应的实验列,清洗程序选择(2bottom,2top),洗液选择2XSSC(0.06%SDS)。清洗完毕,立刻进行DP加样操作。Select the corresponding experiment column on the washer program, select the cleaning program (2bottom, 2top), and choose 2XSSC (0.06% SDS) for the lotion. After the cleaning is completed, the DP loading operation is performed immediately.
3、检测杂交3. Detection of hybridization
3.1DP(检测探针)溶液配制:3.1DP (detection probe) solution preparation:
检测溶液是以PBS作为溶剂,其中酪蛋白的重量百分数为5%,检测探针的浓度为1.5μmol/L,涡旋震荡混匀,离心,备用。The detection solution was PBS as a solvent, wherein the weight percentage of casein was 5%, the concentration of the detection probe was 1.5 μmol/L, vortexed and mixed, centrifuged, and set aside.
3.2加样:3.2 loading:
根据实验设计在对应的孔加入对应DP溶液20μl,加样时枪头贴近孔的底部,但是不接触到底部电极,加完后倾斜或拍打检测孔板使液体在孔里的电极表面均匀覆盖,然后立刻到EFIRM上进行电场操作。According to the experimental design, 20 μl of the corresponding DP solution was added to the corresponding hole. When the sample was applied, the tip of the gun was attached to the bottom of the hole, but the bottom electrode was not touched. After the addition, the tilting or tapping of the detecting plate made the liquid evenly cover the surface of the electrode in the hole. Then immediately go to the EFIRM for electric field operation.
3.3EFIRM电场处理:3.3EFIRM electric field treatment:
在EFIRM软件上选择进行实验的对应列,电场参数设置为:电压A:500mV,1s;电压B:800mV,1s;进行8个循环。电场处理完毕,立刻取出,清洗检测孔板。The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: 500 mV, 1 s; voltage B: 800 mV, 1 s; 8 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
3.4检测孔板清洗:3.4 Detection of orifice cleaning:
在洗板机程序上选择对应的实验列,清洗程序选择(2bottom,2top),洗液选择2XSSC(0.06%SDS)。清洗完毕,立刻进行加样操作。Select the corresponding experiment column on the washer program, select the cleaning program (2bottom, 2top), and choose 2XSSC (0.06% SDS) for the lotion. After the cleaning is completed, the sample loading operation is performed immediately.
4、Reporter Hybridization4, Reporter Hybridization
4.1Poly-HRP溶液配制:4.1Poly-HRP solution preparation:
以PBS作为溶剂,其中酪蛋白的重量百分数为5%,poly-HRP的体积百分数为0.2%(购自thermo fisher,产品名称为PierceTM Streptavidin Poly-HRP,货号为21140,单位规格为0.5mL),涡旋震荡混匀,离心,备用。With PBS as a solvent, wherein the weight percentage of casein is 5%, the volume percentage of poly-HRP 0.2% (available from Thermo Fisher, product name Pierce TM Streptavidin Poly-HRP, item number 21140, the unit size of 0.5 mL) Vortex and mix well, centrifuge, and set aside.
4.2加样:4.2 loading:
根据实验设计在对应的孔加入Poly-HRP溶液20μl,加样时枪头贴近孔的底部,但是不接触到底部电极,加完后倾斜或拍打检测孔板使液体在孔里的电极表面均匀覆盖,然后盖上检测孔板盖子,实验台上室温孵育40min,计时器倒数计时。孵育时间到,立刻清洗检测孔板。According to the experimental design, 20 μl of Poly-HRP solution was added to the corresponding hole. When the sample was applied, the tip of the gun was attached to the bottom of the hole, but the bottom electrode was not touched. After the addition, the tilting or tapping of the detecting plate allowed the liquid to uniformly cover the surface of the electrode in the hole. Then, cover the detection plate cover, incubate for 40 minutes at room temperature on the bench, and count down the timer. The incubation plate is cleaned immediately after the incubation time.
4.3检测孔板清洗:4.3 Detection plate cleaning:
在洗板机程序上选择对应的实验列,清洗程序选择(3bottom,3top),洗液选择PBST(0.15%Tween20)。清洗完毕,立刻进行TMB加样操作。Select the corresponding experiment column on the washing machine program, select the cleaning program (3bottom, 3top), and choose PBST (0.15% Tween20) for the lotion. After the cleaning is completed, the TMB loading operation is performed immediately.
5、Readout5, Readout
5.1加样:5.1 loading:
根据实验设计在对应的孔加入TMB/H2O2溶液(购自thermo fisher,产品货号为34022,名称为According to the experimental design, TMB/H2O2 solution (purchased from thermo fisher, product no. 34022, named as
Turbo TMB底物溶液),每个孔加入80μl,加样时枪头贴近孔的底部,但是不接触到底部电极。加完立刻到EFIRM上进行电场操作。Turbo TMB substrate solution), 80 μl was added to each well, and the tip of the gun was attached to the bottom of the well while the sample was applied, but did not touch the bottom electrode. Immediately after the addition, the electric field operation was performed on the EFIRM.
5.2EFIRM电场读数:5.2EFIRM electric field reading:
在EFIRM软件上选择进行实验的对应列,电场参数设置为:电压A:-300mV,100s,得到电流读数。
Select the corresponding column for the experiment on the EFIRM software. The electric field parameter is set to: voltage A: -300mV, 100s, and the current reading is obtained.
检测结果与基于实施例1记载的步骤所得实施例1-6的结果一致。The test results were in agreement with the results of Examples 1-6 obtained based on the procedure described in Example 1.
替代检测过程二、Alternative detection process
对待检测样本进行检测,具体步骤如下:Test the test sample, the specific steps are as follows:
1、捕获探针固定在检测孔板的板底1. The capture probe is fixed at the bottom of the detection orifice plate.
1.1苯胺与捕获探针(简称CP)的混合液配制:1.1 Mixture of aniline and capture probe (CP for short):
捕获溶液含有以下成分:苯胺的重量百分数为0.1%,NaCl的浓度为0.01mol/L,捕获探针的浓度为0.5μmol/L。The capture solution contained the following components: the weight percentage of aniline was 0.1%, the concentration of NaCl was 0.01 mol/L, and the concentration of the capture probe was 0.5 μmol/L.
1.2加样:1.2 loading:
在96孔的检测孔板上,每个孔加入80μl的已配制好的苯胺与CP混合液,加样时枪头贴近孔的底部,但是不接触到底部电极,加完后倾斜或拍打检测孔板使液体在孔里的电极表面均匀覆盖,然后立刻到EFIRM检测仪上进行电场操作。On a 96-well assay plate, add 80 μl of the prepared aniline and CP mixture to each well. When the sample is applied, the tip of the gun is attached to the bottom of the well, but the bottom electrode is not touched. After the addition, the tilt or tapping detection hole is applied. The plate evenly covers the surface of the electrode in the hole and immediately goes to the EFIRM detector for electric field operation.
1.3EFIRM电场处理:1.3EFIRM electric field treatment:
在EFIRM软件上选择进行实验的对应列,电场参数设置为:电压A:200mV,5s;电压B:800mV,5s;进行3个循环。电场处理完毕,立刻取出,清洗检测孔板。The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: 200 mV, 5 s; voltage B: 800 mV, 5 s; 3 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
1.4检测孔板清洗:1.4 detection orifice cleaning:
在洗板机程序上选择对应的实验列,清洗程序选择(2bottom,2top),洗液选择2XSSC(0.04%SDS)。清洗完毕,立刻进行下一步样本上样操作。Select the corresponding experiment column on the washer program, select the cleaning program (2bottom, 2top), and choose 2XSSC (0.04% SDS) for the lotion. After the cleaning is completed, proceed to the next sample loading operation.
2、样本杂交:2. Sample hybridization:
2.1杂交buffer预处理2.1 hybrid buffer pretreatment
杂交buffer(使用发明内容中的杂交buffer 6)在水浴锅中85℃水浴处理15min,然后室温放置冷却。The hybrid buffer (using the hybrid buffer 6 in the Summary of the Invention) was treated in a water bath at 85 ° C for 15 min in a water bath and then left to cool at room temperature.
2.2样本的配制2.2 Preparation of the sample
样本从-20℃冰箱取出,放进4℃冰箱解冻。完全溶解后,样本与杂交buffer按体积比1:1.5混合,涡旋振荡后离心,即可上样进行检测。The samples were taken out from the -20 ° C refrigerator and thawed in a 4 ° C refrigerator. After complete dissolution, the sample and the hybrid buffer are mixed at a volume ratio of 1:1.5, vortexed and centrifuged, and the sample can be tested.
2.3加样:2.3 loading:
在检测孔板上,在对应的孔里加入空白对照buffer、相应浓度的阴性对照(WT)和阳性对照(MT),上样量20μl。加样时枪头贴近孔的底部,但是不接触到底部电极,加完后倾斜或拍打检测孔板使液体在孔里的电极表面均匀覆盖,然后立刻到EFIRM上进行电场操作。On the detection well plate, a blank control buffer, a corresponding concentration of the negative control (WT) and a positive control (MT) were added to the corresponding wells, and the amount of the sample was 20 μl. When the sample is applied, the tip of the gun is attached to the bottom of the hole, but the bottom electrode is not touched. After the addition, the tilting or tapping of the detecting plate allows the liquid to uniformly cover the surface of the electrode in the hole, and then immediately goes to the EFIMR for electric field operation.
2.4EFIRM电场处理:2.4EFIRM electric field treatment:
在EFIRM软件上选择进行实验的对应列,电场参数设置为:电压A:200mV,5s;电压B:300mV,5s;进行3个循环。电场处理完毕,立刻取出,清洗检测孔板。The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: 200 mV, 5 s; voltage B: 300 mV, 5 s; 3 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
2.5检测孔板清洗:2.5 detection orifice cleaning:
在洗板机程序上选择对应的实验列,清洗程序选择(2bottom,2top),洗液选择2XSSC(0.04%SDS)。清洗完毕,立刻进行DP加样操作。Select the corresponding experiment column on the washer program, select the cleaning program (2bottom, 2top), and choose 2XSSC (0.04% SDS) for the lotion. After the cleaning is completed, the DP loading operation is performed immediately.
3、检测杂交3. Detection of hybridization
3.1DP(检测探针)溶液配制:3.1DP (detection probe) solution preparation:
检测溶液是以PBS作为溶剂,其中酪蛋白的重量百分数为0.1%,检测探针的浓度为0.5μmol/L,涡旋震荡混匀,离心,备用。The detection solution was PBS as a solvent, wherein the weight percentage of casein was 0.1%, the concentration of the detection probe was 0.5 μmol/L, vortexed and mixed, centrifuged, and set aside.
3.2加样:3.2 loading:
根据实验设计在对应的孔加入对应DP溶液30μl,加样时枪头贴近孔的底部,但是不接触到底部电极,加完后倾斜或拍打检测孔板使液体在孔里的电极表面均匀覆盖,然后立刻到EFIRM上进行电场操作。According to the experimental design, 30 μl of the corresponding DP solution was added to the corresponding hole. When the sample was applied, the tip of the gun was attached to the bottom of the hole, but the bottom electrode was not touched. After the addition, the tilting or tapping of the detecting plate made the liquid evenly cover the surface of the electrode in the hole. Then immediately go to the EFIRM for electric field operation.
3.3EFIRM电场处理:3.3EFIRM electric field treatment:
在EFIRM软件上选择进行实验的对应列,电场参数设置为:电压A:200mV,5s;电压B:300mV,5s;进行3个循环。电场处理完毕,立刻取出,清洗检测孔板。The corresponding column for the experiment was selected on the EFIRM software. The electric field parameters were set to: voltage A: 200 mV, 5 s; voltage B: 300 mV, 5 s; 3 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
3.4检测孔板清洗:3.4 Detection of orifice cleaning:
在洗板机程序上选择对应的实验列,清洗程序选择(2bottom,2top),洗液选择2XSSC(0.04%
SDS)。清洗完毕,立刻进行加样操作。Select the corresponding experiment column on the washing machine program, the cleaning program selection (2bottom, 2top), the lotion selection 2XSSC (0.04%)
SDS). After the cleaning is completed, the sample loading operation is performed immediately.
4、Reporter Hybridization4, Reporter Hybridization
4.1Poly-HRP溶液配制:4.1Poly-HRP solution preparation:
以PBS作为溶剂,其中酪蛋白的重量百分数为0.1%,poly-HRP的体积百分数为0.05%(购自thermo fisher,产品名称为PierceTM Streptavidin Poly-HRP,货号为21140,单位规格为0.5mL),涡旋震荡混匀,离心,备用。With PBS as a solvent, wherein the weight percentage of casein 0.1%, the volume percentage of poly-HRP in 0.05% (available from Thermo Fisher, product name Pierce TM Streptavidin Poly-HRP, item number 21140, the unit size of 0.5 mL) Vortex and mix well, centrifuge, and set aside.
4.2加样:4.2 loading:
根据实验设计在对应的孔加入Poly-HRP溶液20-80μl,加样时枪头贴近孔的底部,但是不接触到底部电极,加完后倾斜或拍打检测孔板使液体在孔里的电极表面均匀覆盖,然后盖上检测孔板盖子,实验台上室温孵育20-40min,计时器倒数计时。孵育时间到,立刻清洗检测孔板。According to the experimental design, add 20-80μl of Poly-HRP solution to the corresponding hole. When the sample is applied, the tip of the gun is close to the bottom of the hole, but it does not touch the bottom electrode. After the addition, tilt or tap the surface of the electrode to detect the hole in the hole. Cover evenly, then cover the detection plate cover, incubate for 20-40min at room temperature on the bench, and count down the timer. The incubation plate is cleaned immediately after the incubation time.
4.3检测孔板清洗:4.3 Detection plate cleaning:
在洗板机程序上选择对应的实验列,清洗程序选择(3bottom,3top),洗液选择PBST(0.05%Tween20)。清洗完毕,立刻进行TMB加样操作。Select the corresponding experiment column on the washer program, select the cleaning program (3bottom, 3top), and choose PBST (0.05% Tween20) for the lotion. After the cleaning is completed, the TMB loading operation is performed immediately.
5、Readout5, Readout
5.1加样:5.1 loading:
根据实验设计在对应的孔加入TMB/H2O2溶液(购自thermo fisher,产品货号为34022,名称为According to the experimental design, TMB/H 2 O 2 solution was added to the corresponding well (purchased from thermo fisher, product No. 34022, the name is
Turbo TMB底物溶液),每个孔加入30μl,加样时枪头贴近孔的底部,但是不接触到底部电极。加完立刻到EFIRM上进行电场操作。Turbo TMB substrate solution), add 30 μl to each well, and attach the tip to the bottom of the well while loading, but not the bottom electrode. Immediately after the addition, the electric field operation was performed on the EFIRM.
5.2EFIRM电场读数:5.2EFIRM electric field reading:
在EFIRM软件上选择进行实验的对应列,电场参数设置为:电压A:-100mV,40s,得到电流读数。Select the corresponding column for the experiment on the EFIRM software. The electric field parameter is set to: voltage A: -100mV, 40s, and the current reading is obtained.
同样地,得到的结果与基于实施例1所得的实施例1-6的结果基本一致。Also, the results obtained were substantially in agreement with the results of Examples 1-6 obtained based on Example 1.
本发明的方法中采用的检测孔板,共同特点在于,其中的反应孔的内底部设置有电极,用于接通EFIRM检测仪后对反应孔内溶液施加电场,可以采用现有市售产品,使用时在反应孔内固定捕获探针,或预制好待用或出售。The detecting orifice plate used in the method of the present invention has the common feature that the inner bottom of the reaction hole is provided with an electrode for applying an electric field to the solution in the reaction hole after the EFIRM detector is turned on, and the commercially available product can be used. The capture probe is immobilized in the reaction well during use, or prefabricated for use or for sale.
在本发明的优选实施例中,采用的检测孔板图7所示:包括:反应孔底板101,反应孔底板101包括至少一个检测区域102;工作电极103,工作电极103设置在反应孔底板101上并配置为施加电压以形成电场;以及对置电极104,对置电极104设置在反应孔底板101上并配置为获取检测信号并输出所述检测信号。例如,这里的工作电极103和对置电极104均设置在反应孔底板的同一表面上,因此,工作电极103和对置电极104可以是位于同一平面内。如图7所示,工作电极103包括至少一个宽度均匀的第一线状部1031;对置电极104包括至少一个宽度均匀的第二线状部1041;第一线状部1031和第二线状部1041设置在检测区102内并且相互交替等距间隔设置。需要说明的是,上述的工作电极103和对置电极104的结构相同,因此,位于图1右侧的电极104可配置为施加电压以形成电场的工作电极;位于图7左侧的电极103可配置为获取检测信号并输出所述检测信号的对置电极,本公开在此不作限定。In a preferred embodiment of the present invention, the detection orifice plate is shown in FIG. 7 and includes: a reaction well bottom plate 101, the reaction well bottom plate 101 includes at least one detection region 102, a working electrode 103, and the working electrode 103 is disposed on the reaction well bottom plate 101. And configured to apply a voltage to form an electric field; and the opposite electrode 104, the opposite electrode 104 is disposed on the reaction aperture substrate 101 and configured to acquire a detection signal and output the detection signal. For example, the working electrode 103 and the opposite electrode 104 are both disposed on the same surface of the reaction cell bottom plate, and therefore, the working electrode 103 and the opposite electrode 104 may be in the same plane. As shown in FIG. 7, the working electrode 103 includes at least one first linear portion 1031 having a uniform width; the opposite electrode 104 includes at least one second linear portion 1041 of uniform width; the first linear portion 1031 and the second linear portion 1041 They are disposed within the detection zone 102 and are alternately spaced apart from one another. It should be noted that the above-mentioned working electrode 103 and the opposite electrode 104 have the same structure. Therefore, the electrode 104 located on the right side of FIG. 1 can be configured as a working electrode to apply a voltage to form an electric field; the electrode 103 located on the left side of FIG. The counter electrode is configured to acquire a detection signal and output the detection signal, and the disclosure is not limited herein.
在本实施例提供的检测电极结构中,工作电极103可施加电压产生电场来使标靶物质移动并聚集,例如,工作电极103可施加方波交变电压,先使得待检测液体中包括靶标物质的带电的物质向工作电极103移动富集,使得靶标物质可与工作电极103上探针结合,然后转变电压的极性,使得带电的物质中其他没有与探针结合的物质远离工作电极103(电场对靶标物质的作用力设置为小于靶标物质与探针的结合力);然后,对置电极104可获取关于标靶物质的检测信号并将检测信号输出,例如,与探针结合的靶标物质会与特定的试剂发生反应而产生电流,因此对置电极104可通过检测电流来获取关于标靶物质的检测信号并将检测信号输出;然后,通过分析输出的检测信号可得出关于标靶物质的信息(例如靶标物质的浓度),从而可迅速、准确地进行检测。由于第一线状部1031和第二线状部1041是宽度均匀的线状结构,并且在检测区102内,第一线状部1031和第二线状部1041在检测区102内相互交替等距间隔设置,由此,工作电极103的第一线状部1031可在检测区102产生均匀的电场,对置电极104的第二线状部1041可在检测区102可检测到细微的电流,因此
可提高检测的精度。另外,由于工作电极103的第一线状部1031与第二线状部1041交替间隔设置,从而可控制形成在工作电极103上探针的密度和均匀度,不会使得探针过密,从而给靶标物质与探针的结合提供空间,提高靶标物质与探针的结合的效率,从而既可提高液体活检的反应速度,又可进一步提高检测的精度。In the structure of the detecting electrode provided in this embodiment, the working electrode 103 can apply a voltage to generate an electric field to move and concentrate the target substance. For example, the working electrode 103 can apply a square wave alternating voltage to first include the target substance in the liquid to be detected. The charged substance moves to the working electrode 103 to be enriched, so that the target substance can be combined with the probe on the working electrode 103, and then the polarity of the voltage is changed, so that other substances in the charged substance that are not combined with the probe are away from the working electrode 103 ( The force of the electric field on the target substance is set to be smaller than the binding force of the target substance and the probe; then, the opposite electrode 104 can acquire a detection signal about the target substance and output the detection signal, for example, a target substance bound to the probe. The current reacts with a specific reagent to generate a current, so the opposite electrode 104 can acquire a detection signal about the target substance by detecting the current and output the detection signal; and then, by analyzing the output detection signal, the target substance can be obtained. Information (such as the concentration of the target substance) so that it can be detected quickly and accurately. Since the first linear portion 1031 and the second linear portion 1041 are linear structures having a uniform width, and within the detection region 102, the first linear portion 1031 and the second linear portion 1041 are alternately equidistantly spaced within the detection region 102. It is provided that the first linear portion 1031 of the working electrode 103 can generate a uniform electric field in the detection region 102, and the second linear portion 1041 of the opposite electrode 104 can detect a minute current in the detection region 102, thus
Can improve the accuracy of detection. In addition, since the first linear portion 1031 of the working electrode 103 and the second linear portion 1041 are alternately spaced, the density and uniformity of the probe formed on the working electrode 103 can be controlled, and the probe is not overly dense, thereby giving The binding of the target substance to the probe provides space to increase the efficiency of binding of the target substance to the probe, thereby improving the reaction speed of the liquid biopsy and further improving the accuracy of the detection.
如图7所示,检测孔板中的检测电极结构,检测区102的形状为圆形,工作电极103可包括弧状的第一主体部1030以及与从第一主体部1030延伸而出的多个相互平行的第一线状部1031。对置电极104包括弧状的第二主体部1040以及与从第二主体部1040延伸出的多个相互平行的第二线状部1041。第一主体部1030与第二主体部1040相对设置,多个第一线状部1031与多个第二线状部1041设置在检测区102内并交替间隔等距设置。也就是说,工作电极103和对置电极104呈梳齿状结构,并且工作电极103和对置电极104互相交叉以形成插指状结构。需要说明的是,检测区的范围可包括多个第一线状部与多个第二线状部,还可包括第一主体部和第二主体部,本公开在此不作限制。工作电极可不设置与探针直接接触的固定部,因此第一线状部可做成宽度均匀的线状,从而提供更均匀的电场,使得探针的排列更规则,从而提高检测的效率和精度。As shown in FIG. 7, the detecting electrode structure in the detecting orifice plate has a circular shape, and the working electrode 103 may include an arc-shaped first body portion 1030 and a plurality of extending from the first body portion 1030. The first linear portion 1031 that is parallel to each other. The counter electrode 104 includes an arc-shaped second body portion 1040 and a plurality of second linear portions 1041 that are parallel to each other and extend from the second body portion 1040. The first body portion 1030 is disposed opposite to the second body portion 1040, and the plurality of first linear portions 1031 and the plurality of second linear portions 1041 are disposed in the detection region 102 and are alternately spaced and equidistantly disposed. That is, the working electrode 103 and the opposite electrode 104 have a comb-like structure, and the working electrode 103 and the opposite electrode 104 cross each other to form an interdigitated structure. It should be noted that the range of the detection area may include a plurality of first linear portions and a plurality of second linear portions, and may further include a first main body portion and a second main body portion, which are not limited herein. The working electrode may not be provided with a fixing portion that is in direct contact with the probe, so the first linear portion may be formed into a line having a uniform width, thereby providing a more uniform electric field, so that the arrangement of the probe is more regular, thereby improving the efficiency and accuracy of detection. .
图8示出了另一种检测电极结构的平面示意图,检测区102的形状为圆形,工作电极103包括螺旋状排列的第一线状部1031,对置电极104包括螺旋状排列的第二线状部1041,第一线状部1031与第二线状部1041设置在检测区102内并且交替间隔等距设置。在本实施例一示例提供的检测电极结构中,第一线状部的宽度与第二线状部的宽度相同,从而可提高检测的精度;另外,第一线状部和第二线状部的宽度范围可为3-20mil(千分之一英寸)。8 is a schematic plan view showing another structure of the detecting electrode. The detecting area 102 has a circular shape, the working electrode 103 includes a first linear portion 1031 which is spirally arranged, and the opposite electrode 104 includes a second line which is spirally arranged. The portion 1041, the first linear portion 1031 and the second linear portion 1041 are disposed in the detection region 102 and are alternately spaced and equidistantly disposed. In the detecting electrode structure provided by the example of the embodiment, the width of the first linear portion is the same as the width of the second linear portion, so that the accuracy of the detection can be improved; in addition, the width of the first linear portion and the second linear portion The range can be 3-20 mils (thousandths of an inch).
例如,在本实施例一示例提供的检测电极结构中,第一线状部和第二线状部的间距范围可为3-20mil(千分之一英寸)。For example, in the detecting electrode structure provided in the example of the embodiment, the pitch of the first linear portion and the second linear portion may range from 3 to 20 mils (thousandths of an inch).
例如,在本实施例一示例提供的检测电极结构中,第一线状部的宽度与第二线状部的宽度等于第一线状部和第二线状部的间距。由此,从而可进一步提高电场的均匀度以及检测的均匀度,从而可提高检测的精度。For example, in the detecting electrode structure provided in the example of the embodiment, the width of the first linear portion and the width of the second linear portion are equal to the spacing between the first linear portion and the second linear portion. Thereby, the uniformity of the electric field and the uniformity of detection can be further improved, so that the accuracy of detection can be improved.
例如,图9是另一种检测电极结构的平面示意图,图10是另一种检测电极结构的平面示意图;如图9或图10所示,本实施例一示例提供的检测电极结构还包括设置在检测区边缘的参考电极,由于参考电极设置在检测区边缘,第一线状部和第二线状部的外侧,因此参考电极在获取关于标靶物质的检测信号的过程中可提供对照,可消除工作电极的极性误差,从而进一步提高检测的精度。For example, FIG. 9 is a plan view showing another structure of the detecting electrode, and FIG. 10 is a plan view showing another structure of the detecting electrode. As shown in FIG. 9 or FIG. 10, the detecting electrode structure provided in an example of the embodiment further includes setting. At the reference electrode at the edge of the detection zone, since the reference electrode is disposed at the edge of the detection zone, the outer side of the first linear portion and the second linear portion, the reference electrode can provide a contrast in the process of acquiring the detection signal about the target substance. The polarity error of the working electrode is eliminated, thereby further improving the accuracy of the detection.
例如,在另一实施例中使用的检测孔板的检测电极结构中,工作电极和对置电极的材料包括金。由于金元素化学性质稳定不与待检测液体反应并且具有较低的阻抗,从而可进一步提高检测的精度。当然,包括但不限于此,也可采用其他导电物质,例如铂或氧化铟锡等。For example, in the detecting electrode structure of the detecting orifice plate used in another embodiment, the material of the working electrode and the opposing electrode includes gold. Since the chemical nature of the gold element is stable and does not react with the liquid to be detected and has a lower impedance, the accuracy of the detection can be further improved. Of course, including but not limited to, other conductive materials such as platinum or indium tin oxide may also be used.
图11a和图11b示出了本发明的方法中采用的一种检测孔板,该检测孔板包括:盒体200以及检测电极结构100,盒体200包括多个反应孔211,反应孔的尺寸可参照通常的96孔板的设计,当然,本公开包括但不限于此,反应孔的尺寸可根据待检测液体的浓度和种类进行设计。如图12和13所示,检测电极结构100设置在盒体200的底部,检测电极结构100可为上述实施例一中任一的检测电极结构,并且,检测区102设置在反应孔211的底部并将反应孔211的底部密封。另外,检测电极结构100的反应孔底板101与盒体200可采用同样的材料制作。由此,可在由检测区102和反应孔211组成的容置空间中盛放待检测液体,从而对待检测液体进行检测。例如,工作电极103可施加方波交变电压,以形成垂直于通孔211底面的垂直电场,先使得待检测液体中包括靶标物质的带电的物质从反应孔211的各个位置向反应孔211的底部运动,并向工作电极103移动富集,使得靶标物质可与工作电极103上探针(可与靶标物质结合的物质,例如DNA聚合分子)结合,然后转变电压的极性,使垂直于通孔211底面的垂直电场的方向反转,使得待检测液体中的带电的物质中其他没有与探针结合的物质从反应孔211的底部向反应孔211的上部运动,从而使得带电的物质中其他没有与探针结合的物质远离工作电极103(电场对靶标物质的作用力设置为小于靶标物质与探针的结合力);然后,对置电极104可获取关于标靶物质的检测信号并将检测信号输出,例如,与探针结合的靶标物质会与特定的试剂发生反应而产生电流,因此对置电极104可通过检测电流来获取关于标靶物质的检测信号并将检测信号输出;然后,通过分析输出的检测信号可得出关于标靶物质的信息(例如靶标物质的浓度)。Figures 11a and 11b illustrate a detection orifice plate used in the method of the present invention, the detection orifice plate comprising: a cartridge body 200 and a detection electrode structure 100, the cartridge body 200 comprising a plurality of reaction wells 211, the size of the reaction wells Reference may be made to the design of a conventional 96-well plate. Of course, the present disclosure includes but is not limited thereto, and the size of the reaction well may be designed according to the concentration and kind of the liquid to be detected. As shown in FIGS. 12 and 13, the detecting electrode structure 100 is disposed at the bottom of the casing 200, the detecting electrode structure 100 may be the detecting electrode structure of any of the above-described first embodiment, and the detecting portion 102 is disposed at the bottom of the reaction hole 211. The bottom of the reaction well 211 is sealed. Further, the reaction cell bottom plate 101 of the detecting electrode structure 100 and the casing 200 can be made of the same material. Thereby, the liquid to be detected can be contained in the accommodating space composed of the detection zone 102 and the reaction well 211, thereby detecting the liquid to be detected. For example, the working electrode 103 may apply a square wave alternating voltage to form a vertical electric field perpendicular to the bottom surface of the through hole 211, first causing the charged substance including the target substance in the liquid to be detected from the respective positions of the reaction hole 211 to the reaction hole 211. The bottom moves and moves to the working electrode 103 to enrich, so that the target substance can be combined with the probe on the working electrode 103 (a substance that can bind to the target substance, such as a DNA polymer molecule), and then the polarity of the voltage is converted to make it perpendicular to the pass. The direction of the vertical electric field on the bottom surface of the hole 211 is reversed, so that other substances not charged with the probe in the charged substance in the liquid to be detected move from the bottom of the reaction hole 211 to the upper portion of the reaction hole 211, thereby causing other substances in the charged substance. The substance not bound to the probe is away from the working electrode 103 (the force of the electric field on the target substance is set to be smaller than the binding force of the target substance and the probe); then, the opposite electrode 104 can acquire the detection signal about the target substance and detect The signal output, for example, the target substance bound to the probe reacts with a specific reagent to generate a current, so the opposite electrode 104 can pass the inspection. The current is measured to obtain a detection signal about the target substance and the detection signal is output; then, the information about the target substance (for example, the concentration of the target substance) can be obtained by analyzing the output detection signal.
例如,在本发明提供的检测孔板中,如图11b所示,多个反应孔211成矩阵排列在盒体200中。
如图12或图13所示,多个反应孔211为圆柱形通孔。需要说明的是,反应孔形状包括但不限于此,多个反应孔211的形状还可为方形柱体、三角形柱体或其他柱体。For example, in the detecting orifice plate provided by the present invention, as shown in FIG. 11b, a plurality of reaction wells 211 are arranged in a matrix in the cartridge body 200.
As shown in FIG. 12 or FIG. 13, the plurality of reaction holes 211 are cylindrical through holes. It should be noted that the shape of the reaction well includes, but is not limited to, the shape of the plurality of reaction holes 211 may also be a square cylinder, a triangular cylinder or other cylinders.
例如,在本法采用的检测孔板中,多个反应孔211的数量为四的倍数,如图8所示,四个相邻的反应孔211对应的检测电极结构中的四个工作电极103电性相连。例如,反应孔底板101上形成有导线111以及导线112,导线111将四个工作电极103电性相连;导线112分别与四个对置电极104电性相连以将对置电极104的电信号引出。由此,这四个相邻的通孔可作为一个检测组,在该检测组内,由于四个工作电极电性相连,施加在四个工作电极上的电压一致,四个相邻的通孔可更好地进行对照实验,因此可进一步提高检测精度。当然,还可将任意个数的通孔对应的检测电极结构中的任意个数的工作电极电性相连,以提供一致的电压。For example, in the detection orifice plate used in the present method, the number of the plurality of reaction holes 211 is a multiple of four, as shown in FIG. 8, four adjacent reaction holes 211 correspond to the four working electrodes 103 in the detection electrode structure. Electrically connected. For example, the reaction hole bottom plate 101 is formed with a wire 111 and a wire 112. The wire 111 electrically connects the four working electrodes 103. The wires 112 are electrically connected to the four opposite electrodes 104 to extract the electrical signals of the opposite electrode 104. . Thus, the four adjacent through holes can be used as a detection group. In the detection group, since the four working electrodes are electrically connected, the voltages applied to the four working electrodes are uniform, and four adjacent through holes are The control experiment can be performed better, so the detection accuracy can be further improved. Of course, any number of working electrodes in the detection electrode structure corresponding to any number of through holes may be electrically connected to provide a uniform voltage.
例如,如图15所示,本发明采用的检测孔板还包括:电路板110,电路板与检测极结构电性相连。电路板110上可设置放大电路以放大对置电极或参考电极输出的电信号,以提高检测精度;电路板110上也可设置稳压电路以向工作电极提供稳定的电压,以提高检测精度。当然,本公开包括但不限于此,电路板110上还可设置过流、过压保护电路等。For example, as shown in FIG. 15, the detection aperture board used in the present invention further includes: a circuit board 110 electrically connected to the detection pole structure. An amplification circuit may be disposed on the circuit board 110 to amplify the electrical signal outputted by the opposite electrode or the reference electrode to improve detection accuracy; a voltage stabilization circuit may also be disposed on the circuit board 110 to provide a stable voltage to the working electrode to improve detection accuracy. Of course, the present disclosure includes but is not limited thereto, and an overcurrent, overvoltage protection circuit, or the like may be disposed on the circuit board 110.
如图9所示,电路板110可设置在反应孔底板101下面,从而更合理地利用空间,当然,电路板110也可设置在其他位置,本公开在此不做限制。As shown in FIG. 9, the circuit board 110 can be disposed under the reaction hole bottom plate 101, so that the space can be utilized more reasonably. Of course, the circuit board 110 can also be disposed at other positions, and the disclosure is not limited herein.
综上,本发明提供的病毒基因分型检测试剂盒具有以下优点:In summary, the virus genotyping detection kit provided by the present invention has the following advantages:
1、检测灵敏度高:传统的探针固定方法是把探针的一端固定在平面支持物上,此方法由于支持物表面的疏水性等原因会降低探针与待测靶标DNA的杂交效率,本发明通过电荷吸附作用将捕获探针固定在聚吡咯孔内,可保证捕获探针具有超高活性;传统的核酸杂交过程通过控制杂交温度、盐离子、反应时间等提高杂交效率,本发明增加电场作为第四个控制条件,在电场的作用下提高了捕获探针对目的序列DNA的捕获效率;本方法中通过测定HRP催化TMB氧化过程中产生的电子信号作为检测结果,由于酶的催化效率很高,间接地放大了杂交反应的结果,增加了测定方法的敏感度。瞬间靶标分子捕获、超高活性分子探针固定、捕获分子信号特异放大这三大核心技术保证了本方法具有超高的灵敏性,远高于巴氏涂片、TCT技术和HC2等技术,与聚合酶链反应和基因芯片等技术相当。1. High detection sensitivity: The conventional probe fixing method is to fix one end of the probe on the planar support. This method reduces the hybridization efficiency between the probe and the target DNA to be detected due to the hydrophobicity of the surface of the support, etc. The invention fixes the capture probe in the polypyrrole hole by charge adsorption to ensure the ultra-high activity of the capture probe; the traditional nucleic acid hybridization process improves the hybridization efficiency by controlling the hybridization temperature, the salt ion, the reaction time, etc., and the invention increases the electric field. As a fourth control condition, the capture efficiency of the capture probe to the DNA of the target sequence is improved by the electric field; in this method, the electronic signal generated by the HRP-catalyzed oxidation of TMB is determined as a detection result, since the catalytic efficiency of the enzyme is very high. High, indirect amplification of the results of the hybridization reaction increases the sensitivity of the assay. The three core technologies of instantaneous target molecular capture, ultra-high activity molecular probe immobilization, and capture molecular signal specific amplification ensure that the method has ultra-high sensitivity, far higher than Pap smear, TCT technology and HC2 technology, and Polymerase chain reaction and gene chip and other technologies are equivalent.
2、检测特异性强:本发明中,每一个亚型HPV的检测过程均包含一条捕获探针和一条检测探针,探针长度在25-40bp之间,杂交效率受错配碱基的影响明显,只有目标序列DNA与两条探针同时准确配对后才能有检测信号,大大提高了检测的特异性。2. Strong detection specificity: In the present invention, each subtype of HPV detection process comprises a capture probe and a detection probe, and the probe length is between 25-40 bp, and the hybridization efficiency is affected by the mismatched base. Obviously, only the target sequence DNA and the two probes can be accurately paired at the same time to have a detection signal, which greatly improves the specificity of the detection.
3、操作简便、反应快速:本发明中电场的引入,一方面省略了杂交过程中对样品纯度要求,使得临床样本不经过核酸提取即可直接进行检测;另一方面降低了杂交过程中对反应时间的要求,加快了反应速率。整个检测过程可以在半小时内完成,缩短了总体反应时间,减少门诊病人的等待时间,为患者争取宝贵的治疗时间。3. Simple operation and rapid reaction: The introduction of the electric field in the present invention omits the purity requirement of the sample during the hybridization process, so that the clinical sample can be directly detected without nucleic acid extraction; on the other hand, the reaction in the hybridization process is reduced. The time required to speed up the reaction rate. The entire testing process can be completed in half an hour, shortening the overall response time, reducing waiting time for outpatients, and obtaining valuable treatment time for patients.
4、降低了对临床客户硬件及人员的要求:本发明中检测过程中不需要对样本进行纯化和PCR扩增等前处理,有效避免因环境污染造成的假阳性,且试验操作无需在专门的具有分区的PCR实验室进行,操作人员亦不需要获得临床基因扩增的上岗许可,可以由一般技术人员操作,对实验环境和操作人员的素质要求较低。4. Reduce the requirements for clinical customer hardware and personnel: In the present invention, the sample does not need to be pre-processed such as purification and PCR amplification, thereby effectively avoiding false positives caused by environmental pollution, and the test operation does not need to be specialized. For the PCR laboratory with partitions, the operator does not need to obtain the permission for clinical gene amplification, and can be operated by general technicians, and the requirements for the experimental environment and the quality of the operators are low.
5、成本低:首先,在检测设备方面,目前无论是ARMS-PCR还是数字PCR都采用荧光信号检测,检测设备需配备昂贵的荧光检测系统,荧光定量PCR仪市场售价都在数十万元左右,而数字PCR仪更达100多万元。与之相比,EFIRM平台采用独创的电场引导的释放与测量技术,检测过程利用电场作用,反应快速,最终结果以电信号的形式检测,因而设备的成本大幅降低,仅相当于荧光定量PCR仪的一半左右。5, low cost: First, in the detection equipment, whether it is ARMS-PCR or digital PCR, fluorescent signal detection, detection equipment needs to be equipped with expensive fluorescence detection system, the market price of fluorescent quantitative PCR instrument is hundreds of thousands of dollars Around, and the digital PCR instrument is more than 1 million yuan. In contrast, the EFIRM platform uses an original electric field-guided release and measurement technology. The detection process uses an electric field to react quickly, and the final result is detected in the form of an electrical signal. Therefore, the cost of the device is greatly reduced, which is equivalent to a fluorescence quantitative PCR machine. About half of it.
其次,在检测试剂方面EFIRM技术基于核酸杂交的原理,采用独特设计的电化学技术。本发明中使用的核酸探针长度在25-40bp之间,选择于HPV亚型之间差异较大的E7区或L1区,均采用人工合成寡核苷酸探针,其中CP无需修饰,DP采用较常见的Biotin修饰方法,探针的制备委托商业化的DNA化学合成公司完成,技术难度低,稳定性较好,且部分亚型别共用CP或DP,减少了总探针数量,降低成本。以PCR为基础的荧光定量需要对探针进行两端修饰,且一端为荧光集团,合成成本较高;反向斑点杂交和基因芯片技术大都需要将杂交探针固定到固相载体上,处理工艺复杂,增加了检测成本,且探针经过
固定后活性有一定的降低;HC2使用的探针为全长的RNA探针,长度为7000-8000个碱基,制备工艺复杂,耗时多,成本很高,而且由于探针非常长,杂交效率受错配碱基的影响小,可能出现亚型间的交叉。此外,在检测过程中EFIRM技术节省了DNA提取与纯化的步骤,因此检测试剂成本与其它技术相比相比大幅降低。Secondly, in terms of detection reagents, EFIRM technology is based on the principle of nucleic acid hybridization, using a uniquely designed electrochemical technique. The nucleic acid probe used in the present invention has a length of 25-40 bp, and is selected from the E7 region or the L1 region with a large difference between the HPV subtypes, and artificial synthetic oligonucleotide probes are used, wherein the CP does not need to be modified, DP Using the more common Biotin modification method, the preparation of the probe is completed by a commercial DNA chemical synthesis company, which has low technical difficulty and good stability, and some subtypes share CP or DP, which reduces the total number of probes and reduces the cost. . PCR-based fluorescence quantification requires both ends of the probe to be modified, and one end is a fluorescent group, and the synthesis cost is high; reverse dot blot hybridization and gene chip technology mostly need to immobilize the hybridization probe to the solid phase carrier, and the treatment process Complex, increasing the cost of testing, and the probe passes
The immobilization activity is reduced to a certain extent; the probe used for HC2 is a full-length RNA probe with a length of 7000-8000 bases. The preparation process is complicated, time-consuming, costly, and because the probe is very long, hybridization Efficiency is less affected by mismatched bases, and subtypes may cross. In addition, the EFIRM technology saves the steps of DNA extraction and purification during the detection process, so the cost of the detection reagent is greatly reduced compared to other techniques.
总之,本方法提供的以EFIRM为基础的HPV探针检测方法具有灵敏度高、特异性强、检测耗时短、实验场地要求低、成本低等特点,适用于大量的临床检测和大规模的流行病学筛查。In summary, the EFIRM-based HPV probe detection method provided by the method has the characteristics of high sensitivity, high specificity, short detection time, low experimental site requirement, low cost, and the like, and is suitable for a large number of clinical tests and large-scale epidemics. Pathological screening.
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
The above description is only the preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes can be made to the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the present invention are intended to be included within the scope of the present invention.
Claims (22)
- 一种用于HPV病毒基因分型检测的探针试剂盒,其特征在于,A probe kit for HPV virus genotyping detection, characterized in that包括多条捕获探针,每条所述捕获探针用于结合一种HPV病毒基因型上的目标序列;Including a plurality of capture probes, each of the capture probes being used to bind a target sequence on a HPV viral genotype;所述多个捕获探针包括:第1捕获探针和第2捕获探针、选自第3~11捕获探针的至少一个、以及选自第12~16捕获探针中的至少一个;其中,其中,The plurality of capture probes include: a first capture probe and a second capture probe, at least one selected from the group consisting of the third to 11th capture probes, and at least one selected from the group consisting of the 12th to 16th capture probes; ,among them,第1捕获探针,用于检测HPV 16亚型,碱基序列如SEQ ID NO.1所示;a first capture probe for detecting a HPV 16 subtype, the base sequence of which is set forth in SEQ ID NO.第2捕获探针,用于检测HPV 18亚型,碱基序列如SEQ ID NO.3所示;a second capture probe for detecting a HPV 18 subtype, the base sequence of which is set forth in SEQ ID NO.第3捕获探针,用于检测HPV 31亚型,碱基序列如SEQ ID NO.5所示;a third capture probe for detecting a HPV 31 subtype, the base sequence of which is set forth in SEQ ID NO.第4捕获探针,用于检测HPV 33亚型或52亚型或58亚型的,碱基序列如SEQ ID NO.6所示;a fourth capture probe for detecting HPV 33 subtype or 52 subtype or 58 subtype, the base sequence is shown in SEQ ID NO.第5捕获探针,用于检测HPV 35亚型,碱基序列如SEQ ID NO.7所示;a fifth capture probe for detecting the HPV 35 subtype, the base sequence of which is set forth in SEQ ID NO.第6捕获探针,用于检测HPV 39亚型,碱基序列如SEQ ID NO.8所示;a sixth capture probe for detecting a subtype of HPV 39, the base sequence of which is set forth in SEQ ID NO.第7捕获探针,用于检测HPV 45亚型,碱基序列如SEQ ID NO.9所示;a 7th capture probe for detecting the HPV 45 subtype, the base sequence of which is set forth in SEQ ID NO.第8捕获探针,用于检测HPV 51亚型,碱基序列如SEQ ID NO.10所示;An 8th capture probe for detecting a HPV 51 subtype, the base sequence of which is set forth in SEQ ID NO.第9捕获探针,用于检测HPV 56亚型,碱基序列如SEQ ID NO.11所示;a ninth capture probe for detecting a HPV 56 subtype, the base sequence of which is set forth in SEQ ID NO.第10捕获探针,用于检测HPV 59亚型,碱基序列如SEQ ID NO.12所示;a 10th capture probe for detecting a HPV 59 subtype, the base sequence of which is set forth in SEQ ID NO.第11捕获探针,用于检测HPV 68亚型,碱基序列如SEQ ID NO.13所示,An 11th capture probe for detecting the HPV 68 subtype, the base sequence is set forth in SEQ ID NO.第12捕获探针,用于检测HPV 26亚型,碱基序列如SEQ ID NO.20所示,a 12th capture probe for detecting the HPV 26 subtype, the base sequence is set forth in SEQ ID NO.第13捕获探针,用于检测HPV 53亚型,碱基序列如SEQ ID NO.21所示,a 13th capture probe for detecting the HPV 53 subtype, the base sequence is set forth in SEQ ID NO.第14捕获探针,用于检测HPV 66亚型,碱基序列如SEQ ID NO.22所示a 14th capture probe for detecting the HPV 66 subtype, the base sequence is set forth in SEQ ID NO.第15捕获探针,用于检测HPV 73亚型,碱基序列如SEQ ID NO.23所示,a 15th capture probe for detecting the HPV 73 subtype, the base sequence is set forth in SEQ ID NO.第16捕获探针,用于检测HPV 82亚型,碱基序列如SEQ ID NO.24所示。The 16th capture probe was used to detect the HPV 82 subtype, and the base sequence is shown in SEQ ID NO.
- 根据权利要求1所述的探针试剂盒,其特征在于:还包括多条与所述捕获探针相配合且可结合相应的所述目标序列的检测探针,具体如下:The probe kit according to claim 1, further comprising a plurality of detection probes that cooperate with the capture probe and can bind the corresponding target sequence, as follows:与所述第1捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.2所示,The base sequence of the detection probe complexed with the first capture probe is as shown in SEQ ID NO.与所述第2捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.4所示,The base sequence of the detection probe complexed with the second capture probe is as shown in SEQ ID NO.与所述第3捕获探针或所述第5捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.14所示,The base sequence of the detection probe complexed with the third capture probe or the fifth capture probe is as shown in SEQ ID NO.与所述第4捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.15所示,The base sequence of the detection probe complexed with the fourth capture probe is as shown in SEQ ID NO.与所述第6捕获探针或所述第11捕获探针捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.16所示,The base sequence of the detection probe complexed with the sixth capture probe or the 11th capture probe capture probe is as shown in SEQ ID NO.与所述第7捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.17所示,The base sequence of the detection probe complexed with the seventh capture probe is as shown in SEQ ID NO.与所述第8捕获探针或所述第9捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.18所示,The base sequence of the detection probe complexed with the eighth capture probe or the ninth capture probe is as shown in SEQ ID NO.与所述第10捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.19所示,The base sequence of the detection probe complexed with the 10th capture probe is as shown in SEQ ID NO.与所述第12捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.26所示,The base sequence of the detection probe complexed with the 12th capture probe is as shown in SEQ ID NO.与所述第13捕获探针或所述第14捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.25所示,The base sequence of the detection probe complexed with the 13th capture probe or the 14th capture probe is as shown in SEQ ID NO.与所述第15捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.27所示,The base sequence of the detection probe complexed with the 15th capture probe is as shown in SEQ ID NO.所述第16捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.28所示。The base sequence of the detection probe to which the 16th capture probe is ligated is shown in SEQ ID NO.
- 一种用于中高危HPV病毒基因分型检测的探针试剂盒,其特征在于,A probe kit for genotyping detection of high-risk HPV viruses, characterized in that包括第3~11捕获探针中的至少一个,每条所述捕获探针用于结合一种HPV病毒基因型上的目标序列;Including at least one of the 3rd to 11th capture probes, each of the capture probes being used to bind a target sequence on an HPV viral genotype;其中,among them,第3捕获探针,用于检测HPV 31亚型,碱基序列如SEQ ID NO.5所示;a third capture probe for detecting a HPV 31 subtype, the base sequence of which is set forth in SEQ ID NO.第4捕获探针,用于检测HPV 33亚型或52亚型或58亚型的,碱基序列如SEQ ID NO.6所示;a fourth capture probe for detecting HPV 33 subtype or 52 subtype or 58 subtype, the base sequence is shown in SEQ ID NO.第5捕获探针,用于检测HPV 35亚型,碱基序列如SEQ ID NO.7所示;a fifth capture probe for detecting the HPV 35 subtype, the base sequence of which is set forth in SEQ ID NO.第6捕获探针,用于检测HPV 39亚型,碱基序列如SEQ ID NO.8所示;a sixth capture probe for detecting a subtype of HPV 39, the base sequence of which is set forth in SEQ ID NO.第7捕获探针,用于检测HPV 45亚型,碱基序列如SEQ ID NO.9所示;a 7th capture probe for detecting the HPV 45 subtype, the base sequence of which is set forth in SEQ ID NO.第8捕获探针,用于检测HPV 51亚型,碱基序列如SEQ ID NO.10所示;An 8th capture probe for detecting a HPV 51 subtype, the base sequence of which is set forth in SEQ ID NO.第9捕获探针,用于检测HPV 56亚型,碱基序列如SEQ ID NO.11所示;a ninth capture probe for detecting a HPV 56 subtype, the base sequence of which is set forth in SEQ ID NO.第10捕获探针,用于检测HPV 59亚型,碱基序列如SEQ ID NO.12所示。The 10th capture probe was used to detect the HPV 59 subtype, and the base sequence is shown in SEQ ID NO.
- 根据权利要求3所述的探针试剂盒,还包括 The probe kit according to claim 3, further comprising第1捕获探针,用于检测HPV 16亚型,碱基序列如SEQ ID NO.1所示;a first capture probe for detecting a HPV 16 subtype, the base sequence of which is set forth in SEQ ID NO.和第2捕获探针,用于检测HPV 18亚型,碱基序列如SEQ ID NO.3所示。And a second capture probe for detecting the HPV 18 subtype, the base sequence is shown in SEQ ID NO.
- 根据权利要求3或4所述的探针试剂盒,其特征在于:还包括多条与所述捕获探针相配合且可结合相应的所述目标序列的检测探针,具体如下:The probe kit according to claim 3 or 4, further comprising a plurality of detection probes that cooperate with the capture probe and can bind the corresponding target sequence, as follows:与所述第1捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.2所示,The base sequence of the detection probe complexed with the first capture probe is as shown in SEQ ID NO.与所述第2捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.4所示,The base sequence of the detection probe complexed with the second capture probe is as shown in SEQ ID NO.与所述第3捕获探针或所述第5捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.14所示,The base sequence of the detection probe complexed with the third capture probe or the fifth capture probe is as shown in SEQ ID NO.与所述第4捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.15所示,The base sequence of the detection probe complexed with the fourth capture probe is as shown in SEQ ID NO.与所述第6捕获探针或所述第11捕获探针捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.16所示,The base sequence of the detection probe complexed with the sixth capture probe or the 11th capture probe capture probe is as shown in SEQ ID NO.与所述第7捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.17所示,The base sequence of the detection probe complexed with the seventh capture probe is as shown in SEQ ID NO.与所述第8捕获探针或所述第9捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.18所示,The base sequence of the detection probe complexed with the eighth capture probe or the ninth capture probe is as shown in SEQ ID NO.与所述第10捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.19所示。The base sequence of the detection probe complexed with the 10th capture probe is shown in SEQ ID NO.
- 一种用于高危HPV病毒基因分型检测的探针试剂盒,其特征在于,A probe kit for high-risk HPV virus genotyping detection, characterized in that包括选自第12~16捕获探针中的至少一个,每条所述捕获探针用于结合一种HPV病毒基因型上的目标序列;其中,Included in at least one selected from the group consisting of 12th to 16th capture probes, each of which is for binding to a target sequence on a HPV viral genotype;第12捕获探针,用于检测HPV 26亚型,碱基序列如SEQ ID NO.20所示,a 12th capture probe for detecting the HPV 26 subtype, the base sequence is set forth in SEQ ID NO.第13捕获探针,用于检测HPV 53亚型,碱基序列如SEQ ID NO.21所示,a 13th capture probe for detecting the HPV 53 subtype, the base sequence is set forth in SEQ ID NO.第14捕获探针,用于检测HPV 66亚型,碱基序列如SEQ ID NO.22所示a 14th capture probe for detecting the HPV 66 subtype, the base sequence is set forth in SEQ ID NO.第15捕获探针,用于检测HPV 73亚型,碱基序列如SEQ ID NO.23所示,a 15th capture probe for detecting the HPV 73 subtype, the base sequence is set forth in SEQ ID NO.第16捕获探针,用于检测HPV 82亚型,碱基序列如SEQ ID NO.24所示。The 16th capture probe was used to detect the HPV 82 subtype, and the base sequence is shown in SEQ ID NO.
- 根据权利要求6所述的探针试剂盒,还包括The probe kit according to claim 6, further comprising第1捕获探针,用于检测HPV 16亚型,碱基序列如SEQ ID NO.1所示;a first capture probe for detecting a HPV 16 subtype, the base sequence of which is set forth in SEQ ID NO.和第2捕获探针,用于检测HPV 18亚型,碱基序列如SEQ ID NO.3所示。And a second capture probe for detecting the HPV 18 subtype, the base sequence is shown in SEQ ID NO.
- 根据权利要求6或7所述的探针组合试剂盒,其特征在于:还包括多条与所述捕获探针相配合且可结合相应的所述目标序列的检测探针,具体如下:The probe combination kit according to claim 6 or 7, further comprising a plurality of detection probes that cooperate with the capture probe and can bind the corresponding target sequence, as follows:与所述第1捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.2所示,The base sequence of the detection probe complexed with the first capture probe is as shown in SEQ ID NO.与所述第2捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.4所示,The base sequence of the detection probe complexed with the second capture probe is as shown in SEQ ID NO.与所述第12捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.26所示,The base sequence of the detection probe complexed with the 12th capture probe is as shown in SEQ ID NO.与所述第13捕获探针或所述第14捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.25所示,The base sequence of the detection probe complexed with the 13th capture probe or the 14th capture probe is as shown in SEQ ID NO.与所述第15捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.27所示,The base sequence of the detection probe complexed with the 15th capture probe is as shown in SEQ ID NO.与所述第16捕获探针相配合的所述检测探针的碱基序列如SEQ ID NO.28所示。The base sequence of the detection probe complexed with the 16th capture probe is shown in SEQ ID NO.
- 一种用于HPV病毒基因分型检测的探针试剂盒,其特征在于,包括A probe kit for HPV virus genotyping detection, characterized in that第1捕获探针,用于检测HPV 16亚型,碱基序列如SEQ ID NO.1所示;a first capture probe for detecting a HPV 16 subtype, the base sequence of which is set forth in SEQ ID NO.和第2捕获探针,用于检测HPV 18亚型,碱基序列如SEQ ID NO.3所示。And a second capture probe for detecting the HPV 18 subtype, the base sequence is shown in SEQ ID NO.
- 根据权利要求9所述的探针试剂盒,其特征在于:还包括The probe kit according to claim 9, further comprising检测16型HPV的检测探针,碱基序列如SEQ ID NO.2所示,Detection probe of HPV type 16 is detected, and the base sequence is as shown in SEQ ID NO.检测18型HPV的检测探针,碱基序列如SEQ ID NO.4所示。The detection probe of type 18 HPV was detected, and the base sequence is shown in SEQ ID NO.
- 根据权利要求2,5,8或10所述的探针试剂盒,其特征在于:其中,所述检测探针的3’端或5’端标记有用于结合催化酶的亲和物,所述催化酶用于催化底物产生化学反应形成电子流。The probe kit according to claim 2, 5, 8 or 10, wherein the 3' or 5' end of the detection probe is labeled with an affinity for binding to a catalytic enzyme, Catalytic enzymes are used to catalyze the chemical reaction of a substrate to form a stream of electrons.
- 一种基于EFIRM技术的HPV病毒检测的试剂盒,其特征在于,其包括权利要求11所述的探针试剂盒中的探针。A kit for detecting HPV virus based on EFIRM technology, which comprises the probe in the probe kit of claim 11.
- 根据权利要求12所述的试剂盒,其特征在于,所述试剂盒还包括用于将所述捕获探针固定至检测孔板的固定物,所述固定物包括导电聚合物和离子化合物;The kit according to claim 12, further comprising a fixture for fixing the capture probe to the detection orifice, the fixture comprising a conductive polymer and an ionic compound;所述导电聚合物为选自吡咯、苯胺和噻吩中的任一种;The conductive polymer is any one selected from the group consisting of pyrrole, aniline and thiophene;所述离子化合物为选自氯化钠和氯化钾中的任一种。The ionic compound is any one selected from the group consisting of sodium chloride and potassium chloride.
- 根据权利要求12或13所述的试剂盒,其特征在于,还包括所述催化酶,所述催化酶是带有标记物的辣根过氧化物酶或碱性磷酸酶,所述标记物用于与所述亲和物结合,所述标记物为地高辛抗体、异硫氰酸荧光素抗体和链霉亲和素中的任一种; The kit according to claim 12 or 13, further comprising said catalytic enzyme, said catalytic enzyme being a labeled horseradish peroxidase or alkaline phosphatase, said marker In combination with the affinity, the label is any one of a digoxin antibody, a fluorescein isothiocyanate antibody, and streptavidin;所述检测探针的3’端或5’端标记的所述亲和物是与所述标记物相对应的地高辛、异硫氰酸荧光素和生物素中一种。The affinity labeled with the 3' or 5' end of the detection probe is one of digoxin, fluorescein isothiocyanate and biotin corresponding to the label.
- 根据权利要求14所述的试剂盒,其特征在于,还包括所述催化酶的所述底物;The kit according to claim 14, further comprising said substrate of said catalytic enzyme;当所述催化酶为所述辣根过氧化物酶时,所述底物是TMB、ABTS和OPD中的任一种;When the catalytic enzyme is the horseradish peroxidase, the substrate is any one of TMB, ABTS and OPD;当所述催化酶为所述碱性磷酸酶时,所述底物是BCIP和NBT的组合物、对硝基苯磷酸盐、4-硝基苯磷酸二钠、萘酚AS-BI磷酸盐、萘酚-AS-MX-磷酸盐中的任一种。When the catalytic enzyme is the alkaline phosphatase, the substrate is a combination of BCIP and NBT, p-nitrophenyl phosphate, disodium 4-nitrobenzene phosphate, naphthol AS-BI phosphate, Any of naphthol-AS-MX-phosphate.
- 根据权利要求12-15任一所述的试剂盒,其特征在于,还包括清洗液,所述清洗液包括洗液A和洗液B,所述洗液A是含SDS的SSC缓冲液,所述洗液B是含Tween20的PBS缓冲液。The kit according to any one of claims 12-15, further comprising a cleaning solution comprising a lotion A and a lotion B, the lotion A being an SDS buffer containing SDS, The lotion B is a PBS buffer containing Tween20.
- 根据权利要求12-16所述的试剂盒,其特征在于,还包括所述检测孔板,所述捕获探针按其对应的HPV病毒基因型种类分别固定在所述检测孔板的不同反应孔内;The kit according to any one of claims 12-16, further comprising said detection well plate, said capture probes being respectively fixed in different reaction wells of said detection well plate according to their corresponding HPV virus genotypes Inside;所述检测孔板的反应孔底部设置有工作电极并配置为可施加电压以形成电场。A bottom of the reaction well of the detection orifice is provided with a working electrode and is configured to apply a voltage to form an electric field.
- 根据权利要求17所述的试剂盒,其特征在于,所述反应孔底部还设置有对置电极,所述对置电极设置在所述反应孔底板上并配置为获取检测信号并输出所述检测信号;The kit according to claim 17, wherein the reaction well is further provided with an opposite electrode at the bottom, the opposite electrode is disposed on the reaction well bottom plate and configured to acquire a detection signal and output the detection signal;所述工作电极包括至少一个宽度均匀的第一线状部,所述对置电极包括至少一个宽度均匀的第二线状部,所述第一线状部和所述第二线状部在所述反应孔底部相互交替设置;The working electrode includes at least one first linear portion having a uniform width, the opposite electrode including at least one second linear portion having a uniform width, and the first linear portion and the second linear portion are in the reaction The bottoms of the holes are alternately arranged;至少两个相邻的所述反应孔中的所述工作电极电性相连。The working electrodes of at least two adjacent ones of the reaction wells are electrically connected.
- 一种基于EFIRM技术的HPV病毒基因分型检测的检测孔板,其特征在于,所述检测孔板的反应孔底部设置有工作电极并配置为可施加电压以形成电场;A detection orifice plate for HPV virus genotyping detection based on EFIRM technology, wherein a bottom of a reaction well of the detection orifice plate is provided with a working electrode and configured to apply a voltage to form an electric field;所述检测孔板的反应孔内分配并固定有捕获探针,所述分配并固定的捕获探针选自以下任一组:A capture probe is dispensed and fixed in the reaction well of the detection well plate, and the dispensed and fixed capture probe is selected from any one of the following groups:第1组:包括第1捕获探针和第2捕获探针、选自第3~11捕获探针的至少一个和选自第12~16捕获探针中的至少一个;Group 1 : comprising a first capture probe and a second capture probe, at least one selected from the group consisting of the third to 11th capture probes, and at least one selected from the group consisting of the 12th to 16th capture probes;第2组:包括第3~11捕获探针中的至少一个;Group 2: comprising at least one of the third to eleven capture probes;第3组:包括第12~16捕获探针中的至少一个;Group 3: comprising at least one of the 12th to 16th capture probes;第4组:包括第1捕获探针和第2捕获探针,以及第3~11捕获探针中的至少一个;Group 4: comprising at least one of a first capture probe and a second capture probe, and a third to eleven capture probe;第5组:包括第1捕获探针和第2捕获探针,以及第12~16捕获探针中的至少一个;其中Group 5: comprising at least one of a first capture probe and a second capture probe, and a 12th to 16th capture probe;第1捕获探针,用于检测HPV 16亚型,碱基序列如SEQ ID NO.1所示;a first capture probe for detecting a HPV 16 subtype, the base sequence of which is set forth in SEQ ID NO.第2捕获探针,用于检测HPV 18亚型,碱基序列如SEQ ID NO.3所示;a second capture probe for detecting a HPV 18 subtype, the base sequence of which is set forth in SEQ ID NO.第3捕获探针,用于检测HPV 31亚型,碱基序列如SEQ ID NO.5所示;a third capture probe for detecting a HPV 31 subtype, the base sequence of which is set forth in SEQ ID NO.第4捕获探针,用于检测HPV 33亚型或52亚型或58亚型的,碱基序列如SEQ ID NO.6所示;a fourth capture probe for detecting HPV 33 subtype or 52 subtype or 58 subtype, the base sequence is shown in SEQ ID NO.第5捕获探针,用于检测HPV 35亚型,碱基序列如SEQ ID NO.7所示;a fifth capture probe for detecting the HPV 35 subtype, the base sequence of which is set forth in SEQ ID NO.第6捕获探针,用于检测HPV 39亚型,碱基序列如SEQ ID NO.8所示;a sixth capture probe for detecting a subtype of HPV 39, the base sequence of which is set forth in SEQ ID NO.第7捕获探针,用于检测HPV 45亚型,碱基序列如SEQ ID NO.9所示;a 7th capture probe for detecting the HPV 45 subtype, the base sequence of which is set forth in SEQ ID NO.第8捕获探针,用于检测HPV 51亚型,碱基序列如SEQ ID NO.10所示;An 8th capture probe for detecting a HPV 51 subtype, the base sequence of which is set forth in SEQ ID NO.第9捕获探针,用于检测HPV 56亚型,碱基序列如SEQ ID NO.11所示;a ninth capture probe for detecting a HPV 56 subtype, the base sequence of which is set forth in SEQ ID NO.第10捕获探针,用于检测HPV 59亚型,碱基序列如SEQ ID NO.12所示;a 10th capture probe for detecting a HPV 59 subtype, the base sequence of which is set forth in SEQ ID NO.第11捕获探针,用于检测HPV 68亚型,碱基序列如SEQ ID NO.13所示,An 11th capture probe for detecting the HPV 68 subtype, the base sequence is set forth in SEQ ID NO.第12捕获探针,用于检测HPV 26亚型,碱基序列如SEQ ID NO.20所示,a 12th capture probe for detecting the HPV 26 subtype, the base sequence is set forth in SEQ ID NO.第13捕获探针,用于检测HPV 53亚型,碱基序列如SEQ ID NO.21所示,a 13th capture probe for detecting the HPV 53 subtype, the base sequence is set forth in SEQ ID NO.第14捕获探针,用于检测HPV 66亚型,碱基序列如SEQ ID NO.22所示a 14th capture probe for detecting the HPV 66 subtype, the base sequence is set forth in SEQ ID NO.第15捕获探针,用于检测HPV 73亚型,碱基序列如SEQ ID NO.23所示,a 15th capture probe for detecting the HPV 73 subtype, the base sequence is set forth in SEQ ID NO.第16捕获探针,用于检测HPV 82亚型,碱基序列如SEQ ID NO.24所示,a 16th capture probe for detecting the HPV 82 subtype, the base sequence is set forth in SEQ ID NO.所述分配是指每一种所述捕获探针固定在不同的所述反应孔中。The dispensing means that each of the capture probes is immobilized in a different one of the reaction wells.
- 根据权利要求19所述的检测孔板,其特征在于,所述反应孔底部还设置有对置电极,所述对置电极设置在所述反应孔底板上并配置为获取检测信号并输出所述检测信号;The detecting orifice plate according to claim 19, wherein the bottom of the reaction well is further provided with an opposite electrode, the opposite electrode is disposed on the reaction well bottom plate and configured to acquire a detection signal and output the Detection signal所述工作电极包括至少一个宽度均匀的第一线状部,所述对置电极包括至少一个宽度均匀的第二线状部,所述第一线状部和所述第二线状部在所述反应孔底部相互交替设置;The working electrode includes at least one first linear portion having a uniform width, the opposite electrode including at least one second linear portion having a uniform width, and the first linear portion and the second linear portion are in the reaction The bottoms of the holes are alternately arranged;至少两个相邻的所述反应孔中的所述工作电极电性相连。The working electrodes of at least two adjacent ones of the reaction wells are electrically connected.
- 根据权利要求19或20所述的检测孔板,其特征在于:所述捕获探针是与导电聚合物和离子化合 物混合成混合液后加到所述反应孔中,然后通过所述工作电极施加第一方波电场后固定在所述反应孔内底部表面制成;The detection orifice plate according to claim 19 or 20, wherein the capture probe is combined with a conductive polymer and an ion Mixing the mixture into a mixed solution, adding to the reaction well, and then applying a first square wave electric field through the working electrode and fixing it to the bottom surface of the reaction hole;所述第一电场的参数为:电压A:350mV,1s;电压B:950mV,1s;进行9个循环,The parameters of the first electric field are: voltage A: 350 mV, 1 s; voltage B: 950 mV, 1 s; 9 cycles,所述导电聚合物材料选自苯胺、噻吩和吡咯导电分子单体中的至少一种;The conductive polymer material is selected from at least one of aniline, thiophene and pyrrole conductive molecular monomers;所述盐离子化合物选自氯化盐、硝酸盐、硫酸盐中的至少一种;所述氯化盐为氯化钠、氯化钾、氯化镁、氯化铵中的一种,所述硝酸盐为硝酸钠、硝酸钾、硝酸镁、硝酸铵中的一种,所述硫酸盐为硫酸钠、硫酸钾、硫酸镁、硫酸铵中的一种。The salt ionic compound is at least one selected from the group consisting of a chloride salt, a nitrate salt, and a sulfate salt; the chloride salt is one of sodium chloride, potassium chloride, magnesium chloride, and ammonium chloride, and the nitrate salt It is one of sodium nitrate, potassium nitrate, magnesium nitrate, and ammonium nitrate, and the sulfate is one of sodium sulfate, potassium sulfate, magnesium sulfate, and ammonium sulfate.
- 一种基于EFIRM技术的HPV病毒基因分型检测方法,其特征在于,采用权利要求12-18任一所述的试剂盒,步骤如下:A method for detecting HPV virus genotyping based on EFIRM technology, characterized in that the kit according to any one of claims 12-18 is used, the steps are as follows:(1)采用权利要求19-21任一所述的检测孔板,或所述捕获探针加入到空白检测孔板中,所述反应孔内底部设置有电极,用于接通EFIRM检测仪后对反应孔内溶液施加电场进行聚合反应;接通EFIRM检测仪后对反应孔内溶液施加第一电场进行聚合反应;电场处理完毕,清洗检测孔板所述第一电场处理的参数为:电压200-500mV,1-5s;电压800-1500mV,1-5s;3-10个循环;(1) The detection orifice plate according to any one of claims 19-21, or the capture probe is added to the blank detection orifice plate, and the bottom of the reaction well is provided with an electrode for turning on the EFIRM detector Applying an electric field to the solution in the reaction well to carry out polymerization reaction; after the EFIRM detector is turned on, a first electric field is applied to the solution in the reaction well to carry out polymerization reaction; after the electric field treatment is completed, the parameters of the first electric field treatment of the cleaning detection orifice plate are: voltage 200 -500mV, 1-5s; voltage 800-1500mV, 1-5s; 3-10 cycles;(2)加入待测样本与杂交buffer的混合溶液,对反应孔施加第二电场:电压200-500mV,1-5s;电压300-800mV,1-5s;5-150个循环;清洗检测孔板;(2) adding a mixed solution of the sample to be tested and the hybrid buffer, applying a second electric field to the reaction well: voltage 200-500 mV, 1-5 s; voltage 300-800 mV, 1-5 s; 5-150 cycles; cleaning detection orifice ;(3)加入所述检测探针溶液,在EFIRM上,对反应孔施加第三电场:电压200-500mV,1-5s;电压300-800mV,1-5s;3-10个循环;清洗检测孔板;进行电场操作;(3) adding the detection probe solution, applying a third electric field to the reaction well on the EFIRM: voltage 200-500 mV, 1-5 s; voltage 300-800 mV, 1-5 s; 3-10 cycles; cleaning detection hole Board; performing electric field operation;所述检测探针溶液中检测探针的浓度为0.5-1.5μmol/L;The concentration of the detection probe in the detection probe solution is 0.5-1.5 μmol/L;(4)加入酶液,孵育后清洗;(4) adding the enzyme solution, washing after incubation;(5)加入底物,在电压为-100~-300mV的电场处理下读数,得到电流值。 (5) Adding a substrate and reading under an electric field of a voltage of -100 to -300 mV, and obtaining a current value.
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