WO2018059581A1

WO2018059581A1 - Probe for hpv virus genotyping detection based on efirm technique, kit and use

Info

Publication number: WO2018059581A1
Application number: PCT/CN2017/104829
Authority: WO
Inventors: 廖玮; 莫亚勤; 林晓燕; 张晨光
Original assignee: 广州易活生物科技有限公司
Priority date: 2016-09-30
Filing date: 2017-09-30
Publication date: 2018-04-05

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

Probe, kit and application of HPV virus typing detection based on EFIRM technology

cross reference

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.

Technical field

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.

Background technique

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.

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.

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.

Summary of the invention

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.

The technical problem solved by the present invention is achieved by the following technical solutions.

A probe kit for HPV virus genotyping detection, characterized in that

Including a plurality of capture probes, 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.

a 12th capture probe for detecting the HPV 26 subtype, the base sequence is set forth in SEQ ID NO.

a 13th capture probe for detecting the HPV 53 subtype, the base sequence is set forth in SEQ ID NO.

a 14th capture probe for detecting the HPV 66 subtype, the base sequence is set forth in SEQ ID NO.

a 15th capture probe for detecting the HPV 73 subtype, 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.

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:

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

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;

among them,

The 10th capture probe was used to detect the HPV 59 subtype, and the base sequence is shown in SEQ ID NO.

Preferably, 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 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;

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.

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

And a second capture probe for detecting the HPV 18 subtype, the base sequence is shown in SEQ ID NO.

Preferably, 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.

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.

A kit for detecting HPV virus based on EFIRM technology, characterized in that it comprises a probe in any of the above probe kits.

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;

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.

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;

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.

Preferably, the kit further comprises the substrate of the catalytic enzyme;

When the catalytic enzyme is the horseradish peroxidase, the substrate is any one of TMB, ABTS and OPD;

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.

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.

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.

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;

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 16th capture probe for detecting the HPV 82 subtype, 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.

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;

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.

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;

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:

(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.

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) 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) adding the detection probe solution and performing an electric field operation on the EFIRM;

The concentration of the detection probe in the detection probe solution is 0.5-1.5 μmol/L;

(4) adding the enzyme solution, washing after incubation;

(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.

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.

DRAWINGS

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.

1 is an exemplary detection result of Embodiment 1 of the present invention;

2 is an exemplary detection result of Embodiment 2 of the present invention;

3 is an exemplary detection result of Embodiment 3 of the present invention;

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; FIG.

6 is an exemplary detection result of Embodiment 6 of the present invention;

7 is a schematic plan view showing the structure of a detecting electrode in the detecting orifice plate of the present invention;

8 is a schematic plan view showing another structure of a detecting electrode in the detecting orifice plate of the present invention;

9 is a schematic plan view showing another structure of a detecting electrode in the detecting orifice plate of the present invention;

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;

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.

detailed description

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.

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.

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.

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.

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.

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.

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, 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.

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.

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.

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.

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.

Preferably, 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.

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.

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. .

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.

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.

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.

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.

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.

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.

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.

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.

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 .

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.

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.

The invention provides a method for detecting HPV virus based on EFIRM technology, which comprises:

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;

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.

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.

It should be noted that the detection orifice plate and the current detecting device used in the above method are commercially available.

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:

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.

The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

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.

The positive controls set therein are shown in Table 2.

Example 1

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.

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.

Table 1. Base sequences of detection probes and detection probes for detecting HPV subtypes

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).

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 capture probe fixed

1.1 Preparation of a mixture of pyrrole and CP

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 fixed capture probe

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 electric field treatment

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 board cleaning:

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 sample hybridization

2.1 hybrid buffer pretreatment

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 Preparation of samples to be tested

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 plus sample to be tested

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.)

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.

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.

Table 2. Base sequence of positive oligonucleotides used to detect positive controls for HPV genotypes

2.4EFIRM electric field treatment

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 incubation at room temperature

Cover the E-plate and incubate for 30 min at room temperature on the bench.

2.6E-plate plate cleaning

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 combination

3.1DP solution preparation

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 loading

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 incubation at room temperature

Cover the E-plate and incubate for 30 min at room temperature on the bench.

3.4E-plate plate cleaning

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 streptavidin-labeled horseradish peroxidase (Poly-HRP) combined with biotin

4.1Poly-HRP solution preparation

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 Add enzyme solution

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 incubation at room temperature

Cover the E-plate and incubate for 30 min at room temperature on the bench.

4.4E-plate plate cleaning

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 data reading

5.1 plus substrate

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 electric field reading

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.

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.

Example 2

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.

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.

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.

Example 3

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.

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.

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.

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.

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.

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.

Example 4

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.

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.

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.

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 .

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.

Example 5

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.

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.

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.

Example 6

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.

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. .

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.

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.

Alternative to the testing process:

For the detection of the above Examples 1-6, the following detection steps can be used instead:

Alternative method one.

1. The capture probe is fixed at the bottom of the detection orifice plate.

1.1 Mixture of thiophene and capture probe (CP for short):

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 loading:

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 electric field treatment:

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 detection orifice cleaning:

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. Sample hybridization:

2.1 hybrid buffer pretreatment

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 Preparation of the sample

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 loading:

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 electric field treatment:

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 detection orifice cleaning:

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. Detection of hybridization

3.1DP (detection probe) solution preparation:

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 loading:

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 electric field treatment:

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 Detection of orifice cleaning:

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 Hybridization

4.1Poly-HRP solution preparation:

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 loading:

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 Detection plate cleaning:

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, Readout

5.1 loading:

According to the experimental design, TMB/H2O2 solution (purchased from thermo fisher, product no. 34022, named as

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 electric field reading:

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.

The test results were in agreement with the results of Examples 1-6 obtained based on the procedure described in Example 1.

Alternative detection process

Test the test sample, the specific steps are as follows:

1. The capture probe is fixed at the bottom of the detection orifice plate.

1.1 Mixture of aniline and capture probe (CP for short):

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 loading:

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 electric field treatment:

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 detection orifice cleaning:

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. Sample hybridization:

2.1 hybrid buffer pretreatment

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 Preparation of the sample

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 loading:

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 electric field treatment:

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 detection orifice cleaning:

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. Detection of hybridization

3.1DP (detection probe) solution preparation:

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 loading:

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 electric field treatment:

3.4 Detection of orifice cleaning:

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 Hybridization

4.1Poly-HRP solution preparation:

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 loading:

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 Detection plate cleaning:

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, Readout

5.1 loading:

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 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 electric field reading:

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.

Also, the results obtained were substantially in agreement with the results of Examples 1-6 obtained based on Example 1.

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.

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.

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.

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. .

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).

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).

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.

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.

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.

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.

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.

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.

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.

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.

In summary, the virus genotyping detection kit provided by the present invention has the following advantages:

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. 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. 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. 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, 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.

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.

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.

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

A probe kit for HPV virus genotyping detection, characterized in that

Including a plurality of capture probes, 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.

a 12th capture probe for detecting the HPV 26 subtype, the base sequence is set forth in SEQ ID NO.

a 13th capture probe for detecting the HPV 53 subtype, the base sequence is set forth in SEQ ID NO.

a 14th capture probe for detecting the HPV 66 subtype, the base sequence is set forth in SEQ ID NO.

a 15th capture probe for detecting the HPV 73 subtype, 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 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:

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

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;

among them,

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.
The probe kit according to claim 3, further comprising

a first capture probe for detecting a HPV 16 subtype, the base sequence of which is set forth in SEQ ID NO.

And a second capture probe for detecting the HPV 18 subtype, the base sequence is shown in SEQ ID NO.
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:

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, characterized in that

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;

a 12th capture probe for detecting the HPV 26 subtype, the base sequence is set forth in SEQ ID NO.

a 13th capture probe for detecting the HPV 53 subtype, the base sequence is set forth in SEQ ID NO.

a 14th capture probe for detecting the HPV 66 subtype, the base sequence is set forth in SEQ ID NO.

a 15th capture probe for detecting the HPV 73 subtype, 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 probe kit according to claim 6, further comprising

a first capture probe for detecting a HPV 16 subtype, the base sequence of which is set forth in SEQ ID NO.

And a second capture probe for detecting the HPV 18 subtype, the base sequence is shown in SEQ ID NO.
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:

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.

And a second capture probe for detecting the HPV 18 subtype, the base sequence is shown in SEQ ID NO.
The probe kit according to claim 9, further comprising

Detection probe of HPV type 16 is detected, 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 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.
A kit for detecting HPV virus based on EFIRM technology, which comprises the probe in the probe kit of claim 11.
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;

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 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;

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 according to claim 14, further comprising said substrate of said catalytic enzyme;

When the catalytic enzyme is the horseradish peroxidase, the substrate is any one of TMB, ABTS and OPD;

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.
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.
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;

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.
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;

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.
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;

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.

a 12th capture probe for detecting the HPV 26 subtype, the base sequence is set forth in SEQ ID NO.

a 13th capture probe for detecting the HPV 53 subtype, the base sequence is set forth in SEQ ID NO.

a 14th capture probe for detecting the HPV 66 subtype, the base sequence is set forth in SEQ ID NO.

a 15th capture probe for detecting the HPV 73 subtype, the base sequence is set forth in SEQ ID NO.

a 16th capture probe for detecting the HPV 82 subtype, 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 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

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 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;

The parameters of the first electric field are: voltage A: 350 mV, 1 s; voltage B: 950 mV, 1 s; 9 cycles,

The conductive polymer material is selected from at least one of aniline, thiophene and pyrrole conductive molecular monomers;

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.
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) 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) 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) 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;

The concentration of the detection probe in the detection probe solution is 0.5-1.5 μmol/L;

(4) adding the enzyme solution, washing after incubation;

(5) Adding a substrate and reading under an electric field of a voltage of -100 to -300 mV, and obtaining a current value.