CN106282413B

CN106282413B - Probe combinations, kit and the method for the high-risk strain Genotyping detection of HPV

Info

Publication number: CN106282413B
Application number: CN201610877670.0A
Authority: CN
Inventors: 廖玮; 莫亚勤; 林晓燕; 张晨光
Original assignee: Guangzhou Easy Living Biotechnology Co Ltd
Current assignee: Guangzhou Easy Living Biotechnology Co Ltd
Priority date: 2016-09-30
Filing date: 2016-09-30
Publication date: 2019-11-22
Anticipated expiration: 2036-09-30
Also published as: CN106282413A

Abstract

The invention discloses probe combinations, kit and the methods of the high-risk strain Genotyping detection of HPV, it is related to technical field of gene detection, it includes multiple probes pair, each probe is to including for the capture probe of combining target sequence with detection probe corresponding with capture probe and in combination with target sequence, and the 3 ' of detection probe are held or 5 ' ends are marked with for the affinant in conjunction with catalyzing enzyme.The probe combinations can carry out genotype detection to the virus in sample to be tested, have stronger specificity, higher sensitivity and lower false positive rate, and it is accurate and reliable to detect result.

Description

Probe combinations, kit and the method for the high-risk strain Genotyping detection of HPV

Technical field

The present invention relates to technical field of gene detection, in particular to the probe of the high-risk strain Genotyping detection of HPV Combination, kit and method.

Background technique

Cervical carcinoma is one of most common malignant tumour of female genital tract, and existing research shows high-risk-type human papilloma virus Malicious (Human Papillomavirus, HPV) persistent infection and multiple infection are the one of the major reasons for leading to cervical carcinogenesis, entirely The result of study of ball range shows, 99.7% cervical cancer patient vivo detection to high-risk HPV DNA presence.At present It is found that HPV have more than 200 kinds of types, mainly include low risk and high-risk-type two major classes.The phase of different types and various disease Closing property is also different, and low risk HPV infection may cause genital tract condyloma lesion, high-risk HPV infection then with cervical carcinoma, yin Road cancer is related.The effective treatment means generally acknowledged for HPV are also lacked at present, therefore cervical HPV early discovery, early prevention are to block cancer The key of change.

For many years, the diagnosis of general Cervical intraepitheliaI neoplasia and cervical carcinoma mainly follows " three stageds " diagnosis in the world Program, i.e. cervical cytology, gynecatoptron and histopathological examination.Since HPV is unable in vitro culture, HPV detection method is main Molecule parting based on HPV DNA.HPV nucleic acid detection technique mainly includes hybrid capture, and PCR- fluorescence probe method, transcription are situated between Nucleic acid amplification technologies and PCR- hybrid method for leading etc..

The early screening mode of cervical carcinoma is using Pap smear and improved TCT technology, these methods can only be from The angle of Cytopathic assesses the incidence of cervical carcinoma, that is to say, that needs wait until that the cell by virus infection starts Occur just can detecte out when significant change, specificity and sensitivity are not ideal enough.Lead to cervical carcinoma with to HPV infection Understanding deepen and the development of molecular diagnostic techniques, HPV genetic test obtained more as a kind of screening means of cervical carcinoma It is widely applied.HPV parting detection technique is based primarily upon polymerase chain reaction at present, as fluorescent quantitation, reverse dot blot hybridization and The advantages that biochip technology etc., these Technological expressions go out highly sensitive, high specific, can carry out multiple infection detection, but These technologies are high to experiment condition and reviewer's competency profiling, and testing cost is also higher, are poorly suitable for a large amount of clinical inspection It surveys and large-scale epidemic disease screening.The detection of second generation gene recombination capture technique uses the HC2 skill of U.S. Digene company Art kit, HC2 are a kind of detection method of nucleic acid hybridization that signal amplification is carried out using microwell plate chemiluminescence, are acknowledged as commenting The goldstandard of valence HPV new detecting technique.However, HC2 detection method is expensive, and its method is cumbersome, in operating process It is easy cross contamination and false positive occurs.

Summary of the invention

The first object of the present invention is to provide the HPV probe combinations of high-risk strain Genotyping detection, the probe combinations Genotype detection can be carried out to the HPV high-risk-type virus in sample to be tested, have stronger specificity, higher sensitivity with And lower false positive rate, and it is accurate and reliable to detect result.

The second object of the present invention is to provide the HPV kit of high-risk strain Genotyping detection, which can Genotype detection is carried out to the HPV high-risk-type virus in sample to be tested, with stronger specificity and higher sensitivity, and Detection result is accurate and reliable, false positive rate is low, simple to operate.

The third object of the present invention is to provide the HPV method of high-risk strain Genotyping detection, and this method can be treated HPV high-risk-type virus in test sample sheet carries out genotype detection, has stronger specificity and higher sensitivity, and detect knot Fruit is accurate and reliable, and detection method is simple to operation.

The present invention solves its technical problem and adopts the following technical solutions to realize.

A kind of probe combinations of the high-risk strain Genotyping detection of HPV,

It includes multiple probes pair, and each probe is to including capture probe (Capture for combining target sequence Probe, CP) and detection probe (Detector Probe, DP) corresponding with capture probe and in combination with target sequence, often 3 ' the ends or 5 ' ends of the detection probe of a probe pair are marked with the affinant for combining catalyzing enzyme, and catalyzing enzyme is used for catalysis substrate Generate chemical reaction and form electron stream, multiple probes to include: the 1st probe to, the 2nd probe to, selected from the 3rd~11 probe centering At least one；

Wherein, the base sequence of the capture probe of the 1st probe pair as shown in SEQ ID NO.1, visit by the capture of the 2nd probe pair The base sequence of needle is as shown in SEQ ID NO.3, and the base sequence of the capture probe of the 3rd~11 probe pair is respectively such as SEQ ID Shown in NO.5~13.

A kind of kit of the high-risk strain Genotyping detection of HPV comprising the high-risk strain Genotyping inspection of above-mentioned HPV The probe combinations of survey.

A kind of method of the high-risk strain Genotyping detection of HPV comprising following steps:

Multiple probes pair are provided, each probe to include for combining target sequence capture probe and with capture probe phase Detection probe corresponding and in combination with target sequence, the 3 ' ends or 5 ' ends of the detection probe of each probe pair are marked with for tying The affinant of catalyzing enzyme is closed, catalyzing enzyme generates chemical reaction for catalysis substrate and forms electron stream, and multiple probes are to including: the 1st Probe to, the 2nd probe pair and selected from the 3rd~11 probe at least one of；

First the capture probe for being used for combining target sequence is fixed in the reacting hole of detection orifice plate by electric field action；

Sample to be tested is added to reacting hole；

It is added in corresponding with capture probe and detection probe to reacting hole in combination with target sequence, the 3 ' of detection probe End or 5 ' ends, which are marked with, can be used for combining the affine of the catalyzing enzyme for answering substrate generation chemical reaction to form electron stream for catalytic phase Object；

It is added in catalyzing enzyme and substrate to reacting hole, the electric current in reacting hole is detected by current detection means；

The beneficial effect of probe combinations, kit and method that the high-risk strain Genotyping of HPV provided by the invention detects Fruit is: the probe combinations of the high-risk strain Genotyping detection of HPV provided by the invention include multiple probes pair, and each probe is to packet It includes for the capture probe of combining target sequence and detection probe corresponding with capture probe and in combination with target sequence, Capture probe in conjunction with target sequence, target sequence is captured and is fixed by base complementrity principle, carries out specificity knot for the first time It closes and fixes；Detection probe is specifically bound by base complementrity principle and target sequence second, and detection probe is combined fixation；Inspection 3 ' ends of probing needle or being combined catalyzing enzyme by the affinant of label of 5 ' ends, are catalyzed substrate for enzymatic activity release current signal, The current signal of release is detected, and then detects virogene type.Due to, target sequence only with capture probe and detection probe Simultaneously i.e. by specifically binding twice after accurate pairing, the generation of Cai Nengyou signal, this greatly increases the special of detection Property, so that detection result is accurate and reliable, false positive rate is very low.And the alkali of the capture probe for providing each probe pair of the invention Basic sequence, can capture respectively 16 hypotype of HPV of fixed such as common type, the HPV31 of 18 hypotypes and high-risk-type, 33, 52, the target sequence in 58,35,39,45,51,56,59,68 hypotypes, and then whether can detect that in sample to be tested containing common Type virus such as 16 hypotype of HPV, 18 hypotypes and high-risk-type virus such as HPV31,33,52,58,35,39,45,51,56, 59, one of 68 hypotypes or a variety of virus subtypes.

Detailed description of the invention

In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.

Fig. 1 is the testing result of the embodiment of the present invention 1；

Fig. 2 is the testing result of the embodiment of the present invention 2；

Fig. 3 is the testing result of the embodiment of the present invention 3；

Fig. 4 is the testing result of the embodiment of the present invention 4；

Fig. 5 is the testing result of the embodiment of the present invention 5；

Fig. 6 is the testing result of the embodiment of the present invention 6.

Specific embodiment

It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase Product.

The probe, kit and method of the detection of the HPV of the embodiment of the present invention high-risk strain Genotyping are carried out below It illustrates.

Usually, the probability size occurred according to various HPV hypotypes in cervical cancer patient, under normal circumstances by above-mentioned people Papillomavirus (Human Papillomavirus, HPV) makees following classification.HPV16,18 hypotypes are that clinic is most common, It is the maximum probability that the two types virus occurs in cervical cancer patient；HPV31,33,52,58,35,39,45,51,56,59,68 Hypotype is high state of risk type (can be collectively referred to as high-risk-type group), and the hypotype of HPV26,53,66,73,82 is medium risk type (can be collectively referred to as middle high-risk-type group), carry out probe sequence design of the present invention according to above-mentioned virus subtype, obtained probe groups Conjunction can be used for detecting above-mentioned virus subtype.

The probe combinations of the high-risk strain Genotyping detection of HPV provided by the invention comprising multiple probes pair.Each spy For including detection corresponding for the capture probe of combining target sequence and with capture probe and in combination with target sequence Probe.3 ' the ends or 5 ' ends of the detection probe of each probe pair are marked with the affinant for combining catalyzing enzyme, which uses Electron stream is formed in being catalyzed the generation chemical reaction of its substrate.

Multiple probes to include: the 1st probe to, the 2nd probe to, selected from the 3rd~11 probe at least one of.Below HPV hypotype of each probe to the capture probe of detection and the base sequence of detection probe and its detection is described in detail.

Specifically, for detecting the 1st probe pair of HPV16 hypotype, the base sequence of capture probe such as SEQ ID NO.1 Shown, the base sequence of detection probe is as shown in SEQ ID NO.2.

For detecting the 2nd probe pair of HPV18 hypotype, the base sequence of capture probe as shown in SEQ ID NO.3, The base sequence of detection probe is as shown in SEQ ID NO.4.

For detecting the 3rd probe pair of HPV31 hypotype, the base sequence of capture probe as shown in SEQ ID NO.5, The base sequence of detection probe is as shown in SEQ ID NO.14.

For detecting the 4th probe pair of HPV33 hypotype or 52 hypotypes or 58 hypotypes, the base sequence of capture probe is such as Shown in SEQ ID NO.6, for the base sequence of detection probe as shown in SEQ ID NO.15, which shares the 4th probe It is right.

For detecting the 5th probe pair of HPV35 hypotype, the base sequence of capture probe as shown in SEQ ID NO.7, The base sequence of detection probe is as shown in SEQ ID NO.14.

For detecting the 6th probe pair of HPV39 hypotype, the base sequence of capture probe as shown in SEQ ID NO.8, The base sequence of detection probe is as shown in SEQ ID NO.16.

For detecting the 7th probe pair of HPV45 hypotype, the base sequence of capture probe as shown in SEQ ID NO.9, The base sequence of detection probe is as shown in SEQ ID NO.17.

For detecting the 8th probe pair of HPV51 hypotype, the base sequence of capture probe as shown in SEQ ID NO.10, The base sequence of its detection probe is as shown in SEQ ID NO.18.

For detecting the 9th probe pair of 56 hypotype of HPV, the base sequence of capture probe as shown in SEQ ID NO.11, The base sequence of its detection probe is as shown in SEQ ID NO.18.

For detecting the 10th probe pair of HPV59 hypotype, the base sequence of capture probe as shown in SEQ ID NO.12, The base sequence of its detection probe is as shown in SEQ ID NO.19.

For detecting the 11st probe pair of HPV68 hypotype, the base sequence of capture probe as shown in SEQ ID NO.13, The base sequence of its detection probe is as shown in SEQ ID NO.16.

Preferably, multiple probes are to the probe pair that may also include for high-risk HPV in detecting:

For detecting the 12nd probe pair of HPV26 hypotype, the base sequence of capture probe as shown in SEQ ID NO.20, The base sequence of its detection probe is as shown in SEQ ID NO.26；

For detecting the 13rd probe pair of HPV53 hypotype, the base sequence of capture probe as shown in SEQ ID NO.21, The base sequence of its detection probe is as shown in SEQ ID NO.25；

For detecting the 14th probe pair of HPV66 hypotype, the base sequence of capture probe as shown in SEQ ID NO.22, The base sequence of its detection probe is as shown in SEQ ID NO.25；

For detecting the 15th probe pair of HPV73 hypotype, the base sequence of capture probe as shown in SEQ ID NO.23, The base sequence of its detection probe is as shown in SEQ ID NO.27；

For detecting the 16th probe pair of HPV82 hypotype, the base sequence of capture probe as shown in SEQ ID NO.24, The base sequence of its detection probe is as shown in SEQ ID NO.28.

Wherein, detection probe is corresponding with capture probe refers to, a capture probe and a detection probe form one Probe pair can be used to detect some type of virus.Capture probe and detection probe are all in accordance with the same of same type of virus A conservative gene or Conservative segment of DNA, that is, target sequence are designed, and capture probe and detection probe can be with the virus types The same conservative gene of type or the different zones of Conservative segment of DNA or binding site are combined by base complementrity principle, but are caught It is not complementary with detection probe to obtain probe, base sequence is not also identical, and the two is not overlapped with the binding site of target sequence, not yet Overlapping.In addition, the bond area of capture probe and detection probe and target sequence can be adjacent, it is also possible to be spaced multiple alkali Base.As long as capture probe and detection probe can be with the same conservative genes or same section of conserved dna of same type of virus Segment is combined by base complementrity principle.

It should be noted that the sequence capture probe of above-mentioned each probe pair and the base sequence of detection probe sequence can be Not corresponding to each other, that is to say, that in other examples, the sequence capture probe of each probe pair can be described above Base sequence, and the same DNA that the base sequence of its corresponding detection probe can be taken from same detection subtype virus is conservative The complementation of region others sequence；Or in other examples, the detection probe sequence of each probe pair can be above-mentioned institute The base sequence stated, and the base sequence of its corresponding capture probe can be other sequence selections from same detection hypotype disease The complementary series of the same DNA conservative region others sequence of poison.But capture probe and the inspection for each probe pair that the present invention provides The base sequence of probing needle has many advantages, such as stronger specificity, higher sensitivity, has inspection when it is detected Survey the visible embodiment of result.

In addition, can be grouped detection in the detection of these subtype virus and for example only detect that sample contains some The virus of danger level (such as high-risk-type group or middle high-risk-type group), without detect its contain in that group it is specific certain Type virus.It is of course also possible to carry out the detection of specific hypotype, such as detect in sample specifically containing certain in high-risk-type group Class hypotype such as HPV53 subtype virus.Specifically which kind of degree to obtain the detection of which kind of degree to using which kind of detection method is detected As a result, testing staff can select as the case may be, to provide more reasonable directive significance, the case where specific interblock interference See embodiment.

It certainly, in other examples, can only include the 1st~16 for the probe combinations of viral gene analysis detection Probe to one of or two kinds or three kinds or a variety of probes pair situation, as long as that selects from the 1st~16 probe pair appoints Meaning combination, all belongs to the scope of protection of the present invention.

Furthermore it is preferred that the affinant of 3 ' ends of above-mentioned each detection probe or 5 ' end labels is biotin, biotin Effect is that in conjunction with the catalyzing enzyme of marked by streptavidin, the electric current release generated by catalysis substrate for enzymatic activity detects letter Number.Certainly, in other examples, detection probe can not be combined by biotin-Streptavidin identifying system and be urged Change enzyme, catalyzing enzyme, therefore, affinant can be combined in conjunction with system such as antibody/antigen, ligand/receptor by others It can be with one of antigen/antibody.Such as affinant can be digoxin or fluorescein isothiocynate, accordingly, catalyzing enzyme Upper label has or fluorescein isothiocynate antibody.And affinant can mark 3 ' the end notes in detection probe, It can also mark at its 5 ' end, the two can be with.

In addition, the kit of the high-risk strain Genotyping detection of HPV provided by the invention comprising described above is any One probe combinations.Preferably, each probe is individually present as a solution, such as capture probe is with catching containing capture probe The form for obtaining probe solution exists, and detection probe exists in the form of the detection probe solution containing detection probe.Each probe is molten The concentration and probe concentration that liquid contains is to be arranged according to the actual situation.Preferably, the probe final concentration of 0.5 that each probe solution contains ~1.5 μM.

In addition, the kit of the high-risk strain Genotyping detection of HPV provided by the invention may also include probe to catching Obtain the fixture that probe is fixed to detection orifice plate.Fixture includes conducting polymer and ionic compound.Conducting polymer is selected from One of pyrroles, aniline and thiophene, certainly, conducting polymer are also possible to other conducting polymer materials.Ion combination Object is selected from any one of sodium chloride and potassium chloride.Conducting polymer is positively charged, forms cross-linked network under the action of electric field Structure is deposited over the bottom of reacting hole, and capture probe steadily can be fixed on bottom by cross-linked network, helps to mention The stability and capture ability of high capture probe.

In addition, the kit of the high-risk strain Genotyping detection of HPV provided by the invention may also include detection orifice plate, detect The capture probe of probe pair is fixed in the reacting hole of orifice plate.Detection orifice plate specific structure can refer to such as application No. is Shown in entitled detecting electrode disclosed in 201620769829.2 and the detection orifice plate in detection orifice plate.

In addition, the kit of the high-risk strain Genotyping detection of HPV provided by the invention may also include catalyzing enzyme, catalyzing enzyme It is the horseradish peroxidase with marker, it is preferable that catalyzing enzyme is the horseradish peroxidase with marked by streptavidin Enzyme.Certainly, catalyzing enzyme is also possible to the alkaline phosphatase with marker, and marker is DigiTAb, isosulfocyanic acid fluorescence Any one of plain antibody or Streptavidin, marker is corresponding with affinant, can be according to the affinant in detection probe Classification selected.When affinant is biotin, marker is Streptavidin；When affinant is digoxin, label Object is DigiTAb；When affinant is fluorescein isothiocynate, marker is fluorescein isothiocynate antibody.As long as affine Object is corresponding with marker, can be combined with each other.

In addition, the kit of the high-risk strain Genotyping detection of HPV provided by the invention may also include substrate, the class of substrate It is not selected according to the classification of catalyzing enzyme.

When catalyzing enzyme is horseradish peroxidase, substrate is TMB (Tetramethylbenzidine, tetramethyl biphenyl Amine), ABTS (2,2'-Azinobis- (3-ethylbenzthiazoline-6-sulphonate, 2,2- join (the 3- second of nitrogen-two Any one of base-benzothiazole -6- sulfonic acid) di-ammonium salts) and OPD (o-Phenylenediamine, o-phenylenediamine).TMB, ABTS and OPD is the substrate of horseradish peroxidase, and chromogenic reaction occurs simultaneously under the catalytic action of horseradish peroxidase It is generated with electric current, helps to improve the release of detection signal.

When catalyzing enzyme is alkaline phosphatase, substrate is to BCIP (5-Bromo-4-Chloro-3-Indolyl The chloro- 3- indyl-phosphate of the bromo- 4- of Phosphate, 5-) and NBT (Nitrotetrazolium Blue chloride, tetrazolium Nitro is blue) composition, nitrophenyl phosphate, 4-NPP, naphthols AS-BI phosphate, naphthols-AS-MX- phosphoric acid Any one of salt.

In addition, the kit of the high-risk strain Genotyping detection of HPV provided by the invention may also include cleaning solution, cleaning solution Including washing lotion A and washing lotion B, washing lotion A is the SSC buffer containing SDS, and washing lotion B is the PBS buffer solution containing Tween20.

In addition, the kit of the high-risk strain Genotyping detection of HPV provided by the invention may also include dilution, dilution It is the PBS buffer solution of casein containing protein.

The method of the high-risk strain Genotyping detection of HPV provided by the invention comprising:

Multiple probes pair are provided, each probe to include for combining target sequence capture probe and with capture probe phase Detection probe corresponding and in combination with target sequence, the 3 ' ends or 5 ' ends of the detection probe of each probe pair are marked with for tying The affinant of catalyzing enzyme is closed, catalyzing enzyme generates chemical reaction for catalysis substrate and forms electron stream, and multiple probes are to including: the 1st Probe to, the 2nd probe pair and selected from the 3rd~11 probe at least one of.Wherein, the capture probe of the 1st probe pair Base sequence is as shown in SEQ ID NO.1, and the base sequence of the capture probe of the 2nd probe pair is as shown in SEQ ID NO.3, and the 3rd The base sequence of the capture probe of~11 probes pair is respectively as shown in NO.5~13 SEQ ID.

Capture probe fixing step: the capture probe for being used for combining target sequence is first fixed to detection by electric field action In the reacting hole of orifice plate.Capture probe toward the mobile deposition in reacting hole bottom, and is fixed on reacting hole bottom under electric field action Portion.

Sample hybridization step: sample to be tested is added to reacting hole.Target sequence and capture probe in sample to be tested Reacting hole bottom is fixed on by the way that base complementrity principle is captured.

Detection probe combination step: corresponding with capture probe and detection probe in combination with target sequence is added to reaction In hole, the 3 ' ends or 5 ' ends of detection probe are marked with to can be used for combining answers substrate generation chemical reaction to form electronics for catalytic phase The affinant of the catalyzing enzyme of stream.Detection probe is fixed in reacting hole in conjunction with target sequence by base complementrity principle.

Enzymic catalytic reaction detecting step: being added in catalyzing enzyme and substrate to reacting hole, is detected by current detection means anti- Answer the electric current in hole.Detection probe is through affinant in conjunction with catalyzing enzyme, and catalysis substrate generates electric current release detection to catalyzing enzyme again Signal, which is identified by current detection means amplifies, and then detects result.

It should be noted that detection orifice plate and current detection means employed in the above method can be by purchasing on the market It can buy.

Testing principle of the invention is to utilize electric field induction release and measurement (Electric Field-Induced Release and Measurement, EFIRM) technology quickly detects virogene type, especially to 18 kinds of high-risk HPVs into Row detection.The basic principle of EFIRM is:

Capture probe is fixed on the bottom hole of the reacting hole on detection orifice plate under electric field action；Capture probe is made in electric field With lower target sequence, that is, viral DNA by base pair complementarity principle capture sample to be tested；Inspection with biotin labeling Probing needle is again in conjunction with target sequence；Streptavidin and biotin identification of the catalyzing enzyme by its label, in conjunction with；Toward reacting hole The substrate of catalyzing enzyme is added, generates redox reaction, has electric current generation, instrument sensed current signal, and then judge to be measured Have corresponding target sequence in sample, due to capture probe and detection probe be according to the conservative region of specific virus type into Row design, and then know the type of the virus.

Feature and performance of the invention are described in further detail with reference to embodiments.

Embodiment 1

The present embodiment is for detecting HPV genotype, to the detection of HPV provided in this embodiment high-risk strain Genotyping Kit is illustrated.The present embodiment is to the cell (i.e. sample to be tested) at the cervix opening for being derived from two different cervical cancer patients Its HPV hypotype is detected, the sample to be tested for being derived from two different patients is respectively designated as sample 1 and sample 2, and to sample 1 and sample Originally it 2 detects simultaneously, the sample 1 and sample 2 taken is stored in -20 DEG C.

The kit of gene typing detection provided in this embodiment includes probe combinations, which includes 16 Probe pair for detecting to HPV hypotype, the capture probe and detection probe of each probe pair are individually present in the form of a solution, probe Final concentration be 1 μM.16 probes are to being respectively: for detecting the 1st probe pair of 16 hypotype of HPV, capture probe Base sequence is as shown in SEQ ID NO.1, and the base sequence of detection probe is as shown in SEQ ID NO.2；For detecting HPV 2nd probe pair of 18 hypotypes, the base sequence of capture probe is as shown in SEQ ID NO.3, the base sequence of detection probe As shown in SEQ ID NO.4；For detecting the 3rd probe pair of 31 hypotype of HPV, the base sequence of capture probe such as SEQ ID Shown in NO.5, the base sequence of detection probe is as shown in SEQ ID NO.14；For detect 33 hypotype of HPV or 52 hypotypes or 4th probe pair of 58 hypotypes, the base sequence of capture probe is as shown in SEQ ID NO.6, the base sequence of detection probe As shown in SEQ ID NO.15；For detecting the 5th probe pair of 35 hypotype of HPV, the base sequence of capture probe such as SEQ ID Shown in NO.7, the base sequence of detection probe is as shown in SEQ ID NO.14；For detecting the 6th probe of 39 hypotype of HPV Right, the base sequence of capture probe is as shown in SEQ ID NO.8, the base sequence of detection probe such as SEQ ID NO.16 institute Show；For detecting the 7th probe pair of 45 hypotype of HPV, the base sequence of capture probe is as shown in SEQ ID NO.9, detection The base sequence of probe is as shown in SEQ ID NO.17；For detecting the 8th probe pair of 51 hypotype of HPV, the alkali of capture probe Basic sequence is as shown in SEQ ID NO.10, and the base sequence of detection probe is as shown in SEQ ID NO.18；For detecting HPV 9th probe pair of 56 hypotypes, the base sequence of capture probe is as shown in SEQ ID NO.11, the base sequence of detection probe As shown in SEQ ID NO.18；For detecting the 10th probe pair of 59 hypotype of HPV, the base sequence of capture probe such as SEQ Shown in ID NO.12, the base sequence of detection probe is as shown in SEQ ID NO.19；For detecting the 11st of 68 hypotype of HPV the Probe pair, the base sequence of capture probe is as shown in SEQ ID NO.13, the base sequence of detection probe such as SEQ ID Shown in NO.16；For detecting the 12nd probe pair of 26 hypotype of HPV, the base sequence of capture probe such as SEQ ID NO.20 institute Show, the base sequence of detection probe is as shown in SEQ ID NO.26；For detecting the 13rd probe pair of 53 hypotype of HPV, catch The base sequence of probe is obtained as shown in SEQ ID NO.21, the base sequence of detection probe is as shown in SEQ ID NO.25；With In the 14th probe pair of detection 66 hypotype of HPV, the base sequence of capture probe as shown in SEQ ID NO.22, visit by detection The base sequence of needle is as shown in SEQ ID NO.25；For detecting the 15th probe pair of 73 hypotype of HPV, the alkali of capture probe Basic sequence is as shown in SEQ ID NO.23, and the base sequence of detection probe is as shown in SEQ ID NO.27；For detecting HPV 16th probe pair of 82 hypotypes, the base sequence of capture probe is as shown in SEQ ID NO.24, the base sequence of detection probe Column are as shown in SEQ ID NO.28.The base sequence of each HPV hypotype classification and its detection corresponding capture probe and detection probe It is shown in Table 1.

Table 1. detects the capture probe of HPV hypotype and the base sequence of detection probe

Wherein, detection 33,52 is identical with the capture probe base sequence of 58 hypotypes, detects the detection of 33,52 and 58 hypotypes Probe base sequence is identical, that is to say, that detects shown in the capture probe shown in SEQ ID NO.6 and SEQ ID NO.15 Probe is capable of detecting when the subtype virus of HPV33,52 and 58, and what detection result illustrated is that sample has in the hypotype of HPV33,52 and 58 At least one, but be specifically three kinds of subtype virus in any subtype virus can not detect.39 and 68 hypotypes Detection probe sequence is identical, and 51 is identical with the detection probe sequence of 56 hypotypes, and 53 is identical with the detection probe sequence of 66 hypotypes.This Outside, for detecting 5 ' end label biotins of the detection probe of 16 and 18 hypotypes, 3 ' end labels of the detection probe of remaining hypotype Biotin (as shown in table 1).

Using gene typing detecting reagent kit provided in this embodiment to the specific detecting step of sample 1 and sample 2 It is as follows.

1 capture probe is fixed

1.1 prepare the mixed liquor of pyrroles (pyrrole) and CP

1 1.5mL centrifuge tube is taken, 885 μ l of ultrapure water, 100 μ l 3M KCl of ionic compound are sequentially added, be vortexed concussion It mixes, centrifugation；5 μ l pyrrole of conducting polymer (>=98.0%, be purchased from Sigma, article No. W338605) is added, be vortexed concussion It mixes, centrifugation；10 μ l CP (100 μM) are added and (in the case where interblock interference, 10 μ l are added to detection hole in every kind of CP In)；It is vortexed after concussion mixes and is centrifuged, it is spare.

1.2 immobilized capture probes

Detection orifice plate (E-plate) (its structure and the visible bibliography 201620769829.2 of working principle) in 96 holes On, by its operational manual, it is added the mixed liquor of the prepared pyrrole and CP of 30 μ l toward reacting hole, pipette tips when sample-adding Close to the bottom in hole, but it is not exposed to bottom electrode, adds rear-inclined or pats the electrode table that E-plate makes liquid in hole Face uniform fold, then at once on EFIRM instrument (its working principle and structure can refer to the applying date be August in 2016 11, Application No. is 201610658321.X, the entitled patent document for keeping structure and the detector including holding structure), by its behaviour Book is explained, electric field operation is carried out.

1.3EFIRM electric field treatment

The respective column tested, electric pulse field parameter setting are as follows: voltage A:350mV, 1s are selected on EFIRM software；Voltage B:950mV, 1s；Carry out 9 circulations.Electric field treatment finishes, and takes out at once, cleans E-plate plate.

The cleaning of 1.4E-plate plate:

Corresponding experiment is selected to arrange in board-washing machine program, cleaning procedure selects (2bottom, 2top), cleaning solution selection Washing lotion A.Cleaning finishes, and carries out at once in next step, the operation of sample loading.Wherein, washing lotion A is containing 0.05% (mass percent) 2 × SSC buffer of SDS.

The hybridization of 2 samples

2.1 hybridization buffer pretreatments

Hybridize buffer (being purchased from thermo fisher) 90 DEG C of water bath processing 10min in water-bath, is then placed at room temperature for Cooling 20min.

2.2 prepare sample to be tested

Sample to be tested takes out from -20 DEG C of refrigerators, puts 4 DEG C of refrigerator defrostings into.After being completely dissolved, boiling method or 0.4M are used NaOH pre-processes sample to be tested, and then by sample to be tested, 1:2 is mixed by volume with buffer is hybridized, and is centrifuged after vortex oscillation, Can loading detected.

2.3 add sample to be tested

On E-plate, be added in corresponding hole above-mentioned sample to be tested with hybridize mixed 30 μ of mixed liquor of buffer l.(it is to be noted that pipette tips but are not exposed to bottom electrode close to the bottom in hole when sample-adding, add rear-inclined or beating E-plate makes electrode surface uniform fold of the liquid in hole, then at once to progress electric field operation on EFIRM.)

In the present embodiment, 4 detection groups are set, are 16 hypotype groups, 18 hypotype groups, 11 kinds of high-risk-type groups and 5 respectively High-risk-type group in kind.Each detection group is arranged a Positive control wells, a negative control hole (being repeated 4 times), sample 1 The detection hole of detection hole and a sample 2.Wherein, 16 hypotype groups are (corresponding in each hole of the group to add for detecting HPV16 hypotype Enter the 1st probe to), for 18 hypotype groups for detecting HPV18 hypotype (each hole of the group corresponding be added the 2nd probe to), 11 kinds are high-risk For detecting one of 11 kinds of high-risk HPVs, (each hole of the group is corresponding to be added the 3rd~11 probe pair, high-risk-type in 5 kinds to type group For detecting one of high-risk HPV in 5 kinds (each hole of the group corresponding be added the 12nd~16 probe to).It needs to illustrate It is that the testing result order of accuarcy that the quantity of detection group can be as needed is designed.If only needing to detect that sample to be tested is It is no containing common type HPV, high-risk-type virus and middle high-risk-type virus, then can by the detection group of the present embodiment setting method into Row setting.If only needing to detect whether sample contains HPV, a detection group can be only set, and specific detection method is with reference to real Apply example 2.Detect that sample specifically contains some type of virus if necessary, then settable 18 detection groups, specific detection method Reference implementation example 3.

Wherein, be added in Positive control wells corresponding 30 μ l containing final concentration of 1pM positive oligonucleotides (its can with it is right The capture probe answered and the combination of detection probe complementary pairing) hybridization buffer as positive control (it should be noted that 11 kinds The positive control of high-risk-type group only selects 52 hypotype nucleotide sequences (the 9th row in table 2), and the positive of high-risk-type group is right in 5 kinds According to 26 hypotype nucleotide sequences (the 13rd row in table 2) is only selected, positive control can be played the role of), each HPV hypotype is corresponding The base sequence of positive oligonucleotides be shown in Table 2,30 μ l hybridization buffer is added in negative control hole as negative control.

Table 2. is used to detect the base sequence of the positive oligonucleotides of the positive control of HPV genotype

2.4EFIRM electric field treatment

The respective column tested, electric pulse field parameter setting are as follows: voltage A:300mV, 1s are selected on EFIRM software；Voltage B:500mV, 1s；Carry out 150 circulations.Electric field treatment finishes, and takes out at once, cleans E-plate plate.

2.5 incubation at room temperature

E-plate lid is covered, is incubated at room temperature 30min on experimental bench.

The cleaning of 2.6E-plate plate

Corresponding experiment is selected to arrange in board-washing machine program, cleaning procedure selects (2bottom, 2top), cleaning solution selection Washing lotion A.Cleaning finishes, and carries out DP sample-adding operation at once.

3 DP are combined

3.1DP solution is prepared

Dilution is taken out from 4 DEG C of refrigerators, 1 1.5mL centrifuge tube is taken, the dilution of 990 μ l is added, toward the anti-of each detection group Corresponding 10 μ l DP (100 μM) is added in Ying Kongzhong, and the concussion that is vortexed mixes, and centrifugation is spare.Wherein, dilution is containing 0.1% (matter Measure volume ratio) casein PBS buffer solution (pH7.4).The effect of casein is closing non-specific sites, to improve detection Sensitivity and accuracy.

3.2 sample-adding

Be added corresponding 30 μ l of DP solution in corresponding hole according to experimental design, when sample-adding pipette tips close to hole bottom, still It is not exposed to bottom electrode, add rear-inclined or pats the electrode surface uniform fold that E-plate makes liquid in hole, is then stood It is carved into progress electric field operation on EFIRM.

3.3 incubation at room temperature

The cleaning of 3.4E-plate plate

Corresponding experiment is selected to arrange in board-washing machine program, cleaning procedure selects (2bottom, 2top), cleaning solution selection Washing lotion A.Cleaning finishes, and carries out sample-adding operation at once.

The horseradish peroxidase (Poly-HRP) of 4 marked by streptavidin is in conjunction with biotin

4.1 Poly-HRP solution are prepared

Dilution is taken out from 4 DEG C of refrigerators, 1 1.5mL centrifuge tube is taken, the dilution of 999 μ l is added, the enzyme solution of 1 μ l is added (contains Poly-HRP, concentration 0.5mg/ml, be purchased from thermo fisher, name of product Pierce^TMStreptavidin Poly-HRP, article No. 21140, unit specification are 0.5mL), the concussion that is vortexed mixes, and centrifugation is spare.

4.2 add enzyme solution

Above-mentioned dilution and enzyme solution mixed mixed liquor 30 μ l, Poly-HRP is added in corresponding each hole to mark by it Streptavidin and detection probe on biotin identify and combine.

4.3 incubation at room temperature

The cleaning of 4.4 E-plate plates

Corresponding experiment is selected to arrange in board-washing machine program, cleaning procedure selects (3bottom, 3top), cleaning solution selection Washing lotion B.Cleaning finishes, and carries out TMB sample-adding operation at once.Wherein, washing lotion B is containing 0.1% (mass percent) Tween20's PBS buffer solution.

5 reading data

5.1 add substrate

60 μ l of substrate is added in corresponding each hole, and pipette tips but are not exposed to bottom electrode close to the bottom in hole when sample-adding. It adds at once to progress electric field operation on EFIRM.Wherein, substrate is that the solution containing TMB (is purchased from thermo fisher, product goods Number be 34028, entitled 1-Step^TMUltra TMB-ELISA).The substrate of enzyme is added, redox reaction occurs, generates electricity Stream, detects current value in each hole and completes entire detection process.+ 3 times of standards of current average being repeated 4 times with negative control hole The sum of difference is as positive decision content, and positive decision content=AVG+3 × SD, wherein AVG is that the electric current that negative control hole is repeated 4 times is flat Mean value, SD are the standard deviation that negative control hole is repeated 4 times.If addition has the current value of the detection hole of sample to be tested to be greater than or equal to The positive decision content, then decision bits positive findings, illustrate to contain corresponding HPV subtype virus in sample to be tested.

5.2 EFIRM electric fields reading

The respective column tested, electric pulse field parameter setting are as follows: voltage A:-200mV, 60s are selected on EFIRM software；Electricity Press B:0mV, 0s；Carry out 1 circulation.Electric field treatment finishes, and takes out at once, cleans E-plate plate.

Instrument will be automatically performed detection work, and detection data is automatically uploaded to cloud computing platform.It is drawn according to detection data Histogram, abscissa are the classification of detection group, and ordinate is the current value (Current) of each detection hole in each detection group, unit For Naan (- nA, negative sign indicate direction).The testing result of the present embodiment is as shown in Figure 1.

As shown in Figure 1, in 16 hypotype groups, the testing result of sample 1 is positive, (negative containing HPV16 subtype virus Control current value is 28.67nA, its standard deviation is 5.62, and positive control current value is 130.22nA, and the current value of sample 1 is 192.05nA, the current value of sample 2 are 30.53nA)；In 11 kinds of high-risk-type groups, the testing result of sample 2 is positive (negative Control current value is 24.94nA, its standard deviation is 5.91, and positive control current value is 132.94nA, and the current value of sample 1 is 29.57nA, the current value of sample 2 are 151.30nA), illustrate its contain high-risk HPV 31,33,52,58,35,39,45,51, 56, at least one of 59,68 hypotypes.

Embodiment 2

The kit of the high-risk strain Genotyping detection of HPV provided in this embodiment include probe combinations (with embodiment 1), The horseradish peroxidase (existing in the form of a solution) and its substrate of fixture, marked by streptavidin, fixture are conductive poly- Close object and ionic compound, wherein conducting polymer is pyrroles, and ionic compound is potassium chloride.Substrate is the solution containing TMB. Remaining is the same as embodiment 1.

In the present embodiment, detection group is set as 1, is named as 18 kinds of hypotypes detection group simultaneously, and detection group includes one Positive control wells, a negative control hole (being repeated 4 times), the detection hole of sample 1 and the detection hole of a sample 2.Often 16 kinds of probes pair described in embodiment 1 are all added in a hole, 18 kinds of HPV hypotypes detect simultaneously).Remaining is the same as embodiment 1.If there is The positive detection as a result, the result then detected can illustrate sample to be tested contain HPV16,18,31,33,52,58,35,39,45,51, 56, at least one of 59,68,26,53,66,73,82 hypotypes (as shown in Figure 2).The testing result of the present embodiment such as Fig. 2 institute Show.

As shown in Figure 2, the testing result of sample 1 is positive, and (negative control current value is 26.59nA, its standard deviation is 7.91, positive control current value is 110.46nA, and the current value of sample 1 is 189.66nA), illustrate its contain HPV16,18,31, 33, at least one of 52,58,35,39,45,51,56,59,68,26,53,66,73,82 hypotypes；The testing result of sample 2 Being positive, (negative control current value is 26.59nA, its standard deviation is 7.91, and positive control current value is 110.46nA, sample 2 Current value be 80.60nA), illustrate sample 2 containing HPV16,18,31,33,52,58,35,39,45,51,56,59,68,26, 53, at least one of 66,73,82 hypotypes.

Embodiment 3

The kit of the high-risk strain Genotyping detection of HPV provided in this embodiment include probe combinations (with embodiment 1), The horseradish peroxidase (existing in the form of a solution) and its substrate, cleaning solution of fixture, marked by streptavidin.

Fixture is conducting polymer and ionic compound, wherein conducting polymer is pyrroles, and ionic compound is chlorination Potassium.Substrate is the solution containing TMB.Cleaning solution includes including washing lotion A and washing lotion B, and washing lotion A is the SSC buffer containing SDS, washing lotion B It is the PBS buffer solution containing Tween20.Remaining is the same as embodiment 1.

In the present embodiment, detection group is set as 16 groups, is 16 hypotype groups, 18 hypotype groups, 26 hypotype groups, 31 Asias respectively Type group, 35 hypotype groups, 39 hypotype groups, 45 hypotype groups, 51 hypotype groups, 52 hypotype groups, 53 hypotype groups, 56 hypotype groups, 59 hypotype groups, 66 hypotype groups, 68 hypotype groups, 73 hypotype groups and 82 hypotype groups.Positive control wells (each detection is arranged in each detection group Group is separately added into corresponding positive oligonucleotides shown in table 2), a negative control hole (being repeated 4 times), sample 1 The detection hole of detection hole and a sample 2, each detection hole is interior to be added its corresponding probe pair.Remaining is the same as embodiment 1.If Each detection group has positive detection as a result, the result then detected can then illustrate the corresponding HPV subtype virus that sample to be tested contains. It should be noted that since, the probe of HPV 33,52 and 58 hypotypes is to general, therefore the positive findings that 52 hypotype groups detect Illustrate in sample to be tested containing one or more of hypotype of HPV33,52 and 58.The testing result of the present embodiment is as shown in Figure 3.

From the figure 3, it may be seen that in 16 hypotype groups, the testing result of sample 1 be positive (negative control current value be 28.67nA, Its standard deviation is 5.62, and positive control current value is 130.22nA, and the current value of sample 1 is 192.05nA, the current value of sample 2 For 30.53nA), illustrate that sample 1 contains 16 subtype virus of HPV；In 52 hypotype groups, the testing result of sample 2 is positive (negative Property control current value be 25.58nA, its standard deviation is 5.33, positive control current value is 75.70nA, and the current value of sample 1 is 23.90nA, the current value of sample 2 are 166.10nA), in addition, the testing result of sample 2 is positive (negative in 66 hypotype groups Control current value is 25.92nA, its standard deviation is 6.20, and positive control current value is 115.75nA, and the current value of sample 1 is 34.47nA, the current value of sample 2 are 72.72nA), illustrate sample 2 containing one of 33,52,58 hypotype of HPV and HPV 66 Hypotype.It is sub- that the present embodiment detects the specific virus that the testing result of sample to be tested can be contained by 18 detection groups of setting Type detects that testing result is accurate and reliable.

Embodiment 4

The kit of the high-risk strain Genotyping detection of HPV provided in this embodiment includes probe combinations, detects orifice plate, is different The horseradish peroxidase (existing in the form of a solution) and its substrate (ABTS, in the form of a solution of thiocyanic acid anti-fluorescein antibody label In the presence of), cleaning solution.

Wherein, probe combinations include 9 probes pair, and 9 probes are to being respectively: for detecting the 1st of 16 hypotype of HPV the Probe to, for detect the 2nd probe of 18 hypotype of HPV to, for detect the 3rd probe of 31 hypotype of HPV to, for detecting 4th probe of 33 hypotype of HPV or 52 hypotypes or 58 hypotypes to, for detect the 8th probe of 51 hypotype of HPV to, for detecting 9th probe of 56 hypotype of HPV to, for detect the 12nd probe of 26 hypotype of HPV to, for detecting 53 hypotype of HPV the 13rd Probe is to, the 15th probe pair for detecting 73 hypotype of HPV.3 ' ends of each detection probe have fluorescein isothiocynate.It is each to visit The table 1 in embodiment 1 is seen for the base sequence of corresponding capture probe and detection probe.

The capture probe that above-mentioned probe pair is fixed in the reacting hole of orifice plate is detected, capture probe is fixed to detection orifice plate Method with " 1 capture probe is fixed " step in embodiment 1, certainly, in other examples, adopt with other methods will Capture probe of the invention is fixed to the detection orifice plate obtained on detection orifice plate and also belongs to protection scope of the present invention.

Cleaning solution includes washing lotion A and washing lotion B, and washing lotion A is the SSC buffer containing SDS, and washing lotion B is the PBS containing Tween20 Buffer.Remaining is the same as embodiment 1.

Sample 1 and sample 2 are detected simultaneously using the present embodiment, detecting step and embodiment 1 are almost the same, testing result As shown in Figure 4.

As shown in Figure 4, in 16 hypotype groups, the testing result of sample 1 be positive (negative control current value be 25.22nA, Its standard deviation is 4.43, and positive control current value is 108.10nA, and the current value of sample 1 is 166.03nA), illustrate that sample 1 contains There is HPV16 subtype virus；In 6 kinds of high-risk-type groups, the testing result of sample 2 is positive, and (negative control current value is 24.25nA, its standard deviation are 5.27, and positive control current value is 115.92nA, and the current value of sample 2 is 170.42nA), explanation It contains at least one of high-risk HPV 31,33,51,52,58 and 56 hypotypes.

Embodiment 5

The kit of the high-risk strain Genotyping detection of HPV provided in this embodiment include probe combinations, fixture, it is high The alkaline phosphatase enzyme (existing in the form of a solution) and its substrate of pungent antibody label are (with the solution containing BCIP and the composition of NBT In the presence of), cleaning solution.

Wherein, probe combinations include 4 probes pair, and 4 probes are to being respectively: for detecting the 1st of 16 hypotype of HPV the Probe to, for detect the 2nd probe of 18 hypotype of HPV to, for detecting 33 hypotype of HPV or 52 hypotypes or 58 hypotypes the 4th Probe is to, the 12nd probe pair for detecting 26 hypotype of HPV.5 ' ends of each detection probe have digoxin.Each probe is to corresponding Capture probe and the base sequence of detection probe see the table 1 in embodiment 1.

Fixture is conducting polymer and ionic compound, wherein conducting polymer is thiophene, certainly, other real It applies and can be aniline in example.Ionic compound is sodium chloride.Cleaning solution includes including washing lotion A and washing lotion B, and washing lotion A is containing SDS SSC buffer, washing lotion B are the PBS buffer solution containing Tween20.Remaining is the same as embodiment 1.

Sample 1 and sample 2 are detected simultaneously using the present embodiment, detecting step and embodiment 1 are almost the same, testing result As shown in Figure 5.

As shown in Figure 5, in 16 hypotype groups, the testing result of sample 1 be positive (negative control current value be 26.31nA, Its standard deviation is 5.29, and positive control current value is 118.74nA, and the current value of sample 1 is 185.47nA), illustrate that sample 1 contains There is HPV16 subtype virus；In 3 kinds of high-risk-type groups, the testing result of sample 2 is positive, and (negative control current value is 27.45nA, its standard deviation are 5.15, and positive control current value is 122.62nA, and the current value of sample 2 is 150.07nA), explanation Sample 2 contains at least one of high-risk HPV 33,51,52 and 58 hypotypes.

Embodiment 6

The kit of the high-risk strain Genotyping detection of HPV provided in this embodiment includes probe combinations, fixture, strepto- The horseradish peroxidase (existing in the form of a solution) and its substrate, fixture of Avidin label are conducting polymer and ionization Close object, wherein conducting polymer is pyrroles, and ionic compound is potassium chloride.Substrate is the solution containing TMB.Remaining same embodiment 1。

Wherein, probe combinations only include the 1st probe to, the 2nd probe to, the 3rd probe to, the 4th probe to, the 5th probe to, 6th probe to, the 7th probe to, the 8th probe to, the 9th probe to, the 10th probe pair and the 11st probe to (each probe pair is caught The sequence of probe and detection probe is obtained with embodiment 1).Certainly, in other examples, probe combinations include the 1st probe to, 2nd probe to, selected from the 3rd~11 probe at least one of situation a variety of such as two or three.

Only sample 1 is detected using the kit of the present embodiment, detecting step and embodiment 1 are almost the same, detection As a result as shown in Figure 6.

It will be appreciated from fig. 6 that in 16 hypotype groups, the testing result of sample 1 be positive (negative control current value be 26.37nA, Its standard deviation is 5.62, and positive control current value is 142.22nA, and the current value of sample 1 is 188.55nA), illustrate that sample 1 contains There is HPV16 subtype virus；And in 18 hypotype groups, (negative control current value is 23.21nA, its standard deviation is 5.77, positive control Current value is 123.07nA, and the current value of sample 1 is 13.41nA) and 11 kinds of high-risk-type groups (negative control current value is 25.04nA, its standard deviation are 5.51, and positive control current value is 131.04nA, and the current value of sample 1 is 31.77nA) in, sample This 1 testing result is negative, illustrate in sample 1 without HPV18 hypotype, also without 11 kinds of high-risk HPVs 31,33,52,58, 35, one of 39,45,51,56,59,68 hypotypes or a variety of virus subtypes.The testing result of the present embodiment and embodiment 1 Testing result is almost the same.

To sum up, gene typing detecting reagent kit provided by the invention has the advantage that

1, detection sensitivity is high: traditional probe fixing means is that one end of probe is fixed on plane support, this Method can reduce the hybridization efficiency of probe Yu target DNA to be measured due to hydrophobicity of support surface etc., and the present invention passes through Capture probe is fixed in polypyrrole hole by charge adsorption effect, it is ensured that capture probe has super-active；Traditional nucleic acid Hybrid process improves hybridization efficiency by control hybridization temperature, salt ion, reaction time etc., and the present invention increases electric field as the 4th A control condition improves capture probe to the capture rate of aim sequence DNA under the action of electric field；Pass through survey in this method The electronic signal for determining to generate in HRP catalysis TMB oxidation process is as testing result, since the catalytic efficiency of enzyme is very high, indirectly Be exaggerated hybridization reaction as a result, increasing the susceptibility of measuring method.The capture of moment target molecules, super-active molecular probe Fixed, capture molecule signal, which specifically amplifies this three big core technology, ensure that this method has the sensitivity of superelevation, much higher than bar The technologies such as family name's smear, TCT technology and HC2 are suitable with the technologies such as polymerase chain reaction and genetic chip.

2, detect high specificity: in the present invention, the detection process of each hypotype HPV includes a capture probe and one Detection probe, for probe length between 25-40bp, hybridization efficiency is influenced obvious, only target sequence DNA by base mismatch Can just there be detection signal after accurately matching simultaneously with two probes, substantially increase the specificity of detection.

3, easy to operate, rapid reaction: on the one hand the introducing of electric field in the present invention is omitted pure to sample in hybrid process Degree requires, so that clinical sample can be detected directly without nucleic acid extraction；On the other hand it is right in hybrid process to reduce The requirement in reaction time, accelerates reaction rate.Entire detection process can be completed within half an hour, when shortening general reaction Between, the waiting time of outpatient is reduced, strives for valuable treatment time for patient.

4, it reduces the requirement to clinical client hardware and personnel: not needing to carry out sample in detection process in the present invention Pre-treatments, false positive caused by effectively avoiding because of environmental pollution, and the test operation such as purifying and PCR amplification are not necessarily in special tool There is the PCR Lab of subregion to carry out, operator does not also need to obtain the license that is on duty of Clinic Gene Amplified, can be by general skill Art personnel operation, it is lower to the competency profiling of experimental situation and operator.

5, it is at low cost: firstly, either ARMS-PCR or digital pcr all use fluorescence at present in terms of detection device Signal detection, detection device need to be equipped with expensive fluorescence detecting system, and fluorescence quantitative PCR instrument market price is all left in hundreds of thousands of members The right side, and digital pcr instrument is more up to more than 100 ten thousand yuan.In contrast, release and measurement of the EFIRM platform using the electric field leading of original creation Technology, detection process utilize electric field action, and rapid reaction, final result detects as electronic signals, thus the cost of equipment It is greatly reduced, just corresponds to half of fluorescence quantitative PCR instrument or so.

Secondly, the principle that EFIRM technology is hybridized based on nucleic acid in terms of detection reagent, using the electrochemistry skill of unique design Art.Nucleic acid probe length used in the present invention between 25-40bp, select the area E7 that differs greatly between HPV hypotype or The area L1 is all made of Oligonucleotide probe, and wherein CP uses more typical Biotin method of modifying without modification, DP, The preparation commission commercialized DNA chemical synthesis company of probe completes, and technical difficulty is low, and stability is preferable, and part hypotype is other CP or DP is shared, reduces total number of probes, reduces cost.Fluorescent quantitation based on PCR needs to carry out both ends to probe Modification, and one end is fluorescence group, synthesis cost is higher；Reverse dot blot hybridization and biochip technology require that spy will be hybridized greatly Needle is fixed on solid phase carrier, complex treatment process, increases testing cost, and probe activity after fixation has certain drop It is low；The probe that HC2 is used is the rna probe of overall length, and length is 7000-8000 base, and preparation process is complicated, time-consuming more, at This is very high, and since probe is very long, hybridization efficiency is influenced small by base mismatch, in fact it could happen that the intersection between hypotype.This Outside, the step of EFIRM technology saves DNA extraction and purification in the detection process, therefore detection reagent cost and other technologies It is greatly reduced compared to comparing.

In short, this method provide the HPV Probe-detection methods based on EFIRM have high sensitivity, high specificity, The time-consuming short, experimental site of detection requires the features such as low, at low cost, is suitable for a large amount of clinical detection and large-scale epidemiology Screening.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

Claims

The probe combinations of the high-risk strain Genotyping detection of 1.HPV, which is characterized in that it includes multiple probes pair, each described Probe is to including corresponding for the capture probe of combining target sequence and with the capture probe and in combination with the target The detection probe of sequence, the 3 ' ends or 5 ' ends of the detection probe of each probe pair are marked with for combining the affine of catalyzing enzyme Object, the catalyzing enzyme generate chemical reaction for catalysis substrate and form electron stream, multiple probes to include: the 1st probe to, 2nd probe pair and selected from the 3rd~11 probe at least one of；

Wherein, the base sequence of the capture probe of the 1st probe pair is as shown in SEQ ID NO.1, and the 2nd probe is to catching The base sequence of probe is obtained as shown in SEQ ID NO.3, the base sequence of the capture probe of the 3rd~11 probe pair is respectively such as Shown in NO.5~13 SEQ ID；

The base sequence of the detection probe of 1st probe pair as shown in SEQ ID NO.2, visit by the detection of the 2nd probe pair The base sequence of needle is as shown in SEQ ID NO.4, base sequence of the 3rd probe to the detection probe with the 5th probe pair For column as shown in SEQ ID NO.14, the base sequence of the detection probe of the 4th probe pair is described as shown in SEQ ID NO.15 6th probe to the base sequence of the detection probe with the 11st probe pair as shown in SEQ ID NO.16, the 7th probe pair Detection probe base sequence as shown in SEQ ID NO.17, the 8th probe is to the detection probe with the 9th probe pair Base sequence as shown in SEQ ID NO.18, the base sequence such as SEQ ID NO.19 of the detection probe of the 10th probe pair It is shown.
2. the probe combinations of the high-risk strain Genotyping detection of HPV according to claim 1, which is characterized in that Duo Gesuo State probe to further include the 12nd~16 probe at least one of, the base sequence of the capture probe of the 12nd~16 probe pair Column are as shown in NO.20~24 SEQ ID, the base sequence of the detection probe of the 12nd probe pair such as SEQ ID NO.26 institute Show, the 13rd probe to the base sequence of the detection probe with the 14th probe pair as shown in SEQ ID NO.25, it is described The base sequence of the detection probe of 15th probe pair is as shown in SEQ ID NO.27, the alkali of the detection probe of the 16th probe pair Basic sequence is as shown in SEQ ID NO.28.
The kit of the high-risk strain Genotyping detection of 3.HPV, which is characterized in that it includes any one of claim 1~2 institute The probe combinations of the high-risk strain Genotyping detection of the HPV stated.
4. the kit of the high-risk strain Genotyping detection of HPV according to claim 3, which is characterized in that the reagent Box further include for by the capture probe be fixed to detection orifice plate fixture, the fixture include conducting polymer and from Sub- compound；

The conducting polymer is selected from any one of pyrroles, aniline and thiophene；

The ionic compound is selected from any one of sodium chloride and potassium chloride.
5. the kit of the high-risk strain Genotyping detection of HPV according to claim 3, which is characterized in that the reagent Box further includes the detection orifice plate, is fixed with the capture probe in the reacting hole of the detection orifice plate.
6. the kit of the high-risk strain Genotyping detection of HPV according to claim 3, which is characterized in that the reagent Box further includes the catalyzing enzyme, and the catalyzing enzyme is horseradish peroxidase or alkaline phosphatase with marker, the mark Remember that object is used in conjunction with the affinant, the marker is that DigiTAb, fluorescein isothiocynate antibody and strepto- are affine Any one of element；

The affinant of 3 ' ends of the detection probe or 5 ' end labels is digoxin corresponding with the marker, different sulphur It is a kind of in cyanic acid fluorescein and biotin.
7. the kit of the high-risk strain Genotyping detection of HPV according to claim 6, which is characterized in that the reagent Box further includes the substrate of the catalyzing enzyme；

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

When the catalyzing enzyme is the alkaline phosphatase, the substrate is the composition of BCIP and NBT, p-nitrophenyl phosphoric acid Any one of salt, 4-NPP, naphthols AS-BI phosphate, naphthols-AS-MX- phosphate.
8. the kit of the high-risk strain Genotyping detection of HPV according to claim 3, which is characterized in that the reagent Box further includes cleaning solution, and the cleaning solution includes washing lotion A and washing lotion B, the washing lotion A are the SSC buffers containing SDS, described to wash Liquid B is the PBS buffer solution containing Tween20.
The method of 9.HPV high-risk strain Genotyping detection, the testing goal of the method be for the purpose of the diagnosis of non-disease, It is characterized in that, it includes the following steps:

There is provided multiple probes pair, each probe is to including visiting for the capture probe of combining target sequence and with the capture The detection probe of the corresponding and combinable target sequence of needle, the 3 ' ends or 5 ' ends of the detection probe of each probe pair It is marked with the affinant for combining catalyzing enzyme, the catalyzing enzyme generates chemical reaction for catalysis substrate and forms electron stream, more A probe to include: the 1st probe to, the 2nd probe pair and selected from the 3rd~11 probe at least one of；

First the capture probe for being used for combining target sequence is fixed in the reacting hole of detection orifice plate by electric field action；

Sample to be tested is added to the reacting hole；

It is added in corresponding with the capture probe and detection probe to the reacting hole in combination with the target sequence, it is described 3 ' the ends or 5 ' ends of detection probe are marked with to can be used for combining answers substrate generation chemical reaction to form electron stream for catalytic phase The affinant of catalyzing enzyme；

It is added in the catalyzing enzyme and the substrate to the reacting hole, is detected in the reacting hole by current detection means Electric current；

Wherein, the base sequence of the capture probe of the 1st probe pair is as shown in SEQ ID NO.1, and the 2nd probe is to catching The base sequence of probe is obtained as shown in SEQ ID NO.3, the base sequence of the capture probe of the 3rd~11 probe pair is respectively such as Shown in NO.5~13 SEQ ID；

The base sequence of the detection probe of 1st probe pair as shown in SEQ ID NO.2, visit by the detection of the 2nd probe pair The base sequence of needle is as shown in SEQ ID NO.4, base sequence of the 3rd probe to the detection probe with the 5th probe pair For column as shown in SEQ ID NO.14, the base sequence of the detection probe of the 4th probe pair is described as shown in SEQ ID NO.15 6th probe to the base sequence of the detection probe with the 11st probe pair as shown in SEQ ID NO.16, the 7th probe pair Detection probe base sequence as shown in SEQ ID NO.17, the 8th probe is to the detection probe with the 9th probe pair Base sequence as shown in SEQ ID NO.18, the base sequence such as SEQ ID NO.19 of the detection probe of the 10th probe pair It is shown.