CN108060268A - The application method of the primer and probes of HPV parting detections, kit and kit - Google Patents

Abstract

The invention discloses the application methods of a kind of primer and probe, kit and kit.The primer and probe of the HPV parting detections, the application method of kit and kit can detect a variety of HPV hypotypes simultaneously, by using real-time fluorescent PCR amplification technology, a nucleic acid extraction only need to be carried out, each group also only needs to carry out a PCR detection reaction, the application method of the kit is fast and convenient, and specificity is good, other HPV hypotypes or other microorganisms etc. not will detect that, the problem of being thus less prone to false positive, and high sensitivity, up to 400copies/ml, detection range is 400copies/ml~4.00E+09copies/ml, detection range is wide.

Description

The application method of the primer and probes of HPV parting detections, kit and kit

Technical field

The present invention relates to molecular Biological Detection field, more particularly, to a kind of primer and probe of HPV partings detection, The application method of kit and kit.

Background technology

HPV (Human papillomavirus, HPV, human papilloma virus) is that molecule amount is smaller nonencapsulated Double-stranded cyclic DNA virus, the obligate epithelial cell infected with parasitic human body reproductive organ and other histoorgans.Clinically, According to HPV different subtype pathogenicity sizes or carcinogenic risk, of different sizes can be divided to HPV be two major class of high-risk-type and low risk. Low risk HPV mainly cause skin of anus and the big nymphae of male external genital organs, women, urethral orifice, vagina hypomere it is exophytic Wart class lesion and low cervical intraepithelial neoplasia.High-risk HPV is in addition to it can cause external genital organs wart, it is often more important that cause external Grow device cancer, cervical carcinoma and high-grade cervical intraepithelial neoplasia (cin).

HPV easily Spreading and diffusions in crowd, can be by directly or indirectly contacting cross infection, and infection site is hidden, Incidence of occult is not easy early detection, can cause a variety of proliferative lesions, most common pernicious swollen caused by female reproductive system Knurl is cervical carcinoma.High-risk HPV persistent infections, which are the main pathogenics of cervix cancer, to be understood to the Study of Etiology of cervical carcinoma, There are about 99.7% cervical cancer patient, there are HPV infections.Clinical studies show, from the persistent infection of HPV to general cervical carcinoma before Lesion simultaneously finally develops into cervical carcinoma and takes around 8-10.Treatment of the therapeutic effect of Early pathological changes of uterine cervix more than cervical carcinoma is imitated Fruit is far better, therefore, fast and accurately detects the infection of HPV high-risk-types, and can accurate parting, for early treatment Have great importance with the morbidity and mortality etc. for reducing cervical carcinoma.

Condyloma acuminatum (Genital warts, Condylomata acumlnata, CA) also known as property wart, anus genitals Wart, be as men and women's genitals, the epidermis Tumor like hyperplasia of perineum and anus area caused by some certain types of HPV infections, Mostly occur between twenty and fifty men and women.This disease is popular in the whole world, is the current American-European and most common sexually transmitted disease of African country One of (STD).Incidence of the CA in China is higher, increases sharply again in recent years, becomes the second largest venereal disease for being only second to gonorrhoea.Cause This, fast and accurately detects low risk HPV for the auxiliary diagnosis of condyloma acuminatum, early treatment and prevents its prevalence etc. Have great importance.

The detection of HPV DNA at present is mainly by applied molecular biology method, including nucleic acid hybridization, genetic chip Technology and polymerase chain reaction technology etc..However traditional detection method is often the detection for single HPV hypotypes, it is more The detection process of kind of HPV hypotype mixtures is cumbersome, time-consuming and poor specificity, and sensitivity is not also high.

The content of the invention

Based on this, it is necessary to provide a kind of HPV parting detections for detecting easy, specificity and high sensitivity primer and The application method of probe, kit and kit.

A kind of primer and probe of HPV partings detection, PCR amplification primer pair including multiple HPV hypotypes and corresponding Detection probe, the PCR amplification primer pair of each HPV hypotypes and the sequence of corresponding detection probe are as follows：

The both ends of the detection probe are marked with fluorescent reporter group and fluorescent quenching group, multiple HPV hypotypes point respectively Into fluorescent reporter group difference that is multigroup, being marked in the detection probe of the different HPV hypotypes in same group.

In one of the embodiments, multiple HPV hypotypes are divided into seven groups；Wherein, 16,18,33 and of HPV are included for first group 39 hypotypes；Second group includes HPV 45,59,35 and 66 hypotypes；3rd group includes HPV 51,53,52 and 68 hypotypes；4th group of bag Include HPV 58,31,56 and hypotype；5th group includes HPV 6,40,42 and 55 hypotypes；6th group includes HPV 11,43,44 and 54 Hypotype；7th group includes HPV 67,57 and 73 hypotypes.

In one of the embodiments, the fluorescent reporter group in the detection probe in each group be selected from FAM, HEX, One kind in VIC, CY5 and ROX, the fluorescent quenching group are BHQ1 or BHQ2.

In one of the embodiments, the fluorescent reporter group in the corresponding detection probe in first group be respectively FAM, HEX or VIC, CY5 and ROX；

The fluorescent reporter group in corresponding detection probe in second group is respectively FAM, HEX or VIC, ROX and CY5；

The fluorescent reporter group in corresponding detection probe in 3rd group is respectively FAM, HEX or VIC, CY5 and ROX；

The fluorescent reporter group in corresponding detection probe in 4th group is respectively HEX or VIC, CY5 and FAM；

The fluorescent reporter group in corresponding detection probe in 5th group is respectively FAM, HEX or VIC, ROX and CY5；

The fluorescent reporter group in corresponding detection probe in 6th group is respectively FAM, HEX or VIC, ROX and CY5；

The fluorescent reporter group in corresponding detection probe in 7th group is respectively HEX or VIC, FAM and ROX.

In one of the embodiments, the primer and probe of the HPV partings detection further include internal standard positive quality control and Its PCR amplification primer pair and corresponding detection probe；The internal standard positive quality control is clone's matter containing beta globin gene segment Grain；The sequence of the PCR amplification primer pair of the internal standard positive quality control such as SEQ ID NO:79 and SEQ ID NO:It is corresponding shown in 80 Detection probe sequence such as SEQ ID NO:Shown in 81.

In one of the embodiments, the PCR amplification primer pair of the internal standard positive quality control and corresponding detection probe add It is added in described 4th group, and the fluorescent reporter group marked in the detection probe of the internal standard positive quality control is CY5.

A kind of kit of HPV partings detection, including the primer and probe described in any of the above-described embodiment.

In one of the embodiments, the kit of the HPV partings detection is further included by containing the special of each HPV hypotypes Property genetic fragment cloned plasmids mixing form positive reference substance.

In one of the embodiments, the kit of the HPV partings detection further includes nucleic acid extracting reagent, PCR expands Increase at least one of buffer solution, archaeal dna polymerase and dNTPs reagents.

In one of the embodiments, the nucleic acid extracting reagent includes：Buddhist Sha of 0.01~0.5mM/L is graceful, 50~ The dodecyl sodium sulfate and volumetric concentration that the potassium chloride of 200mM/L, mass-volume concentration are 0.01%~2% be 0.05%~1% ethyl alcohol；And/or

The dNTPs includes dATP, dGTP, dTTP, dCTP and dUTP, and UNG enzymes are further included in the kit.

A kind of application method of HPV parting kits, includes the following steps：

The nucleic acid of sample to be tested is extracted, obtains sample of nucleic acid；

The sample of nucleic acid is divided into it is multigroup, every group using the sample of nucleic acid as template, and with above-mentioned any embodiment Multigroup PCR amplification primer pair and corresponding detection probe carry out real-time fluorescent PCR amplification reaction respectively；

Fluorescence signal is detected in real-time fluorescent PCR amplification reaction process, obtains testing result.

The application method of the primer and probe of above-mentioned HPV partings detection, kit and kit can detect more simultaneously Kind HPV hypotypes by using real-time fluorescent PCR amplification technology, only need to carry out a nucleic acid extraction, each group also only needs carry out one Secondary PCR detection reaction, the application method of the kit is fast and convenient, and specificity is good, to other HPV hypotypes or other Microorganism etc. not will detect that, thus the problem of be less prone to false positive, and high sensitivity, up to 400copies/ml, inspection Survey scope is 400copies/ml~4.00E+09copies/ml, and detection range is wide.

Further, the application method of the kit and kit is utilized by optimizing combination to PCR reaction systems UNG enzymes can degrade DNA chain containing dU the characteristics of, UNG enzymes and dUTP are with the addition of in PCR system, previous PCR production can be prevented The pollution of object prevents pattern detection false positive.

Further, by increasing internal standard positive quality control, adopted by the betaglobulin detected in human epidermal cell to evaluate Whether the sample of collection is suitable for amplified reaction, and whether have PCR mortifier, thus can evaluate core if can monitor in sample to be tested The quality of acid extraction, avoids the problem that PCR false negatives.

It during real-time fluorescent PCR amplification, is gathered by real-time fluorescence, after PCR amplification, passes through curve shape And Ct values can be easy to judge the positive and negative of a variety of HPV hypotypes, testing result can be used for HPV infection auxiliary diagnosis and The observation of curative effect of medication, so as to provide reliable experimental basis for the research of HPV.

Description of the drawings

Fig. 1 is 16 sample positive test symbols of HPV；

Fig. 2 is 18 sample positive test symbols of HPV；

Fig. 3 is 33 sample positive test symbols of HPV；

Fig. 4 is 39 sample positive test symbols of HPV；

Fig. 5 is HPV 16,18,33 and 39 hypotype gradient testing results；

Fig. 6 is HPV 16,18,33 and 39 hypotype minimum detection limit testing results；

Fig. 7 is the pathogen detection result for easily causing the same or similar clinical symptoms；

Fig. 8 is HPV 16,18,33 and 39 hypotype precision R1 testing result figures；

Fig. 9 is HPV 16,18,33 and 39 hypotype precision R2 testing result figures；

Figure 10 is the dilution control amplification for being not added with any substance；

Figure 11 is interference experiment sample amplification curve diagram；

Figure 12 is the testing result without using the amplified production to positive sample and dUTP of dUTP and UNG enzymes；

Figure 13 is the testing result using the amplified production to positive sample and dUTP of dUTP and UNG enzymes.

Specific embodiment

For the ease of understanding the present invention, the present invention is described more fully below with reference to relevant drawings.In attached drawing Give presently preferred embodiments of the present invention.But the present invention can realize in many different forms, however it is not limited to this paper institutes The embodiment of description.On the contrary, the purpose for providing these embodiments is to make the understanding to the disclosure more thorough Comprehensively.

Unless otherwise defined, all of technologies and scientific terms used here by the article is with belonging to technical field of the invention The normally understood meaning of technical staff is identical.Term used in the description of the invention herein is intended merely to description tool The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases The arbitrary and all combination of the Listed Items of pass.

The primer and probe of the HPV parting detections of one embodiment includes the PCR amplification primer pair of multiple HPV hypotypes With corresponding detection probe, the PCR amplification primer pair of each HPV hypotypes and the sequence of corresponding detection probe are as follows：

HPV hypotypes	The sequence (F and R) of PCR amplification primer pair	The sequence (P) of corresponding detection probe
			16	SEQ ID NO:1 and SEQ ID NO:2	SEQ ID NO:3
18	SEQ ID NO:4 and SEQ ID NO:5	SEQ ID NO:6
			31	SEQ ID NO:7 and SEQ ID NO:8	SEQ ID NO:9
33	SEQ ID NO:10 and SEQ ID NO:11	SEQ ID NO:12
			35	SEQ ID NO:13 and SEQ ID NO:14	SEQ ID NO:15
39	SEQ ID NO:16 and SEQ ID NO:17	SEQ ID NO:18
			45	SEQ ID NO:19 and SEQ ID NO:20	SEQ ID NO:21
51	SEQ ID NO:22 and SEQ ID NO:23	SEQ ID NO:24
			52	SEQ ID NO:25 and SEQ ID NO:26	SEQ ID NO:27
53	SEQ ID NO:28 and SEQ ID NO:29	SEQ ID NO:30
			56	SEQ ID NO:31 and SEQ ID NO:32	SEQ ID NO:33
58	SEQ ID NO:34 and SEQ ID NO:35	SEQ ID NO:36
			59	SEQ ID NO:37 and SEQ ID NO:38	SEQ ID NO:39
66	SEQ ID NO:40 and SEQ ID NO:41	SEQ ID NO:42
			68	SEQ ID NO:43 and SEQ ID NO:44	SEQ ID NO:45
6	SEQ ID NO:46 and SEQ ID NO:47	SEQ ID NO:48
			11	SEQ ID NO:49 and SEQ ID NO:50	SEQ ID NO:51
40	SEQ ID NO:52 and SEQ ID NO:53	SEQ ID NO:54
			42	SEQ ID NO:55 and SEQ ID NO:56	SEQ ID NO:57
43	SEQ ID NO:58 and SEQ ID NO:59	SEQ ID NO:60
			44	SEQ ID NO:61 and SEQ ID NO:62	SEQ ID NO:63
54	SEQ ID NO:64 and SEQ ID NO:65	SEQ ID NO:66
			55	SEQ ID NO:67 and SEQ ID NO:68	SEQ ID NO:69
57	SEQ ID NO:70 and SEQ ID NO:71	SEQ ID NO:72
			67	SEQ ID NO:73 and SEQ ID NO:74	SEQ ID NO:75
73	SEQ ID NO:76 and SEQ ID NO:77	SEQ ID NO:78

In the present embodiment, the both ends of detection probe are marked with fluorescent reporter group and fluorescent quenching group respectively, excellent Choosing, fluorescent reporter group is located at 5 ' ends of detection probe, and fluorescent quenching group is located at 3 ' ends of detection probe.Multiple HPV are sub- It is different that type is divided into the fluorescent reporter group marked in the detection probe of multigroup different HPV hypotypes in same group.In an implementation In example, the one kind of the fluorescent reporter group in detection probe in FAM, HEX, VIC, CY5 and ROX in each group, fluorescence is quenched The group that goes out is BHQ1 or BHQ2.

In a specific embodiment, multiple HPV hypotypes are divided into seven groups.Wherein, HPV 16,18,33 is included for first group With 39 hypotypes, the fluorescent reporter group in corresponding detection probe can be but not limited to respectively FAM, HEX or VIC, CY5 and ROX；Second group includes HPV 45,59,35 and 66 hypotypes, the fluorescent reporter group in corresponding detection probe can respectively be but It is not limited to FAM, HEX or VIC, ROX and CY5；3rd group includes HPV 51,53,52 and 68 hypotypes, in corresponding detection probe Fluorescent reporter group can be but not limited to FAM, HEX or VIC, CY5 and ROX respectively；4th group includes 58,31,56 and of HPV Hypotype, the fluorescent reporter group in corresponding detection probe can be but not limited to HEX or VIC, CY5 and FAM respectively；5th group Including HPV 6,40,42 and 55 hypotypes, the fluorescent reporter group in corresponding detection probe can be but not limited to respectively FAM, HEX or VIC, ROX and CY5；6th group includes HPV 11,43,44 and 54 hypotypes, the fluorescence report base in corresponding detection probe Group can be but not limited to FAM, HEX or VIC, ROX and CY5 respectively；7th group includes HPV 67,57 and 73 hypotypes, corresponding Fluorescent reporter group in detection probe can be but not limited to HEX or VIC, FAM and ROX respectively.The PCR amplification primer of each group Pair with detection probe can be directly mixed together as mix reagent participate in subsequent reactions reagent preparation.

Further, in one embodiment, the primer and probe of the HPV parting detections further include internal standard positive quality control and Its PCR amplification primer pair and corresponding detection probe.Internal standard positive quality control is the cloned plasmids containing beta globin gene segment. The sequence of the PCR amplification primer pair of internal standard positive quality control such as SEQ ID NO:79 and SEQ ID NO:Shown in 80, corresponding detection The sequence of probe such as SEQ ID NO:Shown in 81.

In a specific embodiment, the cloned plasmids of the internal standard positive quality control may be employed but be not limited to following methods It is prepared：Corresponding beta globin gene segment is expanded using the primer containing restriction endonuclease sites sequence, and Insertion converts screening positive clone after Escherichia coli using in the empty carrier plasmid of similary digestion with restriction enzyme, extracts matter Grain to obtain the final product.

Further, in one embodiment, the PCR amplification primer pair of the internal standard positive quality control and corresponding detection probe Addition is in above-mentioned 4th group, and the fluorescent reporter group marked in the detection probe of internal standard positive quality control is CY5.By The cloned plasmids of beta globin gene segment are added in reaction system, it is anti-using internal standard monitoring sample collection, DNA extractions and PCR Overall process is answered, whether monitoring reaction system is effective, prevents pattern detection false negative.

Further, present embodiment additionally provides a kind of kit of HPV partings detection, including any of the above-described implementation The primer and probe of example.

In one embodiment, the kit of the HPV parting detections is further included by the specific gene containing each HPV hypotypes The positive reference substance that the cloned plasmids mixing of segment is formed.Preferably, the concentration of each positive HPV hypotypes is in the positive reference substance 1.00~5.00E+05copies/ml.

Further, in one embodiment, the kit of the HPV parting detections further includes negative controls.It is negative right Can be the reagent that various HPV partings are negative according to product, such as in a specific embodiment, which is sterilizing Physiological saline.

In one embodiment, the kit of the HPV parting detections further includes nucleic acid extracting reagent, PCR amplification buffering At least one of liquid, archaeal dna polymerase and dNTPs reagents.

Specifically, which is a kind of nucleic acid releasing agents, including：Buddhist Sha of 0.01~0.5mM/L is graceful, The dodecyl sodium sulfate and volumetric concentration that the potassium chloride of 50~200mM/L, mass-volume concentration are 0.01%~2% be 0.05%~1% ethyl alcohol.

In a specific embodiment, 10 × PCR amplification buffer solution includes the 200mmol/L trihydroxy methyl ammonia of pH 7.5 Methylmethane HCI solution, 20mmol/L magnesium chloride solutions, 500mmol/L Klorvess Liquids, 0.2% (volume/volume) Qula Logical solution and 10% (volume/volume) formamide solution.

Archaeal dna polymerase can be but not limited to hot start Taq polymerase, and concentration is 1U/ μ l~5U/ μ l.

In a specific embodiment, dNTPs includes dATP, dGTP, dTTP, dCTP and dUTP, further, the examination UNG enzymes (uracil dna glycosylase) are further included in agent box, concentration is 0.05U/ μ l~0.2U/ μ l.UNG enzymes have The function for the PCR product containing dU of degrading can play prevention PCR product using the dUTP in UNG enzymes and PCR reaction solution and pollute Effect.Further, in a specific embodiment, can archaeal dna polymerase be mixed composition enzyme mixation with UNG enzymes makes With.

In a preferred embodiment, above-mentioned seven groups can be directed to, prepares real-time fluorescence PCR reaction solution respectively, for Each group, such as can be (each by the dNTPs of 5 μ 10 × PCR amplifications of l buffer solutions, final concentration of 0.05mmol/L~0.2mmol/L The final concentration of dNTP is all 0.05mmol/L~0.2mmol/L), the amplification of each HPV hypotypes of 0.1 μm of ol/L~0.3 μm ol/L Primer, the detection probe of each HPV hypotypes of 0.1 μm of ol/L~0.3 μm ol/L, the internal standard sun of 0.05 μm of ol/L~0.2 μm ol/L The amplimer of property control, the detection probe of internal standard positive quality control of 0.05 μm of ol/L~0.2 μm ol/L mix composition Real-time fluorescence PCR reaction solution subsequently carries out blending reaction with nucleic acid extraction according to preset ratio.

In addition, present embodiment additionally provides a kind of application method of HPV parting kits, include the following steps：

The nucleic acid of sample to be tested is extracted, obtains sample of nucleic acid；

Sample of nucleic acid is divided into it is multigroup, every group using sample of nucleic acid as template, and with multigroup PCR of above-mentioned any embodiment Amplimer pair and corresponding detection probe carry out real-time fluorescent PCR amplification reaction respectively；

Fluorescence signal is detected in real-time fluorescent PCR amplification reaction process, obtains testing result.

The condition of real-time fluorescent PCR amplification reaction can according to the length of buffer solution salt ionic concentration and denaturing nucleic acid and Nucleotide composition, response feature and length nucleic acid etc. are specific to be determined and adjusts, such as in a preferred embodiment, can be according to such as Lower program carries out：

In one embodiment, the application method of the HPV parting kits further include using above-mentioned internal standard positive quality control and/ Or above-mentioned positive reference substance carries out the step of handling similary with sample to be tested.

Further, in one embodiment, the application method of the kit is further included using the moon such as the physiological saline of sterilizing Property reference substance carry out similary with sample to be tested the step of handling.

The application method of the primer and probe of above-mentioned HPV partings detection, kit and kit can detect more simultaneously Kind HPV hypotypes by using real-time fluorescent PCR amplification technology, only need to carry out a nucleic acid extraction, each group also only needs carry out one Secondary PCR detection reaction, the application method of the kit is fast and convenient, and specificity is good, to other HPV hypotypes or other Microorganism etc. not will detect that, thus the problem of be less prone to false positive, and high sensitivity, up to 400copies/ml, inspection Survey scope is 400copies/ml~4.00E+09copies/ml, and detection range is wide.

Further, the application method of the kit and kit is utilized by optimizing combination to PCR reaction systems UNG enzymes can degrade DNA chain containing dU the characteristics of, UNG enzymes and dUTP are with the addition of in PCR system, previous PCR production can be prevented The pollution of object prevents pattern detection false positive.

Further, by increasing internal standard positive quality control, adopted by the betaglobulin detected in human epidermal cell to evaluate Whether the sample of collection is suitable for amplified reaction, and whether have PCR mortifier, thus can evaluate core if can monitor in sample to be tested The quality of acid extraction, avoids the problem that PCR false negatives.

It during real-time fluorescent PCR amplification, is gathered by real-time fluorescence, after PCR amplification, passes through curve shape And Ct values can be easy to judge the positive and negative of a variety of HPV hypotypes, testing result can be used for HPV infection auxiliary diagnosis and The observation of curative effect of medication, so as to provide reliable experimental basis for the research of HPV.

It is specific embodiment part below.

1st, sample collection

The unknown samples such as wart surface cast-off cells, woman uterus epithelial cell, genital secretion.

Internal standard positive quality control：The cloned plasmids of betaglobulin target gene fragment, concentration are 1.00~5.00E+ 05copies/ml.In addition, the internal standard positive quality control of tetra- concentration of A, B, C, D is also sequentially prepared with TE buffer solutions, concentration difference For 1.00~5.00E+07copies/ml (A), 1.00~5.00E+06copies/ml (B), 1.00~5.00E+05copies/ Ml (C), 1.00~5.00E+04copies/ml (D).

Negative controls：The physiological saline of sterilizing.

Positive reference product：The cloned plasmids that each HPV hypotypes are positive mix, and collectively form positive with reference to product.

2nd, sample process

2.1 take out each component in kits, are placed at room temperature for, and treat its equalized temperature to room temperature, spare after mixing.

2.2 take 7 kinds of real-time fluorescence PCR reaction solution n × (38~44 μ l) respectively with 7 parts of n × (1~2 μ l) enzyme mixation (n =+two reference substances of sample number to be checked), fully it is mixed into PCR-mix, it is spare after brief centrifugation.

2.3 sample, quality-control product pre-process

2.3.1 sample to be tested：1ml sterile salines are added in into sample collection tube, mixing are fully vibrated, then complete Portion's liquid (sample elution liquid) pours into 1.5ml sterile centrifugation tubes (cotton swab abandons after being extracted by centrifugation tube wall), brief centrifugation 20~50 μ l are drawn afterwards into another 1.5ml sterile centrifugation tubes, are added in the above-mentioned nucleic acid releasing agents of 20~50 μ l, are made after abundant mixing It is spare for sample to be tested.

2.3.2 negative controls, positive reference substance take 20~50 μ l and 20~50 μ l nucleic acid releasing agent mixings for use respectively.

Traditional generally extracts the nucleic acid of pathogen microorganism using boiling method, i.e., first that secretion sample is dense Contracting, washing, then add lysate, it boils, high speed centrifugation, it is template to take supernatant.For concentrating this step, different institutions often concentrate Effect is different, it can be seen that precipitation, what is had can not see some, and see precipitation is because virus and albumen are all concentrated , it is difficult abundant mixing when can so cause to add in lysate below, and can not see precipitation, it will make operator that can not determine Suction can or can not blow and beat viral nucleic acid when abandoning supernatant.The method of the present embodiment selection nucleic acid release, using strong albuminous degeneration Agent, rapid damage pathogen coat protein structure, releases pathogen nucleic acid, and the release of DNA can be completed without heating and carry It takes, it is simple, quick.

2.4 sample-addings (negative controls, positive reference substance and sample to be tested synchronization process)

2.4.1 treated sample to be tested, negative control, positive control each 4~10 is added in each PCR reaction tubes μ l, need to be repeated 7 times that (i.e. each sample takes the mixed liquor of 4~10 μ l samples and nucleic acid releasing agent to add in same eight connecting leg respectively 7 reaction tubes in, be careful not to obscure.).

2.4.2 it is spaced 10 minutes or more, each sample to be tested, negative controls, 7 reaction tubes of positive reference substance add respectively Enter the above-mentioned prepared 7 kinds of PCR-mix of 40~45 μ l, lid upper tube cap.

3rd, real-time fluorescent PCR amplification is reacted

1) PCR reaction tubes are put into the sample cell of real-time fluorescent PCR amplification instrument, sample to be tested title is set by corresponding order And qualitative reference product concentration.

2) fluorescence detection channel selects：Select FAM passages (Reportere:FAM,Quencher:None) detection detection HPV6、11、16、45、51、56、57-DNA；Select HEX or VIC passages (Reporter:VIC,Quencher:None) detect HPV18、40、43、53、58、59、67-DNA；Select ROX passages (Reporter:ROX,Quencher:None) detect HPV35, 39、42、44、68、73-DNA；Select CY5 passages (Reporter:CY5,Quencher:None) detect HPV31,33,52,54, 55、66-DNA；Reference fluorescent (Passive Reference) is arranged to none.

3) quantitative fluorescent PCR reaction condition is：

4th, interpretation of result

After reaction, instrument automatically save as a result, the software that can be carried using instrument automatically analyzed (can also Initial value, end value and the threshold line value for adjusting baseline manually are analyzed), then record sample Ct values and definite value result. The intersection point of amplification curve and threshold line, being known as Ct, (i.e. cycle threshold refer to the fluorescence signal in PCR reaction tubes and reach and set The cycling numerical value undergone during fixed threshold value)；Instrument software is quantitatively joined according to each sample Ct value sizes by 4 concentration gradients The standard curve of product drafting is examined, the definite value result of each sample can be acquired automatically.And according to the form below carries out result judgement：

To above 1~7 kind of HPV PCR- mixed liquor FAM, HEX, ROX, CY5 passages, there is passage to measure Ct value≤39, and β- The sample of globulin test positive (Ct value≤40) reports that corresponding HPV types are positive.

To above 1~7 kind of HPV PCR- mixed liquor FAM, HEX, ROX, CY5 passages, there is passage to measure Ct value≤39, still Betaglobulin is detected as negative (Ct values>40 or without display) sample, show without cervical epithelial cells in sample, but the patient HPV viruse was contacted in the recent period, whether patient, which infects HPV, not can determine that.It is tested again it is recommended that being resampled to this sample, This phenomenon is such as repeated, then reports that corresponding HPV types are positive.

To above 1~7 kind detection mixed liquor FAM, HEX, ROX, CY5 passage, Ct values are measured all>39, and betaglobulin is examined It surveys as the sample of positive (Ct value≤40), report HPV (26 kinds of types) feminine genders.

To above 1~7 kind detection mixed liquor FAM, HEX, ROX, CY5 passage, Ct values are measured all>39, if betaglobulin is examined Survey Ct values>40 or without display, then the testing result of the sample is invalid, should search and exclude reason, and this sample is carried out again Sampling is tested again.

Using the present invention kit detect National Institute for Food and Drugs Control HPV full-length genome partings reference material, Enterprise work reference material, yin and yang attribute reference material coincidence rate are 100%, and the testing result of sensitivity reference material meets quality standard.

In precision test shows batch and batch between reproducible, the coefficient of variation of testing result Ct values<10%, concentration becomes Different coefficient<50%.

DNA Different Extraction Methods to the HPV-DNA influences detected experiments have shown that：It is sent out by detecting gradient dilution sample simultaneously Existing, the nucleic acid quick release method of the present embodiment and the testing result of boiling method extraction nucleic acid do not have a notable difference, and the present embodiment Method operation is more easy to be quick, without heating.Meanwhile boiling method reagent does not have internal standard monitoring, has PCR in such as DNA of extraction In the presence of mortifier, HPV positive samples can be caused to be detected as feminine gender, that is, false negative, the quick detection reagent of the present embodiment occur Internal standard is added in box, it can be with the presence of effective monitoring false negative.

HPV16, HPV18, HPV33, HPV39 positive sample are chosen, using the reagent of quality inspection qualification, in same SLAN- It is detected on 96P instruments, it is the positive to detect each type, as a result as shown in Figure 1 to 4.

Plasmid gradient detects：HPV16, HPV18, HPV33 and HPV39 type plasmid concentration 1E6copies/ml are chosen, by 4 kinds Plasmid mixing after gradient dilution concentration be about (4.00E+05,4.00E+04,4.00E+03,4.00E+02copies/ml, It is detected on SLAN-96P instruments, detecting each type gradient, the results are shown in Figure 5.

As shown in fig. 6, HPV16, HPV18, HPV33 and HPV39 concentration are 4.00E+02copies/ml, pass through 20 weights Reinspection is looked into, and positive rate is 100%.Therefore, 4.00E+02copies/ml can be as the minimum detection limit of this kit. Further experiment verification shows the detection range of the quick detection kit of the present embodiment up to 400copies/ml~4.00E+ 09copies/ml, detection range are wide.

Specific detection：Easily to cause the pathogen of the same or similar clinical symptoms, (chlamydia trachomatis, solution urea branch are former Body, gonococcus, herpes simplex virus type II, microspironema pallidum, Candida albicans, trichomonas vaginalis, mycoplasma hominis, other 26/61/69/70/71/81/82/83 grade of HPV genotype (national human papilloma virus full-length genome parting reference material) is to be measured Sample is detected using qualified parting kit on same SlAN-96P instrument, by analyzing the yin and yang attribute of testing result, Examine or check the specificity of kit.Specific test shows and common venereal diseases pathogen (CT, NG, HSV, UU etc.), alloytype HPV (61/69/70/71/81/82/83 etc.) no cross reaction.As seen from Figure 7, the same or similar clinical symptoms are easily caused Pathogen (chlamydia trachomatis, Ureaplasma urealyticum, gonococcus, herpes simplex virus type II, microspironema pallidum, Candida albicans, the moon Road trichmonad, mycoplasma hominis, the qualified examination of other HPV genotype (national human papilloma virus full-length genome parting reference material) inspection Agent detects, and internal standard is normal, and type is feminine gender.In conclusion this kit has specificity well, it is anti-that intersection will not be generated It should.

Precision detects：By taking HPV16, HPV18, HPV33 and HPV39 type as an example, it is (1E6copies/ml) to choose concentration Plasmid, be diluted two concentration levels of 2.00E+05copies/ml, 1.00E+03copies/ml respectively as treating test sample This, using the kit of the present embodiment, is detected on SLAN-96P instruments, and each sample is repeated 10 times, and investigates precision Performance.As a result as shown in Figure 8 and Figure 9.

Interference experiment：With minimum detection limit HPV16, HPV18, HPV33 and HPV39 sample, added respectively into dilution Interfering material, hemoglobin, leucocyte, cervical mucus, contraception glue, vaginal douche, antifungal ointment, containing 2% clotrimazole, resists Fungi ointment, containing 4% Miconazole, Astroglide, and not add the dilution of any substance as control.Containing various above-mentioned Detection kit of the sample of interfering material through quality inspection qualification is repeated 3 times, and testing result is as shown in Figure 10 and Figure 11.By above-mentioned knot Fruit understands that it is the positive that detection kit of the sample containing various above-mentioned interfering materials through 3 batches of quality inspection qualifications, which is repeated 3 times detection, And with the Ct value differences of check sample away within 1.96SD, therefore, it can be stated that it is bright under the experiment condition of this kit, in sample Interfering material (2g/L hemoglobins, 1.00E+07cells/mL leucocytes, 10% cervical mucus, 0.5% contraception that may be present Glue, 0.5% vaginal douche, 0.5% antifungal ointment, containing 2% clotrimazole, 0.5% antifungal ointment, containing 4% Miconazole, 10% Astroglide) to the testing result of kit without significantly interfering with.

Further, the dosage of UNG enzymes is respectively 0,0.2U during this experimental formula is per person-portion reaction system, with the expansion containing dUTP Increase production object and clinical sample carry out contrast test, experimental result is as shown in Figures 12 and 13, the results showed that, in PCR reaction systems PCR product pollution can be prevented by adding in suitable UNG enzymes.

Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, the scope that this specification is recorded all is considered to be.

Embodiment described above only expresses the several embodiments of the present invention, and description is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that come for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.

Sequence table

<110>Hunan Shengxiang Biological Technology Co., Ltd.

<120>The application method of the primer and probes of HPV parting detections, kit and kit

<160> 81

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

caagtgtgac tctacgcttc gg 22

<210> 2

<211> 24

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

acaattccta gtgtgcccat taac 24

<210> 3

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

atgtctacgt gtgtgctttg tacgca 26

<210> 4

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

atagaggact ccagtgtggt tacat 25

<210> 5

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

ttgtggaaat agacacagag gtaga 25

<210> 6

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

cctacgttta ctggcacgtc tgggt 25

<210> 7

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

gttatacaag atggggatat ggttg 25

<210> 8

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

gaattacaaa tgtccaaagg aacat 25

<210> 9

<211> 28

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

caggctttgg agctatggat tttactgc 28

<210> 10

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

caacattgaa ctacagtgcg tgg 23

<210> 11

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

agtttacata ttccaaatgg atttcc 26

<210> 12

<211> 28

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

caacgatctg aggtatatga ttttgcat 28

<210> 13

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 13

ccgctgtgtc cagttgaaaa g 21

<210> 14

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 14

tggtttccaa caggacatac acc 23

<210> 15

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 15

cataacatcg gtggacggtg gac 23

<210> 16

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 16

ggtagtagaa gcctcacggg at 22

<210> 17

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 17

ctggtttgca gttgcacacc 20

<210> 18

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 18

acggacacac aaatcctagt gagtcc 26

<210> 19

<211> 24

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 19

acgacggttg tttacaatat caga 24

<210> 20

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 20

tacactgccg ccattttcc 19

<210> 21

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 21

acagttacct gagtctctgc agcttcc 27

<210> 22

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 22

gtattgcatt taacaccaca gactga 26

<210> 23

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 23

cctgtccagc ccgtctttct 20

<210> 24

<211> 29

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 24

cacgcatatt atctacttca tcctcctcc 29

<210> 25

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 25

ccatgtttga ggatccagca ac 22

<210> 26

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 26

cagccttatt tcatgcaccg a 21

<210> 27

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 27

cttccagcac ctcacacaat tcgtg 25

<210> 28

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 28

acagacgagg aaagcaccg 19

<210> 29

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 29

ggcatgtaac aactgctgag c 21

<210> 30

<211> 28

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 30

tgtctccctt tctgcctgtg tagatatt 28

<210> 31

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 31

gttgatggca agcaaacaca g 21

<210> 32

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 32

cagtcccctg tggtaacttg tg 22

<210> 33

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 33

ttggatgtac tcccgctatg ggtgaa 26

<210> 34

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 34

agcaaaaata gtaaaagact gtggc 25

<210> 35

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 35

ccatcatttg ttttttcaca cctac 25

<210> 36

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 36

aagagcagaa aagcgtggta tgacaa 26

<210> 37

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 37

agctagacga gctgaaccac ag 22

<210> 38

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 38

gtaaggctcg caatccgtct 20

<210> 39

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 39

cgtcacaaca ttgtgtgtgt gtgttg 26

<210> 40

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 40

acaattcatt tgatggccta tatga 25

<210> 41

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 41

gcaaaggata atgtagaagg tttaa 25

<210> 42

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 42

atgcaaatat tgatgatgag gcacc 25

<210> 43

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 43

gtgccctaaa acgaaagtat acaga 25

<210> 44

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 44

ggttgtcttg cctgtgtact gc 22

<210> 45

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 45

ttcctgtaat ggcgactttg ctaaagg 27

<210> 46

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 46

acaaaccgct gtgtgaagta gaa 23

<210> 47

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 47

tgttgtccag cagtgtaggc a 21

<210> 48

<211> 24

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 48

cgacccttcc acgtacaatt tagc 24

<210> 49

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 49

cacgcttcat aaaactaaat aaccag 26

<210> 50

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 50

tcctttaggg taacaagtct tccat 25

<210> 51

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 51

atgttgtcca gcagtgtaag caacgac 27

<210> 52

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 52

ctgcgtttat ggacatcatt acc 23

<210> 53

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 53

cccaccctgc taaagcgtat 20

<210> 54

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 54

cccgccgaga cgttaatgc 19

<210> 55

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 55

gatttggtag tggtgtggag ga 22

<210> 56

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 56

aatgctgatc tttcgtagtg tcg 23

<210> 57

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 57

atgcacatgc agcatatgga aagtcc 26

<210> 58

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 58

ggcactttga ctacgcagca t 21

<210> 59

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 59

ctgtactttt tccactggtg ataatg 26

<210> 60

<211> 28

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 60

cacttacagc atctaatgca caaatcaa 28

<210> 61

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 61

gacccgtcca ttgtatcctt g 21

<210> 62

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 62

tctaatatag ctggtgtggt agattca 27

<210> 63

<211> 24

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 63

ttcccatgca ggatttgaaa tcac 24

<210> 64

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 64

tagcacgcac acaggcataa g 21

<210> 65

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 65

aagcacaggt gggacacact att 23

<210> 66

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 66

ctgatgaagc agttcctgca gtacc 25

<210> 67

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 67

ccctactacg caacgcctg 19

<210> 68

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 68

gcccactaat accaacaccc a 21

<210> 69

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 69

cctctaccca cttccaaccc aat 23

<210> 70

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 70

tagaggattt gagaatactg tgcgt 25

<210> 71

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 71

ggaatccctt tctccacact aca 23

<210> 72

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 72

ttcctttact gcaaatgcca gca 23

<210> 73

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 73

gttggacagg acggtgttca g 21

<210> 74

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 74

tgtctccacg catggcttc 19

<210> 75

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 75

acctcaacga acgcagaccc ag 22

<210> 76

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 76

gctgaacgag agtgttacag aatagt 26

<210> 77

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 77

ttttataggt ttctggaaca gttgg 25

<210> 78

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 78

cacagtatgc cttgccattg aaagc 25

<210> 79

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 79

cctgagaact tcagggtgag tctat 25

<210> 80

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 80

attcgtctgt ttcccattct aaact 25

<210> 81

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 81

cccttgatgt tttctttccc cttct 25

Claims (10)

1. a kind of primer and probe of HPV partings detection, which is characterized in that include the PCR amplification primer pair of multiple HPV hypotypes With corresponding detection probe, the PCR amplification primer pair of each HPV hypotypes and the sequence of corresponding detection probe are as follows：

The both ends of the detection probe are marked with fluorescent reporter group and fluorescent quenching group respectively, and multiple HPV hypotypes are divided into more Group, the fluorescent reporter group marked in the detection probe of the different HPV hypotypes in same group are different.

2. the primer and probe of HPV partings detection as described in claim 1, which is characterized in that multiple HPV hypotypes are divided into seven Group；Wherein, HPV 16,18,33 and 39 hypotypes are included for first group；Second group includes HPV 45,59,35 and 66 hypotypes；3rd group Including HPV 51,53,52 and 68 hypotypes；4th group includes HPV 58,31,56 and hypotype；5th group includes 6,40,42 and of HPV 55 hypotypes；6th group includes HPV 11,43,44 and 54 hypotypes；7th group includes HPV 67,57 and 73 hypotypes.

3. the primer and probe of HPV partings detection as claimed in claim 1 or 2, which is characterized in that the inspection in each group The one kind of fluorescent reporter group in FAM, HEX, VIC, CY5 and ROX on probing pin, the fluorescent quenching group are BHQ1 Or BHQ2.

4. the primer and probe of HPV partings detection as claimed in claim 3, which is characterized in that corresponding in first group Fluorescent reporter group in detection probe is respectively FAM, HEX or VIC, CY5 and ROX；

The fluorescent reporter group in corresponding detection probe in second group is respectively FAM, HEX or VIC, ROX and CY5；

The fluorescent reporter group in corresponding detection probe in 3rd group is respectively FAM, HEX or VIC, CY5 and ROX；

The fluorescent reporter group in corresponding detection probe in 4th group is respectively HEX or VIC, CY5 and FAM；

The fluorescent reporter group in corresponding detection probe in 5th group is respectively FAM, HEX or VIC, ROX and CY5；

The fluorescent reporter group in corresponding detection probe in 6th group is respectively FAM, HEX or VIC, ROX and CY5；

The fluorescent reporter group in corresponding detection probe in 7th group is respectively HEX or VIC, FAM and ROX.

5. the primer and probe of HPV partings detection as claimed in claim 4, which is characterized in that further include internal standard positive matter Control and its PCR amplification primer pair and corresponding detection probe；The internal standard positive quality control is gram containing beta globin gene segment Grand plasmid；The sequence of the PCR amplification primer pair of the internal standard positive quality control such as SEQ ID NO:79 and SEQ ID NO:Shown in 80, The sequence of corresponding detection probe such as SEQ ID NO:Shown in 81.

6. the primer and probe of HPV partings detection as claimed in claim 5, which is characterized in that the internal standard positive quality control PCR amplification primer pair and corresponding detection probe add in described 4th group, and the internal standard positive quality control detection visit The fluorescent reporter group marked on pin is CY5.

7. a kind of kit of HPV partings detection, which is characterized in that draw including such as according to any one of claims 1 to 6 Object and probe.

8. the kit of HPV partings detection as claimed in claim 7, which is characterized in that further include nucleic acid extracting reagent, At least one of PCR amplification buffer solution, archaeal dna polymerase and dNTPs reagents.

9. the kit of HPV partings detection as claimed in claim 8, which is characterized in that the nucleic acid extracting reagent includes： Buddhist Sha of 0.01~0.5mM/L is graceful, 50~200mM/L potassium chloride, the dodecyl that mass-volume concentration is 0.01%~2% Sodium sulfonate and the ethyl alcohol that volumetric concentration is 0.05%~1%；And/or

The dNTPs includes dATP, dGTP, dTTP, dCTP and dUTP, and UNG enzymes are further included in the kit.

10. a kind of application method of HPV parting kits, which is characterized in that include the following steps：

The nucleic acid of sample to be tested is extracted, obtains sample of nucleic acid；

The sample of nucleic acid is divided into it is multigroup, every group using the sample of nucleic acid as template, and with any in claim 7~9 Multigroup PCR amplification primer pair and corresponding detection probe described in carry out real-time fluorescent PCR amplification reaction respectively；

Fluorescence signal is detected in real-time fluorescent PCR amplification reaction process, obtains testing result.