CN107641662A

CN107641662A - For detecting the primer and probe and kit of high-risk human mammilla papillomavirus oncogene E6/E7mRNA partings

Info

Publication number: CN107641662A
Application number: CN201710628952.1A
Authority: CN
Inventors: 顾月清; 张万存; 张嘉琪
Original assignee: China Pharmaceutical University
Current assignee: BEIJING ZHONGSHENG JINYU DIAGNOSIS TECHNOLOGY CO., LTD.
Priority date: 2017-07-28
Filing date: 2017-07-28
Publication date: 2018-01-30

Abstract

The present invention relates to for detecting a kind of viral primer and probe and kit, the primer and probe and kit of high-risk human mammilla papillomavirus oncogene E6/E7 mRNA partings are more particularly, to detected, belongs to technological field of biochemistry.The present invention cleverly designs specific upstream and downstream primer and corresponding specific molecular beacon probe by using oncogene E6/E7 and internal reference the β Actin to 13 kinds of high-risk HPVs, carries out specific amplification to 13 kinds of high-risk HPVs and internal reference β Actin and high specific detects.The present invention can be used to carry out HPV oncogene E6/E7 mRNA real-time quantitative and parting detects, and molecular beacon probe can also be used 13 kinds of high-risk HPVs are carried out with the detection of qualitative and parting in PCR terminals.HPV16,18,31,33,35,39,45,51,52,56,58,59,68 that 13 kinds of high-risk HPVs refer in the present invention.The present invention is used as detection target using HPV oncogene E6/E7 mRNA, it can not only effectively overcome traditional using HPV L1 DNA as the false positive and false negative result caused by detection method, while the molecular beacon probe utilized in the present invention has that detection sensitivity is high, specificity is high, simple operation and other advantages.

Description

For detecting drawing for high-risk human mammilla papillomavirus oncogene E6/E7 mRNA partings Thing and probe and kit

Technical field

The present invention relates to for detecting a kind of viral primer and probe and kit, high-risk-type is more particularly, to detected The primer and probe and kit of human papilloma virus E6/E7 mRNA partings, belong to technological field of biochemistry.

Background technology

Cervical carcinoma (cervical carcinoma) is one of women common cancer, and it is uniquely to find out so far Nosogenetic cancer, it is by single hypotype high-risk-type human papilloma virus (Human Papilloma virus, abbreviation HPV) caused by persistent infection.

HPV belongs to the papilloma vacuolating virus A category of papovaviridae, is spherical DNA viruses, can cause human body skin The scaly epithelium propagation of mucous membrane.Research show more than 99% cervical carcinoma be due to infection HPV caused by.HPV presses infection portion Position classification can be divided into epitheliated type and mucosal pattern, and wherein epitheliated type includes 1,5,8,14,20,21,25,47 types, and mucous membrane type includes 6th, 11,16,18,31,33,35,39,41,45,51,52,56,58,59,68,70 type；It can be divided into according to degree of danger high-risk Type and low risk, wherein low risk include 6,11,41,42,43,44,66 types, high-risk-type includes 16,18,31,33,35,39, 45th, 51,52,56,58,59,68 type, easily causes cervical carcinoma.HPV include six early stage controlling genes (E1, E2, E4, E5, E6, ) and two late genes (L1, L2) E7.Early gene participates in the functions such as duplication, transcription, translation and the cell transformation of viral DNA, And the function of late gene is the capsid protein of coding virus.Wherein two early genes of E6, E7 inhibit the suppression of tumour respectively Oncogene P53 and Rb and direct relation be present in generation with cervical carcinoma, development.

Research shows that at least 50% adult for having sexuality once infects HPV in life at it, and at least 80% women exists HPV is once infected before 50 years old.But 80%HPV the infected can voluntarily disappear in 2 years, if this groups of people is used as inspection using HPV DNA Survey target, it will be positive.Only 20% HPV infection person can further develop, i.e. E6/E7 gene integrations to human genome By being translated as E6/E7 albumen after transcribing out E6/E7 mRNA, precancerous lesion and cervical carcinoma are induced.So the present invention is with HPV E6/E7 mRNA are as detection target.

The probe commonly used in quantitative fluorescent PCR is TaqMan probe and molecular beacon probe.Wherein TaqMan probe is deposited Relatively low in Tm values, heat endurance is weak, the problems such as hybrid specificities are low, easily causes quantitative fluorescent PCR final detection result and vacation occurs Positive situation.At present, a domestic only HPV E6/E7 mRNA kits manufacturer, should using hybrid capture The method operating time is grown, and sensitivity is low and cannot be distinguished by specific type.

The content of the invention

The purpose of the present invention is to be directed to the defects of testing result existing for prior art is inaccurate, and sensitivity is low, proposes to use In the primer and probe and kit of detection high-risk human mammilla papillomavirus E6/E7 mRNA partings, testing result has preferable Clinical reference value.

Present invention firstly provides be primer and spy for detecting high-risk human mammilla papillomavirus E6/E7 mRNA partings Pin group, the primer include 13 kinds of high-risk HPVs and internal reference β-Actin upstream and downstream primer and molecular beacon probe probe groups, It is specific as follows：

HPV16 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer Such as the SEQ ID NO.1 in sequence table, the SEQ ID NO.2 in anti-sense primer such as sequence table, the SEQ in probe such as sequence table ID NO.3；Second group, the SEQ ID NO.4 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.6 in NO.5, probe such as sequence table；3rd group, the SEQ ID NO.7 in sense primer such as sequence table, downstream SEQ ID NO.8 in primer such as sequence table, the SEQ ID NO.9 in probe such as sequence table；

HPV18 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer Such as the SEQ ID NO.10 in sequence table, the SEQ ID NO.11 in anti-sense primer such as sequence table, the SEQ in probe such as sequence table ID NO.12；Second group, the SEQ ID NO.13 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.15 in NO.14, probe such as sequence table；3rd group, the SEQ ID NO.16 in sense primer such as sequence table, under Swim the SEQ ID NO.17 in primer such as sequence table, the SEQ ID NO.18 in probe such as sequence table；

HPV31 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer Such as the SEQ ID NO.19 in sequence table, the SEQ ID NO.20 in anti-sense primer such as sequence table, the SEQ in probe such as sequence table ID NO.21；Second group, the SEQ ID NO.22 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.24 in NO.23, probe such as sequence table；3rd group, the SEQ ID NO.25 in sense primer such as sequence table, under Swim the SEQ ID NO.26 in primer such as sequence table, the SEQ ID NO.27 in probe such as sequence table；

HPV33 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer Such as the SEQ ID NO.28 in sequence table, the SEQ ID NO.29 in anti-sense primer such as sequence table, the SEQ in probe such as sequence table ID NO.30；Second group, the SEQ ID NO.31 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.33 in NO.32, probe such as sequence table；3rd group, the SEQ ID NO.34 in sense primer such as sequence table, under Swim the SEQ ID NO.35 in primer such as sequence table, the SEQ ID NO.36 in probe such as sequence table；

HPV35 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer Such as the SEQ ID NO.37 in sequence table, the SEQ ID NO.38 in anti-sense primer such as sequence table, the SEQ in probe such as sequence table ID NO.39；Second group, the SEQ ID NO.40 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.42 in NO.41, probe such as sequence table；3rd group, the SEQ ID NO.43 in sense primer such as sequence table, under Swim the SEQ ID NO.44 in primer such as sequence table, the SEQ ID NO.45 in probe such as sequence table；

HPV39 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer Such as the SEQ ID NO.46 in sequence table, the SEQ ID NO.47 in anti-sense primer such as sequence table, the SEQ in probe such as sequence table ID NO.48；Second group, the SEQ ID NO.49 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.51 in NO.50, probe such as sequence table；3rd group, the SEQ ID NO.52 in sense primer such as sequence table, under Swim the SEQ ID NO.53 in primer such as sequence table, the SEQ ID NO.54 in probe such as sequence table；

HPV45 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer Such as the SEQ ID NO.55 in sequence table, the SEQ ID NO.56 in anti-sense primer such as sequence table, the SEQ in probe such as sequence table ID NO.57；Second group, the SEQ ID NO.58 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.60 in NO.59, probe such as sequence table；3rd group, the SEQ ID NO.61 in sense primer such as sequence table, under Swim the SEQ ID NO.62 in primer such as sequence table, the SEQ ID NO.63 in probe such as sequence table；

HPV51 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer Such as the SEQ ID NO.64 in sequence table, the SEQ ID NO.65 in anti-sense primer such as sequence table, the SEQ in probe such as sequence table ID NO.66；Second group, the SEQ ID NO.67 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.69 in NO.68, probe such as sequence table；3rd group, the SEQ ID NO.70 in sense primer such as sequence table, under Swim the SEQ ID NO.71 in primer such as sequence table, the SEQ ID NO.72 in probe such as sequence table；

HPV52 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer Such as the SEQ ID NO.73 in sequence table, the SEQ ID NO.74 in anti-sense primer such as sequence table, the SEQ in probe such as sequence table ID NO.75；Second group, the SEQ ID NO.76 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.78 in NO.77, probe such as sequence table；3rd group, the SEQ ID NO.79 in sense primer such as sequence table, under Swim the SEQ ID NO.80 in primer such as sequence table, the SEQ ID NO.81 in probe such as sequence table；

HPV56 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer Such as the SEQ ID NO.82 in sequence table, the SEQ ID NO.83 in anti-sense primer such as sequence table, the SEQ in probe such as sequence table ID NO.84；Second group, the SEQ ID NO.85 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.87 in NO.86, probe such as sequence table；3rd group, the SEQ ID NO.88 in sense primer such as sequence table, under Swim the SEQ ID NO.89 in primer such as sequence table, the SEQ ID NO.90 in probe such as sequence table；

HPV58 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer Such as the SEQ ID NO.91 in sequence table, the SEQ ID NO.92 in anti-sense primer such as sequence table, the SEQ in probe such as sequence table ID NO.93；Second group, the SEQ ID NO.94 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.96 in NO.95, probe such as sequence table；3rd group, the SEQ ID NO.97 in sense primer such as sequence table, under Swim the SEQ ID NO.98 in primer such as sequence table, the SEQ ID NO.99 in probe such as sequence table；

HPV59 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer Such as the SEQ ID NO.100 in sequence table, the SEQ ID NO.101 in anti-sense primer such as sequence table, probe is as in sequence table SEQ ID NO.102；Second group, the SEQ ID NO.103 in sense primer such as sequence table, anti-sense primer is as in sequence table SEQ ID NO.104, the SEQ ID NO.105 in probe such as sequence table；3rd group, the SEQ ID in sense primer such as sequence table SEQ ID NO.107 in NO.106, anti-sense primer such as sequence table, the SEQ ID NO.108 in probe such as sequence table；

HPV68 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer Such as the SEQ ID NO.109 in sequence table, the SEQ ID NO.110 in anti-sense primer such as sequence table, probe is as in sequence table SEQ ID NO.111；Second group, the SEQ ID NO.112 in sense primer such as sequence table, anti-sense primer is as in sequence table SEQ ID NO.113, the SEQ ID NO.114 in probe such as sequence table；3rd group, the SEQ ID in sense primer such as sequence table SEQ ID NO.116 in NO.115, anti-sense primer such as sequence table, the SEQ ID NO.117 in probe such as sequence table；

SEQ ID NO.118 in β-Actin sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.120 in NO.119, probe such as sequence table.

The probe groups also include the reverse complementary sequence in all molecular beacon probe areas.

The present invention further provides the kit for detecting high-risk human mammilla papillomavirus E6/E7 mRNA partings, institute State kit and contain the μ L of reverse transcription premixed liquid 300；QRT-PCR premixed liquids 1.6mL；13 kinds of high-risk HPVs and internal reference β-Actin Upstream and downstream primer and probe groups, each of which HPV hypotypes and internal reference β-Actin upstream and downstream primer are 10 μ Μ, 160 μ L, Molecular beacon probe is 10 μ Μ, 100 μ L；The μ L of strong positive quality-control product 500；The μ L of weakly positive quality-control product 500 and negative controls 500μL。

The reverse transcription premixed liquid in mentioned reagent box includes 100-300nM dNTP, 5-20U/ μ L reverse transcription Enzyme, 0.3-1U/ μ L RNase inhibitors, random primer and the sulphur of poly T primers and 50mM pH8.3 Tris-HCl, 6mM bis- Soviet Union Sugar alcohol, wherein 40mM KCl, 0.5mM dTTP composition, random primer are that tetra- kinds of bases of A, G, C, T form at random 6 aggressiveness, poly T is 18-25 poly T base composition.

The qRT-PCR premixed liquids by 200-500nM dNTP, 2-10mM Mg²⁺, concentration be 0.1-0.5U/ μ L Taq archaeal dna polymerases, concentration be 0.01-0.02U/ μ L UNG enzymes, 20mM pH8.3 Tris-Hcl and 100mM KCl groups Into.

The primer and probe of the kit can be visited by HPV16 primed probes group, HPV18 primed probes group, β-Actin primers Pin group and a kind of zero to ten other high-risk HPVs (HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68) random combine as needed.

The fluorophor of the 5' ends mark of the molecular beacon is appointing in organic fluorescent dye or quantum dot inorganic dyestuff One kind, 3' ends mark quencher are the nano-particles such as any or gold nano of organic dyestuff.

The organic fluorescent dye is FAM, VIC, HEX, TRT, Cy3, Cy5, ROX and JOE, and the organic dyestuff is TAMRA, DABCYL, MGB, BHQ-1, BHQ-2 and BHQ-3.

Positive quality control product is by the high-risk HPV DNA fragmentation in acknowledged detection range containing kit is sequenced The RNA or its complementary series that plasmid transcription goes out, the wherein concentration of strong positive quality-control product are 1 × 10⁷Copies/ μ L, weakly positive matter The concentration of control product is 1 × 10³ copies/μL。

The negative controls are the water of DEPC processing.

For the present invention by the use of random primer and poly T as the primer of reverse transcription, it is cDNA that can reverse total mRNA, is only needed Once reverse the detection for being applicable to all models other HPV and reference gene β-Actin.Detection is used as using HPV E6/E7 mRNA Target, there is more preferable clinical reference value compared with using HPV DNA as detection target object detection method.Simultaneously by skilful Wonderful design HPV oncogene E6/E7 and internal reference β-Actin specific upstream and downstream primer and corresponding specific molecular letter Probe is marked, parting detection is carried out to high-risk HPV using qRT-PCR as detection instrument.Molecular beacon probe can be fine simultaneously The shortcomings that overcoming qRT-PCR often to use probe Taqman probes (Tm values are relatively low, and heat endurance is weak, and hybrid specificities are low etc.).This The invention kit advantage such as have high sensitivity, specificity, precision, simple to operate and cost low, is suitable for pushing away on a large scale Wide application.

Brief description of the drawings

Fig. 1 is HPV16 amplification curve diagram (A)；HPV16 canonical plotting (B).

Fig. 2 is that HPV18 mRNA detect amplification curve diagram in Hela cells.

Fig. 3 this kit Precision Experiment figure (A) amplification curve diagram；(B) Ct values summary view.

Embodiment

A kind of 13 kinds of high-risk human mammilla papillomavirus mRNA detection kits are provided in the present embodiment, can be to 13 kinds of height Danger type human papilloma virus E6/E7 mRNA are detected, and testing result can be used for the auxiliary diagnosis and uterine neck of clinical HPV infection The early screening of cancer.By taking HPV16 as an example, other hypotypes prepare identical the present embodiment with HPV16, repeat no more.

Specifically carry out according to the following steps：

1. pair 13 kinds of high-risk HPV E6/E7 gene orders inquiries：

It is high-risk by 13 kinds of National Center for Biotechnology Information (NCBI) inquiries Type HPV E6/E7 gene orders, names of the wherein HPV16 in GenBank are NC_001526.4, and HPV16 E6 correspond to base Position corresponds to base positions in 7604-7900 in 7125-7601, HPV16 E7；HPV18 names in GenBank are AY262282., HPV18 E6 correspond to base positions and correspond to base positions in 590-907 in 105-581, HPV18 E7；HPV31 Name is J04353.1 in GenBank, and HPV31 E6 correspond to base positions and correspond to base positions in 108-557, HPV31 E7 In 560-856；Wherein HPV33 names in GenBank are M12732.1, and HPV33 E6 correspond to base positions in 109-558, HPV33 E7 correspond to base positions in 573-866；HPV35 names in GenBank are M74117.1, and HPV35 E6 correspond to alkali Base location corresponds to base positions in 562-861 in 110-559, HPV35 E7；HPV39 names in GenBank are KC470245.1, HPV39 E6 correspond to base positions and correspond to base positions in 592-921 in 107-583, HPV39 E7；HPV45 Name is KC470260.1 in GenBank, and HPV45 E6 correspond to base positions and correspond to base in 102-578, HPV45 E7 Position is in 587-907；HPV51 names in GenBank are M62877.1, and HPV51 E6 correspond to base positions in 70-536, HPV51 E7 correspond to base positions in 554-831；HPV52 names in GenBank are HQ537751.1, and HPV52 E6 are corresponding Base positions correspond to base positions in 553-852 in 102-548, HPV52 E7；HPV56 names in GenBank are X74483.1, HPV56 E6 correspond to base positions and correspond to base positions in 572-889 in 102-566, HPV56 E7；HPV58 Name is D90400.1 in GenBank, and HPV58 E6 correspond to base positions and correspond to base positions in 110-559, HPV58 E7 In 574-870；HPV59 names in GenBank are X77858.1, and HPV59 E6 correspond to base positions in 55-537, HPV59 E7 corresponds to base positions in 542-865；HPV68 names in GenBank are DQ080079.1, and HPV68 E6 correspond to base position Put and correspond to base positions in 484-816 in 1-477, HPV68 E7.

2. pair 13 kinds of high-risk HPV E6/E7 upstream and downstream primers and molecular beacon probe design：

Using Beacon Designer, Primer Premier5 and NCBI Photographing On-lines software to 13 kinds of high-risk HPVs And reference gene β-Actin upstream and downstream primer and molecular beacon probe are designed, specific design flow is as follows：

(1) using Primer Premier5 and NCBI Photographing On-line softwares to 13 kinds of high-risk HPVs and reference gene β- Actin upstream and downstream primer is designed.(2) according to upstream and downstream design of primers molecular beacon sequences, and RNA is utilized Structure, OligoAnalyzer 3.1 and mfold is predicted to its structure；(3) NCBI checking primers and probe are utilized Specificity.

Upstream and downstream primer is both needed to meet following condition：(1) between amplified production length 60-250bp；(2) primer length 15-25bp；(3) 3 ' ends of primer avoid high GC or high AT concentration areas；(4) it is G or C that primer 3 ', which holds last base, Avoid using T；(5) the Tm values of forward primer and reverse primer, which preferably differ, does not exceed 5 DEG C.Tm values are adjusted to 52 DEG C -62 ℃；(6) G/C content of primer is controlled between 30%-70% preferably；(7) primer A, G, C, T overall distributions are as far as possible uniform, keep away Exempt from, using the high region of GC or TA contents, especially 3 ' ends, to avoid the uneven region of G/C content；(8) please during design of primers T/C or A/G continuous structure are avoided as far as possible；Design of primers finishes retrieves verification primer specificity with NCBI BLAST functions, To avoid non-specific amplification from producing.

Molecular beacon design follows following principle：(1) probe length control is in 18-35bp；(2) the Tm value ratios of probe draw The Tm values of thing are about high 5-15 DEG C；(3) (G+C) % of probe is between 25%-70%；(4) two level knot is not present in probe region in itself Structure；

Upstream and downstream primer and the probe groups for obtaining 13 kinds of high-risk HPVs and internal reference β-Actin are as follows：

Above-mentioned probe groups also include the reverse complementary sequence in all molecular beacon probe areas.

The acquisition of 3 positive reference product

Target gene conversion Escherichia coli (by taking HPV16 as an example)

Detection of high risk human papillomavirus HPV16 E6/E7 sequences are inserted into plasmid pUC57 construction recombination plasmids, the recombinant plasmid It is transformed into Escherichia coli top10.Above section hands over Sangon Biotech (Shanghai) Co., Ltd.) limited company's completion.Wherein HPV16 E6/E7 gene orders (SEQ ID NO:121) it is as follows:

Escherichia coli are cultivated

Escherichia coli are in solid LB media (tryptone 10g/L, yeast extract 5 g/L, sodium chloride 10g/L, ammonia benzyl Penicillin 50 μ g/ml, agar 15g/L) 37 DEG C be incubated overnight, picking single bacterium fall on LB liquid medium (tryptone 10g/L, Yeast extract 5g/L, sodium chloride 10g/L, the μ g/ml of ampicillin 50) culture 5-8h.12000r/min centrifugations 1min is collected Coli somatic.

Plasmid extraction in Escherichia coli

Plasmid extraction is extracted using the small extraction reagent kit of Tiangeng DP103-02 plasmids, and extraction step refers to its specification portion Point.Quantitative detection is carried out to the plasmid of extraction using One Drop.The copy number calculating method of target sequence is as follows：

Copy number=quality (g) × 6.23 × 1023/324.5 of target sequence × DNA length

HPV16 target gene (being used as template using the plasmid of extraction) concrete operations only praise T7High with reference to promise in amplification plasmid Yield RNA Transcription Kit require to carry out.

Upstream primer sequence (such as sequence table SEQ ID NO:122) it is as follows: TAATACGACTCACTATAGGGATGCACCAAAAGAGAACTGC

Downstream primer sequence (such as sequence table SEQ ID NO:123) it is as follows:CTGAGAACAGATGGGGCAC

System such as table 1：

Table 1

Reagent	Volume (μ L)
		QRT-PCR premixed liquids	10
Sense primer 10	0.4
		Anti-sense primer	0.4
Template	2
		DEPC water	Mend to 20

QRT-PCR premixed liquids by 200-500nM dNTP, 2-10mM Mg²⁺, concentration be 0.1-0.5U/ μ L taq DNA Polymerase, concentration be 0.01-0.02U/ μ L UNG enzymes, 20mM pH8.3 Tris-Hcl and 100mM KCl compositions.

Amplification condition such as table 2：

Table 2

Gel electrophoresis analysis

Enter row agarose gel electrophoresis point to PCR primer using 2% agarose gel electrophoresis (voltage 110V, 20min) Analysis, the band about at 750bp is isolated, obtain PCR purified products.

In-vitro transcription

System such as table 3：

Table 3

Component	Volume (μ L)
		10×Reaction Buffer	2
ATP Solution	2
		GTP Solution	2
UTP Solution	2
		CTP Solution	2
PCR purified products	2
		T7RNA Polymerase Mix	2
RNase-free H2O	Supply 20

Gently mix each component with pipettor, and it is of short duration be collected by centrifugation, 37 DEG C incubation 2h.

1 μ l DNase I, 37 DEG C of incubation 15min are added in reaction system, digest the DNA profiling of transcription.

The RNA of synthesis carries out purifying using phenol/chloroform method of purification and is used to remove removing protein and most of free nucleotide.Specifically It is as follows.

A. 160 μ L RNase-free H are added₂O is by product dilution to 180 μ L.

B. 20 μ L 3M sodium acetate (pH 5.2) is added into the product after dilution, is fully mixed with pipettor.

C. 200 μ L phenol/chloroform mixed liquor (1 is added:1) it is stripped, room temperature 10000rpm centrifugation 5min, by upper strata Solution (aqueous phase) is transferred in new RNase-free EP pipes.

D. add the chloroform isometric with aqueous phase 2 times, collect upper strata aqueous phase.

E. absolute ethyl alcohol and the mixing of 2 times of volumes, -20 DEG C of incubations at least 30min, 4 DEG C of 15000rpm centrifugations are added 15min。

F. abandon supernatant and add the 70% ethanol washing RNA precipitate of 500 μ L precoolings, 4 DEG C of 15000rpm centrifugations, abandon supernatant.

G. uncap dry 2min, adds 20-50 μ L RNase-free H2O or other buffer solution RNA precipitates.

It is positive reference product that h.-80 DEG C, which preserves RNA sample,.

Copy number=quality (g) × 6.23 × 1023/324.5 of positive quality control product × target RNA length

The same HPV16 of construction method of other positive reference materials, is repeated no more.

Wherein positive quality control product is made up of HPV16 and HPV18 positive reference product.2 HPV16 upstream and downstream primers and point Sub- beacon probe detects positive quality control product sensitivity analysis

(exemplified by HPV16) specific as follows is analyzed using this kit detection sensitivity

This kit contains the μ L of reverse transcription premixed liquid 300；QRT-PCR premixed liquids 1.6mL；13 kinds of high-risk HPVs and internal reference β-Actin upstream and downstream primers and probe groups, each of which HPV hypotypes and internal reference β-Actin upstream and downstream primer are 10 μ Μ, 160 μ L, every HPV hypotypes and internal reference β-Actin molecular beacon probe are 10 μ Μ, 100 μ L；Strong positive quality-control product 500μL；The μ L of the weakly positive quality-control product 500 and μ L of negative controls 500.

Contain the μ L of reverse transcription premixed liquid 300；QRT-PCR premixed liquids 1.6mL；13 kinds of high-risk HPVs and internal reference β-Actin Upstream and downstream primer sets, each of which HPV hypotypes and internal reference β-Actin upstream and downstream primer and probe groups are upstream and downstream primer 10 μ Μ, 160 μ L；13 kinds of high-risk HPVs and internal reference β-Actin detections molecular beacon probe group, each of which HPV hypotypes And internal reference β-Actin molecular beacon probe is 10 μ Μ, 100 μ L；The μ L of strong positive quality-control product 500；The μ of weakly positive quality-control product 500 The L and μ L of negative controls 500.

This kit also provides strong positive quality-control product, weakly positive quality-control product and negative control simultaneously, specific as follows：Positive matter Control product are gone out by the plasmid in-vitro transcription for the high-risk HPV DNA fragmentation being sequenced in acknowledged detection range containing kit MRNA or its complementary series, the wherein concentration of strong positive quality-control product are 1 × 10⁷Copies/ μ L, the concentration of weakly positive quality-control product For 1 × 10³Copies/ μ L, negative controls are the water of DEPC processing.

The kit can be by HPV16 primed probes group, HPV18 primed probes group, β-Actin primed probes groups and zero To a kind of ten other high-risk HPVs (HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68) need to be combined according to experiment.

Take 20 μ L HPV16 positive references product to add the water of 180 μ L DEPC processing, carry out gradient dilution successively, obtain not With the positive quality control product of gradient.It is followed successively by 1 × 10⁹ copies/μL、1×10⁸copies/μL、1×10⁷copies/μL、1× 10⁶copies/ μL、1×10⁵copies/μL、1×10⁴copies/μL、1×10³copies/μL、 1×10²copies/μL。

Reverse transcription system such as table 4：

Table 4

Reverse transcription premixed liquid includes 100-300nM dNTP, 5-20U/ μ L reverse transcriptase, the suppression of 0.3-1U/ μ L RNases Preparation, random primer and poly T primers and 50mM pH8.3 Tris-HCl, 6mM dithiothreitol (DTT)s, 40mM KCl, 0.5mM tri- Phosphoric acid AZT forms, and wherein random primer is 6 aggressiveness that tetra- kinds of bases of A, G, C, T form at random, and poly T is 18-25 Poly T base compositions.

Reaction condition such as table 5：

Table 5

- 20 DEG C of preservations of reverse transcription product (cDNA).

The cDNA of upstream and downstream primer and molecular beacons detection positive reference product is verified using qRT-PCR.

QRT-PCR reaction systems such as table 6：

Table 6

HPV16 upstream primer sequences are as follows：CACTGTGTCC TGAAGAAAAG C

HPV16 upstream primer sequences are as follows：TGGGTTTCTC TACGTGTTCT TG

HPV16 molecular beacon probe sequences are as follows：FAM-CGCCACCGGT CGATGTATGT CTTGTTGCAG ATTGGCG-BHQ1

The direction of all sequences is from left to right 5 ' to 3 ' directions of sequence in the present invention, and above-mentioned HPV16 molecular beacons are visited Pin shows to be same as above, repeat no more in its 5 ' terminal modified FAM, 3 ' terminal modified BHQ1, the method for being presented below.

QRT-PCR reaction conditions such as table 7：

Table 7

As shown in Figure 2, HPV16 is minimum can detect 1 × 10 for present invention detection²Copies/ μ L, and when HPV16 is positive Quality-control product is 1 × 10⁹Copies/ μ L to 1 × 10²The preferable range of linearity is presented in the range of copies/ μ L.

3 Hela cell HPV18 mRNA are detected

Because Hela cells are the positive cell strains of HPV18 gene integrations, in order to verify the feasible of the primer of design and probe Property, the HPV18 mRNA in Hela cells are detected.Specific detecting step and result are as follows：

Hela cell culture

Hela cell culture is in DMEM nutrient solutions (100kU/L containing penicillin, the μ of streptomysin 100 containing 10% hyclone G/m L), 37 DEG C, 5% concentration carbon dioxide, continuously cultivate under conditions of saturated humidity environment.

Hela cell mRNAs are extracted

Extracted using Trizol mRNAs total to Hela cells, it is specific as follows:

(1) Hela cells are put into 1.5mL centrifuge tubes, then add 1mL Trizol, 15-30 DEG C of standing after piping and druming for several times 5min。

(2) 200 μ L chloroforms are added into 1.5mL centrifuge tubes, turn upside down 15-30 DEG C of standing 3-5min for several times.

(3) 4 DEG C, 12000r/min centrifugation 15min, taking-up are placed on ice.

(4) the μ L of upper strata 700 are drawn into new 1.5mL centrifuge tubes, add isometric isopropanol, -20 DEG C of standings are put in mixing 10min。

(5) 4 DEG C, 12000r/min centrifugations 10min abandon supernatant, each centrifuge tube adds the ethanol of 1mL 75%, appropriate to mix.

(6) 7500r/min centrifuges 5min and abandons supernatant, adds 20 μ L DEPC water, and piping and druming is completely dissolved RNA for several times, and -80 DEG C preserve.

Reverse transcription

Reverse transcription system such as table 8：

Table 8

Composition	Volume (μ L)
		Extract total mRNA	8
Reverse transcription premixed liquid	2

Reaction condition such as table 9：

Table 9

Temperature (DEG C)	Time (min)
		25	10
50	30
		85	5

- 20 DEG C of preservations of reverse transcription product (cDNA).

QRT-PCR detects to HPV18 mRNA cDNA

Reaction system such as table 10：

Table 10

Wherein 13 kinds of HPV upstream and downstream primers and probe set sequences are as follows：

HPV16 upstream primer sequences are as follows：CCAGAGACAA CTGATCTCTA CTG

HPV16 downstream primer sequences are as follows：GTCCGGTTCT GCTTGTCC

HPV16 molecular beacon probes are as follows：FAM-CCGCGAGACA GCTCAGAGGA GGAGGATGAA ATAGTCGCGG-BHQ1

HPV18 upstream primer sequences are as follows：CAACATTTACCAGCCCGAC

HPV18 downstream primer sequences are as follows：GCTGGAATGCTCGAAGG

HPV18 molecular beacon probes are as follows：VIC-CGCGAATGTT GTGTATGTGT TGTAAGTGTG AAGCCATCGC G-BHQ1

HPV31 upstream primer sequences are as follows：TGTGTACAGA GCACACAAGT AG

HPV31 downstream primer sequences are as follows：TTACAGTCTA GTAGAACAGT TGG

HPV31 molecular beacon probes are as follows：Texas Red-CGCGATGTTA ATGGGCTCAT TTGGAATCGT GTGTCGCG-Dabycle

HPV33 upstream primer sequences are as follows：AGCAATTAAG TGACAGCTC

HPV33 downstream primer sequences are as follows：CAAGTGTGAC AACAGGTTAC

HPV33 molecular beacon probes are as follows：Cy5-CGCGAGGACC GGCCAGATGG ACAAGTCGCG-BHQ3

HPV35 upstream primer sequences are as follows：ACCCGAGGCA ACTGACCTAT AC

HPV35 downstream primer sequences are as follows：TGTACACACA GACGTAGTGT C

HPV35 molecular beacon probes are as follows：FAM-CGCGAGACAA GCAAAACCAG ACACCTCCAA TTCGCG- BHQ1

HPV39 upstream primer sequences are as follows：TGAACTACAG CCGGTTGAC

HPV39 downstream primer sequences are as follows：CAGCTGCAGT GTGTTGTTAC

HPV39 molecular beacon probes are as follows：VIC-CCGCGAGAGC AATTAGGAGA GTCAGAGGAT GAAATCGCGG-BHQ1

HPV45 upstream primer sequences are as follows：CAGAAACCAT TGAACCCAG

HPV45 downstream primer sequences are as follows：TCTTTCTTGC CGTGCCTG

HPV45 molecular beacon probes are as follows：FAM-CGCGAAAAAC GTAGACACCT TAAGGACAAA CGATCGCG- BHQ1

HPV51 upstream primer sequences are as follows：TGGTAAAAGT ATAGAAGAAC

HPV51 downstream primer sequences are as follows：CGTTCAAAGC TTCACATAAT TC

HPV51 molecular beacon probes are as follows：VIC-CGCGAGAAGA CAAGAGGGAA AGACCACGAT CGCG-BHQ1

HPV52 upstream primer sequences are as follows：AGGATCCAGC AACACGAC

HPV52 downstream primer sequences are as follows：TTTTACACTG CACACACTGC

HPV52 molecular beacon probes are as follows：FAM-CGCGATGAGG TGCTGGAAGA ATCGGTTCGC G-BHQ1

HPV56 upstream primer sequences are as follows：GCCACAGCAA GCTAGACAAG

HPV56 downstream primer sequences are as follows：TAACGCACCC ATAAGCAGC

HPV56 molecular beacon probes are as follows：VIC-CGCGAGTTGT GAGTGTAAGT TTGTGGTGCA GTTGGTCGCG-BHQ1

HPV58 upstream primer sequences are as follows：AGCAATTATG TGACAGCTCA G

HPV58 downstream primer sequences are as follows：ATACACAAAC GAACCGTGG

HPV58 molecular beacon probes are as follows：FAM-CGCGAGGACG GGCCAGATGG ACAAGTCGCG-BHQ1

HPV59 upstream primer sequences are as follows：TGGCACGCTT TGAGGATC

HPV59 upstream primer sequences are as follows：CTCTTTCTTG CAGTTCCCC

HPV59 molecular beacon probes are as follows：VIC-CGCCAACGAC CATACAAACT GCCTGATTTG AGTGGCG- BHQ1

HPV68 upstream primer sequences are as follows：TGTATGTCAC GAGCAATTAG G

HPV68 upstream primer sequences are as follows：GTTACACTTA CAACACAGAC AC

HPV68 molecular beacon probes are as follows：FAM-CGCGAATCAC CACCAACATC TACTACTAGC CATCGCG- BHQ1

QRT-PCR reaction conditions such as table 11：

Table 11

HPV18 is in good amplification curve in Fig. 3, illustrates that kit of the present invention can detect HPV18 in Hela cells MRNA and with preferably specificity.

5 precision detect

According to above-mentioned HPV16 qRT-PCR reaction systems and reaction condition, HPV16 positive quality control product is taken, repeats to examine Survey six times.The present invention has preferable precision as shown in Figure 4.

6. clinical sample is analyzed

6.1. the total mRNA extractions of cervical exfoliated cell

Extracted using Trizol mRNAs total to cervical exfoliated cell.Carried out using Trizol mRNAs total to clinical sample Extraction, it is specific as follows：

(1) clinical sample is put into 1.5mL centrifuge tubes, then adds 1mL Trizol, 15-30 DEG C of standing after piping and druming for several times 5min。

(3) 4 DEG C, 12000r/min centrifugation 15min, taking-up are placed on ice.

(5) 4 DEG C, 12000r/min centrifugations 10min abandon supernatant, each centrifuge tube adds the ethanol of 1mL 75%, appropriate mixed It is even.

6.1.2 the total mRNA reverse transcriptions of cervical exfoliated cell are cDNA

It is using the reverse transcription mixed liquor of this kit that the total mRNA reverse transcriptions of the cervical exfoliated cell of extraction is inverse for cDNA. Transcription system such as table 12：

Table 12

Reaction condition such as table 13：

Table 13

Temperature (DEG C)	Time (min)
		25	10
50	30
		85	5

- 20 DEG C of preservations of reverse transcription product (cDNA).

6.1.3 qRT-PCR carries out qualitative detection to cDNA.

HPV mRNA are entered using the qRT-PCR reaction solutions and qRT-PCR upstream and downstream primer and qRT-PCR of this kit Row detection

Reaction system such as table 14：

Table 14

The upstream and downstream primer and probe groups of 13 kinds of HPV and internal reference are as follows：

HPV16 upstream primer sequences are as follows：CCAGAGACAA CTGATCTCTA CTG

HPV16 downstream primer sequences are as follows：GTCCGGTTCT GCTTGTCC

HPV18 upstream primer sequences are as follows：CAACATTTACCAGCCCGAC

HPV18 downstream primer sequences are as follows：GCTGGAATGCTCGAAGG

HPV31 upstream primer sequences are as follows：TGTGTACAGA GCACACAAGT AG

HPV31 downstream primer sequences are as follows：TTACAGTCTA GTAGAACAGT TGG

HPV33 upstream primer sequences are as follows：AGCAATTAAG TGACAGCTC

HPV33 downstream primer sequences are as follows：CAAGTGTGAC AACAGGTTAC

HPV35 upstream primer sequences are as follows：ACCCGAGGCA ACTGACCTAT AC

HPV35 downstream primer sequences are as follows：TGTACACACA GACGTAGTGT C

HPV39 upstream primer sequences are as follows：TGAACTACAG CCGGTTGAC

HPV39 downstream primer sequences are as follows：CAGCTGCAGT GTGTTGTTAC

HPV45 upstream primer sequences are as follows：CAGAAACCAT TGAACCCAG

HPV45 downstream primer sequences are as follows：TCTTTCTTGC CGTGCCTG

HPV51 upstream primer sequences are as follows：TGGTAAAAGT ATAGAAGAAC

HPV51 downstream primer sequences are as follows：CGTTCAAAGC TTCACATAAT TC

HPV52 upstream primer sequences are as follows：AGGATCCAGC AACACGAC

HPV52 downstream primer sequences are as follows：TTTTACACTG CACACACTGC

HPV56 upstream primer sequences are as follows：GCCACAGCAA GCTAGACAAG

HPV56 downstream primer sequences are as follows：TAACGCACCC ATAAGCAGC

HPV58 upstream primer sequences are as follows：AGCAATTATG TGACAGCTCA G

HPV58 downstream primer sequences are as follows：ATACACAAAC GAACCGTGG

HPV59 upstream primer sequences are as follows：TGGCACGCTT TGAGGATC

HPV59 upstream primer sequences are as follows：CTCTTTCTTG CAGTTCCCC

HPV68 upstream primer sequences are as follows：TGTATGTCAC GAGCAATTAG G

HPV68 upstream primer sequences are as follows：GTTACACTTA CAACACAGAC AC

β-Actin upstream primer sequences are as follows：AGTGCTGTCT GGCGGC

β-Actin upstream primer sequences are as follows：TCTTCATTGT GCTGGGTGC C

β-Actin molecular beacon probe sequences are as follows：FAM-CGCGAGCCGA CAGGATGCAG AAGGAGATTC GCG- BHQ1

QRT-PCR reaction conditions such as table 15：

Table 15

Testing result such as table 16：

Table 16

Table 16 shows that the detection method of the present invention is consistent with existing clinical detection result, illustrates the feasible of this method detection Property.

Sensitivity analysis and clinical detection result with reference to patent of the present invention, the result interpretation method of this kit are as follows：

1. think that the experiment is invalid if reference gene is without amplified production.

2. if reference gene has amplification, the cycle values of target sequence within 30 circulations and have good amplification bent Line, then it is assumed that amplification is effective.

3. if reference gene has amplification, the cycle values of target sequence in 30-35 circulation and have good amplification curve, It need to repeat to test, if result is consistent with result of the test above, then it is assumed that amplification is effective.If without amplification, then it is assumed that amplification knot Fruit is invalid, in the absence of target sequence.

In addition to above-mentioned implementation, the present invention can also have other embodiment.It is all to be formed using equivalent substitution or equivalent transformation Technical scheme, all fall within the protection domains of application claims.

Sequence table

<110>China Medicine University

<120>For detecting the primer and probe and kit of high-risk human mammilla papillomavirus oncogene E6/E7 mRNA partings

<130> 2017

<160> 123

<210> SEQ ID NO:1

<211> 23

<212> DNA

<213>Artificial sequence

<400> 1

AATGTGTGTA CTGCAAGCAA CAG

<210> SEQ ID NO:2

<211> 22

<212> DNA

<213>Artificial sequence

<400> 2

ACAGCATATG GATTCCCATC TC

<210> SEQ ID NO:3

<211> 38

<212> DNA

<213>Artificial sequence

<400> 3

CGCGATGCGA CGTGAGGTAT ATGACTTTGC TTTTCGCG

<210> SEQ ID NO:4

<211> 23

<212> DNA

<213>Artificial sequence

<400> 4

CCAGAGACAA CTGATCTCTA CTG

<210> SEQ ID NO:5

<211> 18

<212> DNA

<213>Artificial sequence

<400> 5

GTCCGGTTCT GCTTGTCC

<210> SEQ ID NO:6

<211> 40

<212> DNA

<213>Artificial sequence

<400> 6

CCGCGA GACAGCTCAGAGGAGGAGGATGAAATAG TCGCGG

<210> SEQ ID NO:7

<211> 21

<212> DNA

<213>Artificial sequence

<400> 7

CACTGTGTCC TGAAGAAAAG C

<210> SEQ ID NO:8

<211> 22

<212> DNA

<213>Artificial sequence

<400> 8

TGGGTTTCTC TACGTGTTCT TG

<210> SEQ ID NO:9

<211> 37

<212> DNA

<213>Artificial sequence

<400> 9

CGCCACCGGT CGATGTATGT CTTGTTGCAG ATTGGCG

<210> SEQ ID NO:10

<211> 22

<212> DNA

<213>Artificial sequence

<400> 10

GAATTGAGCT AGTAGTAGAA AG

<210> SEQ ID NO:11

<211> 20

<212> DNA

<213>Artificial sequence

<400> 11

CGGACACACA AAGGACAGGG

<210> SEQ ID NO:12

<211> 36

<212> DNA

<213>Artificial sequence

<400> 12

CCGCGAGACC TTCGAGCATT CCAGCAGCTG TCGCGG

<210> SEQ ID NO:13

<211> 19

<212> DNA

<213>Artificial sequence

<400> 13

CAACATTTAC CAGCCCGAC

<210> SEQ ID NO:14

<211> 17

<212> DNA

<213>Artificial sequence

<400> 14

GCTGGAATGC TCGAAGG

<210> SEQ ID NO:15

<211> 41

<212> DNA

<213>Artificial sequence

<400> 15

CGCGAATGTT GTGTATGTGT TGTAAGTGTG AAGCCATCGC G

<210> SEQ ID NO:16

<211> 21

<212> DNA

<213>Artificial sequence

<400> 16

GGTGCCAGAA ACCGTTGAAT C

<210> SEQ ID NO:17

<211> 20

<212> DNA

<213>Artificial sequence

<400> 17

GTGTTTCTCT GCGTCGTTGG

<210> SEQ ID NO:18

<211> 36

<212> DNA

<213>Artificial sequence

<400> 18

CGCGACACAA CATAGCTGGG CACTATAGAG GTCGCG

<210> SEQ ID NO:19

<211> 22

<212> DNA

<213>Artificial sequence

<400> 19

TGTGTACAGA GCACACAAGT AG

<210> SEQ ID NO:20

<211> 23

<212> DNA

<213>Artificial sequence

<400> 20

TTACAGTCTA GTAGAACAGT TGG

<210> SEQ ID NO:21

<211> 38

<212> DNA

<213>Artificial sequence

<400> 21

CGCGATGTTA ATGGGCTCAT TTGGAATCGT GTGTCGCG

<210> SEQ ID NO:22

<211> 21

<212> DNA

<213>Artificial sequence

<400> 22

GTGTCAAAGA CCGTTGTGTC C

<210> SEQ ID NO:23

<211> 22

<212> DNA

<213>Artificial sequence

<400> 23

ACACTTGGGT TTCAGTACGA GG

<210> SEQ ID NO:24

<211> 38

<212> DNA

<213>Artificial sequence

<400> 24

CGCCAACAAC ATAGGAGGAA GGTGGACAGG ACGTGGCG

<210> SEQ ID NO:25

<211> 21

<212> DNA

<213>Artificial sequence

<400> 25

CATGAACTAA GCTCGGCATT G

<210> SEQ ID NO:26

<211> 22

<212> DNA

<213>Artificial sequence

<400> 26

ACCTCTGTTT CTGTTAACTG AC

<210> SEQ ID NO:27

<211> 44

<212> DNA

<213>Artificial sequence

<400> 27

CCGCCACCCT ACGATGAACT AAGATTGAAT TGTGTCTACG GCGG

<210> SEQ ID NO:28

<211> 19

<212> DNA

<213>Artificial sequence

<400> 28

AGCAATTAAG TGACAGCTC

<210> SEQ ID NO:29

<211> 20

<212> DNA

<213>Artificial sequence

<400> 29

CAAGTGTGAC AACAGGTTAC

<210> SEQ ID NO:30

<211> 30

<212> DNA

<213>Artificial sequence

<400> 30

CGCGAGGACC GGCCAGATGG ACAAGTCGCG

<210> SEQ ID NO:31

<211> 25

<212> DNA

<213>Artificial sequence

<400> 31

CCAACGTTAA AGGAATATGT TTTAG

<210> SEQ ID NO:32

<211> 20

<212> DNA

<213>Artificial sequence

<400> 32

CTGAGCTGTC ACTTAATTGC

<210> SEQ ID NO:33

<211> 35

<212> DNA

<213>Artificial sequence

<400> 33

CGCGATCCTG AACCAACTGA CCTATACTGC TCGCG

<210> SEQ ID NO:34

<211> 22

<212> DNA

<213>Artificial sequence

<400> 34

GGACACAAGC CAACGTTAAA GG

<210> SEQ ID NO:35

<211> 19

<212> DNA

<213>Artificial sequence

<400> 35

GTGGCTGGTT GTGCTTGTC

<210> SEQ ID NO:36

<211> 34

<212> DNA

<213>Artificial sequence

<400> 36

CGCGAGATGA GGATGAAGGC TTGGACCGGT CGCG

<210> SEQ ID NO:37

<211> 21

<212> DNA

<213>Artificial sequence

<400> 37

CCGCTGTGTC CAGTTGAAAA G

<210> SEQ ID NO:38

<211> 21

<212> DNA

<213>Artificial sequence

<400> 38

ACACCTCGGT TTCTCTACGT G

<210> SEQ ID NO:39

<211> 36

<212> DNA

<213>Artificial sequence

<400> 39

CGCGAGTGGA CGGTGGACAG GTCGGTGTAT GTCGCG

<210> SEQ ID NO:40

<211> 22

<212> DNA

<213>Artificial sequence

<400> 40

ACCCGAGGCA ACTGACCTAT AC

<210> SEQ ID NO:41

<211> 21

<212> DNA

<213>Artificial sequence

<400> 41

TGTACACACA GACGTAGTGT C

<210> SEQ ID NO:42

<211> 36

<212> DNA

<213>Artificial sequence

<400> 42

CGCGAGACAA GCAAAACCAG ACACCTCCAA TTCGCG

<210> SEQ ID NO:43

<211> 19

<212> DNA

<213>Artificial sequence

<400> 43

GACAAGCAAA ACCAGACAC

<210> SEQ ID NO:44

<211> 20

<212> DNA

<213>Artificial sequence

<400> 44

GGGGCACACT ATTCCAAATG

<210> SEQ ID NO:45

<211> 37

<212> DNA

<213>Artificial sequence

<400> 45

CGCGATGTAA CGTCCTGTTG TAAATGTGAG GCTCGCG

<210> SEQ ID NO:46

<211> 19

<212> DNA

<213>Artificial sequence

<400> 46

TGAACTACAG CCGGTTGAC

<210> SEQ ID NO:47

<211> 20

<212> DNA

<213>Artificial sequence

<400> 47

CAGCTGCAGT GTGTTGTTAC

<210> SEQ ID NO:48

<211> 40

<212> DNA

<213>Artificial sequence

<400> 48

CCGCGAGAGC AATTAGGAGA GTCAGAGGAT GAAATCGCGG

<210> SEQ ID NO:49

<211> 19

<212> DNA

<213>Artificial sequence

<400> 49

ACGAGCAATT AGGAGAGTC

<210> SEQ ID NO:50

<211> 19

<212> DNA

<213>Artificial sequence

<400> 50

TGTATTGTGT GACGCTGTG

<210> SEQ ID NO:51

<211> 39

<212> DNA

<213>Artificial sequence

<400> 51

CCGCGAAATCA CCAACATCAA CTACTAGCCA GACTCGCGG

<210> SEQ ID NO:52

<211> 20

<212> DNA

<213>Artificial sequence

<400> 52

TACTCGGACT CGGTGTATGC

<210> SEQ ID NO:53

<211> 23

<212> DNA

<213>Artificial sequence

<400> 53

TCTTCGTTTG CTATTTAGGT GTC

<210> SEQ ID NO:54

<211> 38

<212> DNA

<213>Artificial sequence

<400> 54

CGCGAATAAG GTGCATGTGT TGTCTGAAAC CGCTCGCG

<210> SEQ ID NO:55

<211> 19

<212> DNA

<213>Artificial sequence

<400> 55

CAGAAACCAT TGAACCCAG

<210> SEQ ID NO:56

<211> 18

<212> DNA

<213>Artificial sequence

<400> 56

TCTTTCTTGC CGTGCCTG

<210> SEQ ID NO:57

<211> 38

<212> DNA

<213>Artificial sequence

<400> 57

CGCGAAAAAC GTAGACACCT TAAGGACAAA CGATCGCG

<210> SEQ ID NO:58

<211> 20

<212> DNA

<213>Artificial sequence

<400> 58

TGACCTGTTG TGTTACGAGC

<210> SEQ ID NO:59

<211> 20

<212> DNA

<213>Artificial sequence

<400> 59

TTTTGTGACG CTGTGGTTCG

<210> SEQ ID NO:60

<211> 37

<212> DNA

<213>Artificial sequence

<400> 60

CGCGAAGAGG AGGAAAACGA TGAAGCAGAT GGTCGCG

<210> SEQ ID NO:61

<211> 22

<212> DNA

<213>Artificial sequence

<400> 61

TGTTGTGTTA CGAGCAATTA AG

<210> SEQ ID NO:62

<211> 19

<212> DNA

<213>Artificial sequence

<400> 62

CATCTGCCGA GCTCTCTAC

<210> SEQ ID NO:63

<211> 30

<212> DNA

<213>Artificial sequence

<400> 63

CGCGATGCAC AACTACCAGC CCGACTCGCG

<210> SEQ ID NO:64

<211> 20

<212> DNA

<213>Artificial sequence

<400> 64

TGGTAAAAGT ATAGAAGAAC

<210> SEQ ID NO:65

<211> 22

<212> DNA

<213>Artificial sequence

<400> 65

CGTTCAAAGC TTCACATAAT TC

<210> SEQ ID NO:66

<211> 34

<212> DNA

<213>Artificial sequence

<400> 66

CGCGAGAAGA CAAGAGGGAA AGACCACGAT CGCG

<210> SEQ ID NO:67

<211> 20

<212> DNA

<213>Artificial sequence

<400> 67

GGCTACGTGT TACAGAATTG

<210> SEQ ID NO:68

<211> 20

<212> DNA

<213>Artificial sequence

<400> 68

CCCATTAACA TCTGCTGTAC

<210> SEQ ID NO:69

<211> 30

<212> DNA

<213>Artificial sequence

<400> 69

CGCCAGGCAG TGGAAAGCAG TGGAGTCGCG

<210> SEQ ID NO:70

<211> 21

<212> DNA

<213>Artificial sequence

<400> 70

ATATGCGTGA CCAGCTACCA G

<210> SEQ ID NO:71

<211> 21

<212> DNA

<213>Artificial sequence

<400> 71

AAGGGTGTCT CCACTGCTTT C

<210> SEQ ID NO:72

<211> 33

<212> DNA

<213>Artificial sequence

<400> 72

CGCGTTGAAG CTCCGTGTTG CAGGTGTTAC GCG

<210> SEQ ID NO:73

<211> 22

<212> DNA

<213>Artificial sequence

<400> 73

ACAACTGACC TACACTGCTA TG

<210> SEQ ID NO:74

<211> 20

<212> DNA

<213>Artificial sequence

<400> 74

TTGCTTGTGG CTTGTTCTGC

<210> SEQ ID NO:75

<211> 38

<212> DNA

<213>Artificial sequence

<400> 75

CCGCGATAGG TGACAGCTCA GATGAGGAGG ATTCGCGG

<210> SEQ ID NO:76

<211> 18

<212> DNA

<213>Artificial sequence

<400> 76

AGGATCCAGC AACACGAC

<210> SEQ ID NO:77

<211> 20

<212> DNA

<213>Artificial sequence

<400> 77

TTTTACACTG CACACACTGC

<210> SEQ ID NO:78

<211> 31

<212> DNA

<213>Artificial sequence

<400> 78

CGCGATGAGG TGCTGGAAGA ATCGGTTCGC G

<210> SEQ ID NO:79

<211> 22

<212> DNA

<213>Artificial sequence

<400> 79

GGGAAAACAT TAGAAGAGAG GG

<210> SEQ ID NO:80

<211> 21

<212> DNA

<213>Artificial sequence

<400> 80

CTACACTTGG GTCACAGGTC G

<210> SEQ ID NO:81

<211> 32

<212> DNA

<213>Artificial sequence

<400> 81

CGCGAAGGGC GCTGTTCAGA GTGTTGGTCG CG

<210> SEQ ID NO:82

<211> 22

<212> DNA

<213>Artificial sequence

<400> 82

ATGACTATTC AGTGTATGGA GC

<210> SEQ ID NO:83

<211> 22

<212> DNA

<213>Artificial sequence

<400> 83

CTAGGTTCTC TAGATGTTTG TC

<210> SEQ ID NO:84

<211> 30

<212> DNA

<213>Artificial sequence

<400> 84

CGCGAGGTCA TGTTTGGGGT GCTGGTCGCG

<210> SEQ ID NO:85

<211> 20

<212> DNA

<213>Artificial sequence

<400> 85

GCCACAGCAA GCTAGACAAG

<210> SEQ ID NO:86

<211> 19

<212> DNA

<213>Artificial sequence

<400> 86

TAACGCACCC ATAAGCAGC

<210> SEQ ID NO:87

<211> 40

<212> DNA

<213>Artificial sequence

<400> 87

CGCGAGTTGT GAGTGTAAGT TTGTGGTGCA GTTGGTCGCG

<210> SEQ ID NO:88

<211> 20

<212> DNA

<213>Artificial sequence

<400> 88

TACACGTACC TTGTTGTGAG

<210> SEQ ID NO:89

<211> 19

<212> DNA

<213>Artificial sequence

<400> 89

TGTTAACGCA CCCATAAGC

<210> SEQ ID NO:90

<211> 33

<212> DNA

<213>Artificial sequence

<400> 90

CGCGAGTGGT GCAGTTGGAC ATTCAGAGTC GCG

<210> SEQ ID NO:91

<211> 21

<212> DNA

<213>Artificial sequence

<400> 91

AGCAATTATG TGACAGCTCA G

<210> SEQ ID NO:92

<211> 19

<212> DNA

<213>Artificial sequence

<400> 92

ATACACAAAC GAACCGTGG

<210> SEQ ID NO:93

<211> 30

<212> DNA

<213>Artificial sequence

<400> 93

CGCGAGGACG GGCCAGATGG ACAAGTCGCG

<210> SEQ ID NO:94

<211> 22

<212> DNA

<213>Artificial sequence

<400> 94

GATTTACATC CTGAACCAAC TG

<210> SEQ ID NO:95

<211> 22

<212> DNA

<213>Artificial sequence

<400> 95

CAATGTAGTA ATTAGCTGTG GC

<210> SEQ ID NO:96

<211> 34

<212> DNA

<213>Artificial sequence

<400> 96

CGCGAGAGGA TGAAATAGGC TTGGACGGGT CGCG

<210> SEQ ID NO:97

<211> 18

<212> DNA

<213>Artificial sequence

<400> 97

AGAGGAGAAA CCACGGAC

<210> SEQ ID NO:98

<211> 21

<212> DNA

<213>Artificial sequence

<400> 98

ATACCTCAGA TCGCTGCAAA G

<210> SEQ ID NO:99

<211> 32

<212> DNA

<213>Artificial sequence

<400> 99

CGCGAGATTT GTGTCAGGCG TTGGAGATCG CG

<210> SEQ ID NO:100

<211> 33

<212> DNA

<213>Artificial sequence

<400> 100

TACACAACGA CCATACAAAC TGC

<210> SEQ ID NO:101

<211> 22

<212> DNA

<213>Artificial sequence

<400> 101

GCTGCATACG GTGTACAGTC TC

<210> SEQ ID NO:102

<211> 39

<212> DNA

<213>Artificial sequence

<400> 102

CGCCAGCAAA GGGGAACTGC AAGAAAGAGA GGTATGGCG

<210> SEQ ID NO:103

<211> 18

<212> DNA

<213>Artificial sequence

<400> 103

TGGCACGCTT TGAGGATC

<210> SEQ ID NO:104

<211> 19

<212> DNA

<213>Artificial sequence

<400> 104

CTCTTTCTTG CAGTTCCCC

<210> SEQ ID NO:105

<211> 37

<212> DNA

<213>Artificial sequence

<400> 105

CGCCAACGAC CATACAAACT GCCTGATTTG AGTGGCG

<210> SEQ ID NO:106

<211> 22

<212> DNA

<213>Artificial sequence

<400> 106

TATTCCTCTG CATGATATTC GC

<210> SEQ ID NO:107

<211> 20

<212> DNA

<213>Artificial sequence

<400> 107

TTTCAGACAC GCTGCATACG

<210> SEQ ID NO:108

<211> 40

<212> DNA

<213>Artificial sequence

<400> 108

CCGCGACAAA GGGGAACTGC AAGAAAGAGA GGTATCGCGG

<210> SEQ ID NO:109

<211> 22

<212> DNA

<213>Artificial sequence

<400> 109

TATGAATTTG CCTTTAGTGA CC

<210> SEQ ID NO:110

<211> 22

<212> DNA

<213>Artificial sequence

<400> 110

ATCGTAGTTC CCGTATTTTA GC

<210> SEQ ID NO:111

<211> 38

<212> DNA

<213>Artificial sequence

<400> 111

CGCGATAGTG TATAGAGACG GGGTACCATT TGCTCGCG

<210> SEQ ID NO:112

<211> 22

<212> DNA

<213>Artificial sequence

<400> 112

ATTGGACACT ACATTGCATG AC

<210> SEQ ID NO:113

<211> 19

<212> DNA

<213>Artificial sequence

<400> 113

TGGTACCCCG TCTCTATAC

<210> SEQ ID NO:114

<211> 40

<212> DNA

<213>Artificial sequence

<400> 114

CGCGAACTAC AACGGACAGA GGTATATGAA TTTGCTCGCG

<210> SEQ ID NO:115

<211> 21

<212> DNA

<213>Artificial sequence

<400> 115

TGTATGTCAC GAGCAATTAG G

<210> SEQ ID NO:116

<211> 22

<212> DNA

<213>Artificial sequence

<400> 116

GTTACACTTA CAACACAGAC AC

<210> SEQ ID NO:117

<211> 37

<212> DNA

<213>Artificial sequence

<400> 117

CGCGAATCAC CACCAACATC TACTACTAGC CATCGCG

<210> SEQ ID NO:118

<211> 16

<212> DNA

<213>Artificial sequence

<400> 118

AGTGCTGTCT GGCGGC

<210> SEQ ID NO:119

<211> 20

<212> DNA

<213>Artificial sequence

<400> 119

TCTTCATTGT GCTGGGTGCC

<210> SEQ ID NO:120

<211> 33

<212> DNA

<213>Artificial sequence

<400> 120

CGCGAGCCGA CAGGATGCAG AAGGAGATTC GCG

<210> SEQ ID NO:121

<211> 776

<212> DNA

<213>Artificial sequence

<400> 121

ATGCACCAAA AGAGAACTGC AATGTTTCAG GACCCACAGG AGCGACCCAG AAAGTTACCA CAGTTATGCA CAGAGCTGCA AACAACTATA CATGATATAA TATTAGAATG TGTGTACTGC AAGCAACAGT TACTGCGACG TGAGGTATAT GACTTTGCTT TTCGGGATTT ATGCATAGTA TATAGAGATG GGAATCCATA TGCTGTATGT GATAAATGTT TAAAGTTTTA TTCTAAAATT AGTGAGTATA GACATTATTG TTATAGTTTG TATGGAACAA CATTAGAACA GCAATACAAC AAACCGTTGT GTGATTTGTT AATTAGGTGT ATTAACTGTC AAAAGCCACT GTGTCCTGAA GAAAAGCAAA GACATCTGGA CAAAAAGCAA AGATTCCATA ATATAAGGGG TCGGTGGACC GGTCGATGTA TGTCTTGTTG CAGATCATCA AGAACACGTA GAGAAACCCA GCTGTAATCA TGCATGGAGA TACACCTACA TTGCATGAAT ATATGTTAGA TTTGCAACCA GAGACAACTG ATCTCTACTG TTATGAGCAA TTAAATGACA GCTCAGAGGA GGAGGATGAA ATAGATGGTC CAGCTGGACA AGCAGAACCG GACAGAGCCC ATTACAATAT TGTAACCTTT TGTTGCAAGT GTGACTCTAC GCTTCGGTTG TGCGTACAAA GCACACACGT AGACATTCGT ACTTTGGAAG ACCTGTTAAT GGGCACACTA GGAATTGTGT GCCCCATCTG TTCTCAGAAA CCATAA

<210> SEQ ID NO:122

<211> 40

<212> DNA

<213>Artificial sequence

<400> 122

TAATACGACT CACTATAGGG ATGCACCAAA AGAGAACTGC

<210> SEQ ID NO:123

<211> 19

<212> DNA

<213>Artificial sequence

<400> 123

CTGAGAACAG ATGGGGCAC

Claims

1. primer and probe groups for detecting high-risk human mammilla papillomavirus E6/E7mRNA partings, it is characterised in that

The primer and probe groups include 13 kinds of high-risk HPVs and internal reference β-Actin upstream and downstream primer and probe groups：

HPV16 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer such as sequence SEQ ID NO.1 in list, the SEQ ID NO.2 in anti-sense primer such as sequence table, the SEQ ID in probe such as sequence table NO.3；Second group, the SEQ ID NO.4 in sense primer such as sequence table, the SEQ ID NO.5 in anti-sense primer such as sequence table, SEQ ID NO.6 in probe such as sequence table；3rd group, the SEQ ID NO.7 in sense primer such as sequence table, anti-sense primer is such as SEQ ID NO.8 in sequence table, the SEQ ID NO.9 in probe such as sequence table；

HPV18 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer such as sequence SEQ ID NO.10 in list, the SEQ ID NO.11 in anti-sense primer such as sequence table, the SEQ ID in probe such as sequence table NO.12；Second group, the SEQ ID NO.13 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.15 in NO.14, probe such as sequence table；3rd group, the SEQ ID NO.16 in sense primer such as sequence table, under Swim the SEQ ID NO.17 in primer such as sequence table, the SEQ ID NO.18 in probe such as sequence table；

HPV31 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer such as sequence SEQ ID NO.19 in list, the SEQ ID NO.20 in anti-sense primer such as sequence table, the SEQ ID in probe such as sequence table NO.21；Second group, the SEQ ID NO.22 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.24 in NO.23, probe such as sequence table；3rd group, the SEQ ID NO.25 in sense primer such as sequence table, under Swim the SEQ ID NO.26 in primer such as sequence table, the SEQ ID NO.27 in probe such as sequence table；

HPV33 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer such as sequence SEQ ID NO.28 in list, the SEQ ID NO.29 in anti-sense primer such as sequence table, the SEQ ID in probe such as sequence table NO.30；Second group, the SEQ ID NO.31 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.33 in NO.32, probe such as sequence table；3rd group, the SEQ ID NO.34 in sense primer such as sequence table, under Swim the SEQ ID NO.35 in primer such as sequence table, the SEQ ID NO.36 in probe such as sequence table；

HPV35 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer such as sequence SEQ ID NO.37 in list, the SEQ ID NO.38 in anti-sense primer such as sequence table, the SEQ ID in probe such as sequence table NO.39；Second group, the SEQ ID NO.40 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.42 in NO.41, probe such as sequence table；3rd group, the SEQ ID NO.43 in sense primer such as sequence table, under Swim the SEQ ID NO.44 in primer such as sequence table, the SEQ ID NO.45 in probe such as sequence table；

HPV39 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer such as sequence SEQ ID NO.46 in list, the SEQ ID NO.47 in anti-sense primer such as sequence table, the SEQ ID in probe such as sequence table NO.48；Second group, the SEQ ID NO.49 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.51 in NO.50, probe such as sequence table；3rd group, the SEQ ID NO.52 in sense primer such as sequence table, under Swim the SEQ ID NO.53 in primer such as sequence table, the SEQ ID NO.54 in probe such as sequence table；

HPV45 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer such as sequence SEQ ID NO.55 in list, the SEQ ID NO.56 in anti-sense primer such as sequence table, the SEQ ID in probe such as sequence table NO.57；Second group, the SEQ ID NO.58 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.60 in NO.59, probe such as sequence table；3rd group, the SEQ ID NO.61 in sense primer such as sequence table, under Swim the SEQ ID NO.62 in primer such as sequence table, the SEQ ID NO.63 in probe such as sequence table；

HPV51 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer such as sequence SEQ ID NO.64 in list, the SEQ ID NO.65 in anti-sense primer such as sequence table, the SEQ ID in probe such as sequence table NO.66；Second group, the SEQ ID NO.67 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.69 in NO.68, probe such as sequence table；3rd group, the SEQ ID NO.70 in sense primer such as sequence table, under Swim the SEQ ID NO.71 in primer such as sequence table, the SEQ ID NO.72 in probe such as sequence table；

HPV52 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer such as sequence SEQ ID NO.73 in list, the SEQ ID NO.74 in anti-sense primer such as sequence table, the SEQ ID in probe such as sequence table NO.75；Second group, the SEQ ID NO.76 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.78 in NO.77, probe such as sequence table；3rd group, the SEQ ID NO.79 in sense primer such as sequence table, under Swim the SEQ ID NO.80 in primer such as sequence table, the SEQ ID NO.81 in probe such as sequence table；

HPV56 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer such as sequence SEQ ID NO.82 in list, the SEQ ID NO.83 in anti-sense primer such as sequence table, the SEQ ID in probe such as sequence table NO.84；Second group, the SEQ ID NO.85 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.87 in NO.86, probe such as sequence table；3rd group, the SEQ ID NO.88 in sense primer such as sequence table, under Swim the SEQ ID NO.89 in primer such as sequence table, the SEQ ID NO.90 in probe such as sequence table；

HPV58 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer such as sequence SEQ ID NO.91 in list, the SEQ ID NO.92 in anti-sense primer such as sequence table, the SEQ ID in probe such as sequence table NO.93；Second group, the SEQ ID NO.94 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.96 in NO.95, probe such as sequence table；3rd group, the SEQ ID NO.97 in sense primer such as sequence table, under Swim the SEQ ID NO.98 in primer such as sequence table, the SEQ ID NO.99 in probe such as sequence table；

HPV59 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer such as sequence SEQ ID NO.100 in list, the SEQ ID NO.101 in anti-sense primer such as sequence table, the SEQ in probe such as sequence table ID NO.102；Second group, the SEQ ID NO.103 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.105 in NO.104, probe such as sequence table；3rd group, the SEQ ID in sense primer such as sequence table SEQ ID NO.107 in NO.106, anti-sense primer such as sequence table, the SEQ ID NO.108 in probe such as sequence table；

HPV68 upstream and downstream primer and probe can be following three groups in any group, specific first group, sense primer such as sequence SEQ ID NO.109 in list, the SEQ ID NO.110 in anti-sense primer such as sequence table, the SEQ in probe such as sequence table ID NO.111；Second group, the SEQ ID NO.112 in sense primer such as sequence table, the SEQ ID in anti-sense primer such as sequence table SEQ ID NO.114 in NO.113, probe such as sequence table；3rd group, the SEQ ID in sense primer such as sequence table SEQ ID NO.116 in NO.115, anti-sense primer such as sequence table, the SEQ ID NO.117 in probe such as sequence table；

2. it is used for the primer and probe groups for detecting high-risk human mammilla papillomavirus E6/E7mRNA partings according to claim 1, It is characterized in that：The probe groups also include the reverse complementary sequence in all molecular beacon probe areas.

3. for detecting high-risk human mammilla papillomavirus E6/E7mRNA and the kit of parting, it is characterised in that：The kit Contain the μ L of reverse transcription premixed liquid 300；QRT-PCR premixed liquids 1.6mL；13 kinds of high-risk HPVs and internal reference β-Actin upstream and downstream are drawn Thing and probe groups, each of which HPV hypotypes and internal reference β-Actin upstream and downstream primer are 10 μ Μ, 160 μ L, every HPV sub- Type and internal reference β-Actin molecular beacon probe are 10 μ Μ, 100 μ L；The μ L of strong positive quality-control product 500；Weakly positive quality-control product The 500 μ L and μ L of negative controls 500.

4. it is used for the kit for detecting high-risk human mammilla papillomavirus E6/E7mRNA partings, its feature according to claim 3 It is：The reverse transcription premixed liquid includes 100-300nM dNTP, 5-20U/ μ L reverse transcriptase, the suppression of 0.3-1U/ μ L RNases Preparation, random primer and poly T primers and 50mM pH8.3 Tris-HCl, 6mM dithiothreitol (DTT)s, 40mM KCl, 0.5mM tri- Phosphoric acid AZT forms, and wherein random primer is 6 aggressiveness that tetra- kinds of bases of A, G, C, T form at random, and poly T is 18-25 Poly T base compositions.

5. it is used for the kit for detecting high-risk human mammilla papillomavirus E6/E7mRNA partings, its feature according to claim 3 It is：The qRT-PCR premixed liquids by 200-500nM dNTP, 2-10mM Mg²⁺, concentration be 0.1-0.5U/ μ L taq Archaeal dna polymerase, concentration be 0.01-0.02U/ μ L UNG enzymes, 20mM pH8.3 Tris-Hcl and 100mM KCl compositions.

6. it is used for the kit for detecting high-risk human mammilla papillomavirus E6/E7mRNA partings, its feature according to claim 3 It is：The kit can be by HPV16 primed probes group, HPV18 primed probes group, β-Actin primed probes groups and zero to ten A kind of other high-risk HPVs (HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68) composition.

7. it is used for the kit for detecting high-risk human mammilla papillomavirus E6/E7mRNA partings, its feature according to claim 3 It is：The fluorophor of the 5' ends mark of the molecular beacon is any of organic fluorescent dye or inorganic dyestuff, 3' ends Quencher is marked as nano-particles such as any or gold nanos of organic dyestuff.

8. it is used for the kit for detecting high-risk human mammilla papillomavirus E6/E7mRNA partings, its feature according to claim 7 It is：The organic fluorescent dye is FAM, VIC, HEX, TRT, Cy3, Cy5, ROX and JOE, and the inorganic dyestuff is quantum dot, The organic dyestuff is TAMRA, DABCYL, MGB, BHQ-1, BHQ-2 and BHQ-3.

9. it is used for the kit for detecting high-risk human mammilla papillomavirus E6/E7mRNA partings, its feature according to claim 3 It is：Positive quality control product is the plasmid by the high-risk HPV DNA fragmentation in acknowledged detection range containing kit is sequenced The mRNA or its complementary series that in-vitro transcription goes out, the wherein concentration of strong positive quality-control product are 1 × 10⁷Copies/ μ L, weakly positive The concentration of quality-control product is 1 × 10³copies/μL。

10. being used for the kit for detecting high-risk human mammilla papillomavirus E6/E7mRNA partings according to claim 3, it is special Sign is：The negative controls are the water of DEPC processing.