CN104450954B

CN104450954B - Detect the fluorescent PCR kit and its method of 13 kinds of hypotypes of human papilloma virus

Info

Publication number: CN104450954B
Application number: CN201310446102.1A
Authority: CN
Inventors: 沈国成
Original assignee: NABI GENMED Inc
Current assignee: NABI GENMED Inc
Priority date: 2013-09-25
Filing date: 2013-09-25
Publication date: 2018-08-17
Anticipated expiration: 2033-09-25
Also published as: CN104450954A

Abstract

The invention discloses a kind of fluorescent PCR kits and its method of detection 13 kinds of hypotypes of human papilloma virus.The kit includes：PCR reaction solution, PCR mixed liquors, positive criteria product, internal reference product and negative reference product.Present invention employs unique quadruple fluorescent quantitative PCR techniques, separately design the corresponding PCR amplification primer and exploratory probe of 13 kinds of hypotypes of human papilloma virus, be divided into 2 pipes can disposable parting quantitatively detect 6 kinds of high-risk hypotypes of human papilloma virus, merge parting detection uncommon 7 kinds of high-risk hypotypes in Chinese population.

Description

Detect the fluorescent PCR kit and its method of 13 kinds of hypotypes of human papilloma virus

Technical field

The present invention relates to a kind of fluorescent PCR kits and its method of detection 13 kinds of hypotypes of human papilloma virus, belong to The detection field of human papilloma virus hypotype.The present invention relates to application multiple real time fluorescence quantifying PCR technology is anti-in two PCR Ying Guanzhong simultaneously rapidly shape quantitatively detect 6 kinds of high-risk hypotypes human papilloma virus (HPV-16,18,31,33,52, 58) and merge the uncommon high-risk hypotype human papilloma virus of 7 kinds of Chinese of sizing detection (HPV-35,39,45,51,56, 59,68) method.

Background technology

Human papilloma virus (Human Papilloma Virus, HPV)

It is a kind of double-stranded DNA virus, belongs to Papillomavirus, is a kind of specific infection skin or mucous membrane stratified epithelium Virus.Currently, HPV it has been found that hypotype has close to 200 kinds, is not shown bright after the wherein most infection mankind There are pathological manifestations in aobvious symptom, a small number of parts, some may cause mankind's wart, such as be grown near genitals skin and mucous membrane On mankind's verruca vulgaris, condyloma acuminatum.Some high-risk hypotypes can cause the tumour of the mankind, lead to cervical carcinoma, carcinoma of vagina, anus The generation of cancer, carcinoma of penis etc..Propagation wherein more than 30 kinds of HPV hypotypes is the lesion that passability contacted and caused genital area.

HPV infection is the direct cause of disease of cervical carcinoma, and the generation from infection to final cervical carcinoma has for Most patients One slow incubation period.Therefore, in time periodically to cervical exfoliated cell carry out HPV detection cervical carcinoma early screening, Auxiliary diagnosis and assessment etc. is particularly important in links more afterwards.Early stage is sent out in cervical carcinoma early symptom unobvious, screening early warning Existing cervical carcinoma, it is particularly important to reduce the death rate.The diagnosis of HPV at present places one's entire reliance upon the detection of HPV DNA in clinical samples. It is different carcinogenic that HPV partings and clinical binding have confirmed that different HPV genotype and different virus loads have Property, thus the parting of HPV DNA quantitatively detects forecast before teiology and cancer to cervical carcinoma and female genital tract tumor has weight The meaning wanted.

Since HPV cannot still be cultivated in vitro so far, and without suitable experimental animal, thus it is detected and is depended on Morphological Identification and molecular Biological Detection technology.Mainly cell pathology detection, spot print HPV detection methods in a organized way at present Mark method, fluorescence in situ hybridization, Southern hybrid methods, polymerase chainreaction method (Polymerase Chain Reaction, PCR) and hybrid capture etc..Cytology method susceptibility and specificity are relatively low, and dot blotting has radioactivity. The higher method of susceptibility has in situ hybridization and normal PCR method, but ribozyme trace hybridization in situ is complicated, and normal PCR method is special Property it is very low, false positive rate is higher, and unsuitable larger scale clinical uses.

At present HC-II be one by Food and Drug Administration (FDA) ratify can Clinical practice detection HPVDNA Technology, but it cannot carry out specific parting to HPV, and there are problems that crisscrossing.

Multiple fluorescence quantitative PCR technology, which can not only detect specific type, can also detect all types, be one have compared with The method of great development foreground.The shortcomings that for existing method and deficiency, we have various detection methods by continuous effort Machine combines, and learns from other's strong points to offset one's weaknesses, and develops simple, fast and easy, inexpensive, and sensibility, the very high HPV detection methods of specificity are simultaneously Applied in the screening of cervical carcinoma.

Real-time fluorescent PCR technology based on fluorescence labeling probe, using the most in current domestic clinical diagnosis Extensively.In the real-time fluorescence PCR reaction system using sonde method, according to the type of detected pathogen, it is further divided into list Weight or multiple real time fluorescence round pcr frequently include one or more pairs of Specific PCR primers and probe in reaction system, pass through Condition in R＆D process is groped, and probe and primer are only combined with corresponding template specificity, and binding site is at two Between primer.5 ' ends of probe are marked with reporter group (Reporter, R), such as FAM, VIC, ROX, and 3 ' ends are marked with fluorescence Quenching group (Quencher, Q), such as BHQ1, BHQ2, TAMRA.When probe is complete, reporter group is emitted glimmering Light energy is quenched group absorptions, and instrument can't detect signal.With the progress of PCR, Taq archaeal dna polymerases are in chain extension process In encounter the probe combined with template, 5 ' → 3 ' exonuclease activities will cut off probe, and reporter group is far from being quenched Group, energy cannot be absorbed, that is, generate fluorescence signal.So often passing through a PCR cycle, fluorescence signal is also and purpose Segment is the same, and there are one the processes that sync index increases.The key that each hypotypes of HPV are detected using real-time fluorescent PCR technology is to need The PCR primer and probe of each hypotype can specifically, sensitively be detected by designing.

Having two class HPV diagnostic kit one kind currently on the market is examined with PCR fluorescence methods and single fluorescent dye production HPV Disconnected kit, since probe molecule all in product all uses identical fluorescent dye, this product that cannot tell the Asias HPV Type classification can not also detect the amount of HPV different subtypes.Another kind of product is produced with gene chips, this product energy A variety of different HPV hypotypes classifications are told, but cannot detect their amount.In addition the kit of gene chips, operation It is complicated, the testing time is long, it is of high cost, be not easy to promote.

Invention content

The main object of the present invention, which is to provide one kind, can disposably detect 13 kinds of hypotype fluorescent PCRs of human papilloma virus Kit and its method.The invention is for normal to 6 kinds of high-risk Chinese in genitourinary tract secretion and cervical exfoliated cell The humam papillomavirus genotype seen carries out hypotype resolution and quantitative determination, uncommon virus subtype high-risk to 7 kinds merge Qualitative detection.

The fluorescence PCR detecting method of 13 kinds of hypotypes of human papilloma virus includes the following steps：

(1) design of primers：The human papilloma virus of 13 kinds of hypotypes of retrieval acquisition in gene pool, i.e. HPV-16,18,31, The conservative gene of 33,35,39,45,51,52,56,58,59,68 specificity determines the height of each hypotype by online sequence alignment Conserved sequence area is spent, special primer is designed as target gene specific nucleic acid sequence to be checked；

(2) design of fluorescence probe：According to the target gene specific nucleic acid sequence to be detected of 13 kinds of hypotypes in step (1) Design fluorescence probe；

(3) structure of standard molecule：According to the primer in step (1), builds 6 kinds using gene clone technology and contain respectively HPV-16, the standard plasmid molecule in 18,31,33,52,58 highly conserved sequence areas；

(4) composition of multiple fluorescence PCR reaction system：Using in the primer and step (2) in step (1) probe and PCR reaction solution, detected sample form multiple fluorescence PCR reaction system;

(5) optimization and foundation of multiple fluorescence PCR reaction system：By the group for adjusting the primer and probe of various concentration It closes, forms different multiple fluorescence quantitative PCR reaction systems, final PCR reaction systems are determined according to reaction efficiency and curve;

(6) prepared by standard curve：Using the multiple fluorescence PCR reaction system in step (5), by containing for known copy number The recombinant plasmid of purpose amplified fragments continuously presses 10 times of doubling dilution, and it is anti-to be added to quantitative fluorescent PCR as detected sample Ying Zhong, each item data that composite analyser provides set rational threshold value (Threshold) and baseline (Baseline), into Row interpretation of result, prepares standard curve；

(7) clinical samples detect：Use the clinical samples of known HPV genotype and virus load extract viral DNA as Template further carries out multiple fluorescence quantitative PCR amplification with (4) and (5) method and condition in claim.

The conservative gene of the specificity refers to capsid protein code area (L1) gene.

The gene specific nucleotide sequence to be detected is referred to for specific to each subtype virus gene One section of gene order.

The specificity fluorescent quantification PCR primer refers to that length is the oligonucleotide chain of 20-30bp, with target to be detected Gene specific nucleic acid sequence is identical or complementary.

The specificity fluorescent quantitative PCR fluorescence probe, refer to length be 20-30bp oligonucleotide chain with it is to be checked Survey target gene specific nucleic acid sequence is identical or complementary, and the 5 ' ends and 3 ' ends of all probes are marked using fluorescence.

Primer and its specific quantification PCR fluorescence probe sequences described in step (1), (2) is as follows：

HPV-16：

Forward primer sequence is：

SEQ ID NO：1(5'-CACGTAGTAGGTACTCCTTAAAGTT-3’)

Reverse primer sequences are：

SEQ ID NO：2(5'-CTATTCTTGATACTACACGCAGTAC-3’)

Fluorescence probe sequence A is：

SEQ ID NO：3(5’-FAM-CTTCCGTACCTACTTCAGAAMCTAC-BHQ-3’)

HPV-18：

Forward primer sequence is：

SEQ ID NO：4(5'-TTAGTCACTGTGGTAGATACTAC-3’)

Reverse primer sequences are：

SEQ ID NO：5(5'-GAGAACTAAACTGTAAATCATATTC-3’)

Fluorescence probe sequence B is：

SEQ ID NO：6(5'-HEX-TTATAGACGGTYTCCTGTACCTGG-BHQ-3’)

HPV-31：

Forward primer sequence is：

SEQ ID NO：7(5'-GGACAGTCAGTTATTTGTTACTGT-3’)

Reverse primer sequences are：

SEQ ID NO：8(5’-AAGTGTAAATCAAATTCCTCA-3’)

Fluorescence probe sequence C is：

SEQ ID NO：9(5’-HEX-TACTAGCAGATGTCTGTKTGTGCTGCAA-BHQ-3’)

HPV-33：

Forward primer sequence is：

SEQ ID NO：10(5’-GGTCACTCAGTTfATTGTTACTGT-3’)

Reverse primer sequences are：

SEQ ID NO：11(5’-GAGAATTAGACTGTAAATCATATTCTTC-3’)

Fluorescence probe sequence D is：

SEQ ID NO：12(5’-ROX-TGCCTCTAGGCCCACAGTAACAGAGAAAG-BHQ-3’)

HPV-35：

Forward primer sequence is：

SEQ ID NO：13(5’-GCATAGCCGGTCGTTTGTTAGTGAAG-3’)

Reverse primer sequences are：

SEQ ID NO：14(5’-GACAAGTACACTGTAATCATAATCATC-3’)

Fluorescence probe sequence E is：

SEQ ID NO：15(5’-FAM-TCAGTCTGTCTGCTGTGTCTWCTTGTCAC-BHQ-3’)

HPV-39：

Forward primer sequence is：

SEQ ID NO：16(5’-AAGCATTTATTCTTACTGATGAGG-3’)

Reverse primer sequences are：

SEQ ID NO：17(5’-ATGAACTGTAAAT℃ATACACRACC-3’)

Fluorescence probe sequence F is：

SEQ ID NO：18(5’-FAM-CCGACATTGCATATCTACCTCTATAGAGACTACC-BHQ-3’)

HPV-45：

Forward primer sequence is：

SEQ ID NO：19(5’-GCATACCGAGTTGTTTGTTAGTGAAG-3’)

Reverse primer sequences are：

SEQ ID NO：20(5’-AATTCGTATTCTCCACTTGACT-3’)

Fluorescence probe sequence G is：

SEQ ID NO：21(5’-FAM-TTCTGCGCATCTACACAAMTCGTGAGC-BHQ-3’)

HPV-51：

Forward primer sequence is：

SEQ ID NO：22(5’-CCGGTCTTGATACTACCACAACTAC-3’)

Reverse primer sequences are：

SEQ ID NO23：(5’-ATCAAGTGCAAATCATACACRACC-3’)

Fluorescence probe sequence H is：

SEQ ID NO24：(5’-FAM-TCTAAGTATTAGCTGCCACAGCAGC-BHQ-3’)

HPV-52：

Forward primer sequence is：

SEQ ID NO25：(5’-GAGAACTAGATTGTAAAT℃TAAAT℃-3’)

Reverse primer sequences are：

SEQ ID NO26：(5’-TTCGTGACAGTTGTGGAAACGAC-3’)

Fluorescence probe sequence I is：

SEQ ID NO27：(5’-CY5-TCACCGTGCGAAGTATACCATA-BHQ-3’)

HPV-56：

Forward primer sequence is：

SEQ ID NO28：(5’-GGACACTCGGTGATTTGTAACAGT-3’)

Reverse primer sequences are：

SEQ ID NO29：(5’-AACTCGTACTCGTCCACTTGACT-3’)

Fluorescence probe sequence J is：

SEQ ID NO30：(5’-FAM-TGGTACTTTGCGTGCATCATATTACATATCT-BHQ-3’)

HPV-58

Forward primer sequence is：

SEQ ID NO31：(5’-GACAAGCATACTGTAAGTCTTAATC-3’)

Reverse primer sequences are：

SEQ ID NO32：(5’-TTCGTCACCGTGGATGAAACGAC-3’)

Fluorescence probe sequence K is：

SEQ ID NO33：(5’-ROX-TTGTATACAGTYTCCTGTTCCAGG-BHQ-3’)

HPV-59：

Forward primer sequence is：

SEQ ID NO34：(5’-ATGCTCCTGGCAGCACGAAACT-3’)

Reverse primer sequences are：

SEQ ID NO35：(5’-GACATGTACACTGCAAATCTAAATC-3’)

Fluorescence probe sequence L is：

SEQ ID NO36：(5’-FAM-TCGACTTGCCTGCATATTCYTTTAATCT-BHQ-3’)

HPV-68：

Forward primer sequence is：

SEQ ID NO37：(5’-GGTATCAT℃GATTATTTCTAACAGT-3’)

Reverse primer sequences are：

SEQ ID NO38：(5’-AACTGCATATCAAATACCACA-3’)

Fluorescence probe sequence M is：

SEQ ID NO39：(5’-FAM-ATCGTACYATATGCCTTAATTTATTACGAACA-BHQ-3’)

Primer, the probe N of internal reference plasmid for control：

Forward primer sequence is：

SEQ ID NO40：(5’-CAATGCCAAGCTTGTCTGTAAGC-3’)

Forward primer sequence is：

SEQ ID NO41：(5’-TTGAATGTCGACATTCCTTTT-3’)

Fluorescence probe sequence N is：(5’-CY5-TCTGTACCATCAGACAGCGACCGGTA-BHQ-3’)

CY5, FAM, HEX, ROX 5 ' holds four kinds of different fluorescent reporter groups of label, 3 ' label BHQ fluorescent quenching bases Group.

The multiple fluorescence quantitative PCR reaction system is referred to by four groups of primers, four kinds of probes and PCR reaction solution group At quadruple quantitative fluorescent PCR system；And nine heavy quantitative fluorescent PCRs being made of with PCR reaction solution nine groups of primers, nine kinds of probes Reaction system.

The standard molecule refers to that the artificial recombination plasmid using gene clone technology structure, each plasmid only weigh A kind of special target-gene sequence to be checked of group.

The multiple fluorescence quantitative PCR reaction normal curve refers to：The mark of the hypotype of HPV-16,18,31,33,52,58 Quasi- plasmid difference dilution detects gained, the r of standard curve with quantitative fluorescent PCR reaction system²It is all higher than 0.97, meets fluorescence The requirement of quantitative PCR standard curve.

The present invention logs in GenBank and obtains correlated series, answer according to the conserved genetic sequences of 13 kinds of hypotypes of HPV viruse All sequences are compared with DNAMAN softwares and are analyzed, most conservative gene order is selected and is set with Primer Premier5.0 softwares Primer is counted, NCBI is logged in and analysis is compared by BLAST, the HPV of each hypotype filters out the strongest primer of a pair of of specificity and phase The specific probe answered, (can detect 6 kinds of fluorescence simultaneously) carries out single in Bioer LineGene9600 fluorescence quantitative PCR instruments Weight and multiple fluorescence PCR experiment, optimize multiple fluorescence quantitative PCR reaction system, determine the sensitivity of this method, specificity, again Renaturation etc..

The main purpose of invention is achieved through the following technical solutions：A kind of 13 kinds of Asias of detection human papilloma virus The fluorescent PCR kit of type, including following each component：PCR reaction solution；PCR mixed liquors;Negative reference product;Positive criteria product；It is interior Reference material.The PCR mixed liquors contain 14 kinds of probes：Probe A for detecting 16 hypotype of human papilloma virus；For examining Survey the probe B of HPV type 18 hypotype；Probe C for detecting 52 hypotype of human papilloma virus；For detecting people The probe D of 58 hypotype of papillomavirus；Probe E for detecting 31 hypotype of human papilloma virus；For detecting human milk head The probe F of 33 hypotype of shape tumor virus；Probe G for detecting 35 hypotype of human papilloma virus；For detecting people's papilloma The probe H of viral 39 hypotypes；Probe I for detecting 45 hypotype of human papilloma virus；For detecting human papilloma virus The probe J of 51 hypotypes；Probe K for detecting 56 hypotype of human papilloma virus；It is sub- for detecting human papilloma virus 59 The probe L of type；Probe M for detecting 68 hypotype of human papilloma virus;The probe N of internal reference product.The PCR reaction solution Including Taq archaeal dna polymerases, Buffer, MgCl₂、dNTPs；The internal reference product are the standard plasmid DNA for detecting human body gene, The negative reference product are sterile water, and positive criteria product is the HPV Plasmid DNA of known copy number.

The mixed liquor of the probe and upstream and downstream primer composition is sub-packed in first and second liang of pipes, wherein contains 0.5- in first pipe The probe B and its each 1.5- of upstream and downstream primer of each 1.5-2.5 μ l, 0.5-1.5 μ l of probe A and its upstream and downstream primer of 1.5 μ l Probe D and its upstream and downstream of the probe C and its each 1.5-2.5 μ l, 0.7-1.7 μ l of upstream and downstream primer of 2.5 μ l, 1.0-2.0 μ l Each 1.5-2.5 μ l of primer and sterilizing ultra-pure water is mended to 100 μ l；Probe E containing 0.4-1.4 μ l in second pipe and its upstream and downstream The probe G of the probe F and its each 1.5-2.5 μ l, 0.5-1.5 μ l of upstream and downstream primer of each 1.5-2.5 μ l, 0.62-1.62 μ l of primer And its probe H and its upstream and downstream primer each 1.5-2.5 μ l, 0.3-1.3 of each 1.5-2.5 μ l, 0.5-1.5 μ l of upstream and downstream primer The probe J and its each 1.5-2.5 μ of upstream and downstream primer of each 1.5-2.5 μ l, 0.5-1.5 μ l of probe I and its upstream and downstream primer of μ l The probe L and its upstream and downstream primer of each 1.5-2.5 μ l, 0.7-1.7 μ l of probe K and its upstream and downstream primer of l, 0.3-1.3 μ l The probe N of the probe M and its each 1.5-2.5 μ l, 0.1-1.1 μ l of upstream and downstream primer of each 1.5-2.5 μ l, 0.5-1.5 μ l and its Each 0.7-1.7 μ l of upstream and downstream primer and sterilizing ultra-pure water is mended to 100 μ l.

Each ingredient in reaction solution, the PCR reaction solution are stored in the third pipe as an optimization, by the Taq DNA of 6-10 μ l The MgCl of polymerase, the Buffer of 60-100 μ l, 80-100 μ l₂, 15-25 μ l dNTPs composition.

Detection method for the fluorescent PCR kit for detecting 13 kinds of hypotypes of human papilloma virus, steps are as follows：

(1) PCR amplification solution is prepared：5 μ of mixed liquor of probe A-D and its upstream and downstream primer composition is pipetted from first pipe L pipettes 5 μ l of PCR reaction solution from the third pipe, and the 8 μ l of distilled water of 2 μ l of sample to be tested, sterilizing are put into PCR pipe (1)；Simultaneously with 2 μ l Distilled water does negative reference, is designated as PCR pipe (2).The mixed liquor 5 of probe E-N and its upstream and downstream primer composition is pipetted from second pipe μ l, from the third pipe pipettes PCR reaction solution 5 μ l and 2 μ l of sample to be tested, sterilizing 8 μ l of distilled water are put into PCR pipe (3)；It uses simultaneously 2ul distilled waters do negative reference, are designated as PCR pipe (4).

(2) fluoroscopic examination：Prepared PCR pipe is placed on real-time fluorescence PCR instrument, setting reaction condition is：First Step, reacts 5min by 37 DEG C；Second step, reacts 5min by 94 DEG C；Third walks, 94 DEG C, reacts 15s;4th step, 50 DEG C, reaction 40s；Third walks and the 4th step cycle carries out 42 times, last reads fluorescence.

The main innovation of the present invention is：The present invention has multiple Taq Man probes, for each HPV hypotype, designs Probe contain that there are one unique fluorescent dyes corresponding thereto, therefore various HPV hypotypes can be told.Different from current city Similar product on field, our PCR amplification are Multiplex real-time PCR amplifications, and the intensity of various fluorescent dyes reflects in real time The PCR amplification amount of its corresponding HPV hypotype, the content of various hypotypes is gone out so as to quantitative analysis.The exclusive use of this kit Quadruple fluorescent quantitative PCR technique, separately designs the correspondent probe of 13 kinds of hypotypes of human papilloma virus and upper and lower primer, point 13 kinds of hypotype human papilloma viruses can be disposably detected for 2 pipes, often it is sub- to be divided into tetra- channels differentiations of FAM, HEX, ROX, CY5 for pipe Type.

The evaluation of the present invention：(1) accuracy in detection is high.The present invention be it is a kind of can early stage quickly, easy, effective detection The HPV viruse of clinical most common 6 kinds of high-risk hypotypes and 7 kinds belong to the more of high-risk hypotype but the uncommon HPV viruse of Chinese Weight real-time fluorescence quantitative PCR detection method.HPV-16,18,31,33,52,58 use parting quantitative analysis, HPV-35,39,45, 51, it 59,68 is analyzed using sizing is merged, and different subtype detection is not interfere with each other, it was demonstrated that its specificity is preferably；Random detection 50 A clinical sample is compared with the result of triumphant general similar product detection kit, and degree of conformity reaches 98.5%, and is tied with sequencing Fruit is consistent；By same sample (virus load 10⁴Copy/μ l) 10 PCR amplifications are carried out, result is the positive, due to using Fluorescein label probe, makes its detection sensitivity greatly improve, detection time greatly shortens, easy to operate, and can be special Large batch of sample analysis detection is carried out, more existing substance fluorescent quantitative PCR technique has greatly in terms of detecting energy It improves, improves a lot and improve in detection sensitivity, time-consuming, quantitative etc. compared with HC-II technologies.(2) safe to use Property is good.The present invention is external diagnosis reagent, is free of any harmful components, with subject without being in direct contact, securely and reliably.Kit Employed in internal reference product be Plasmid DNA, negative reference product be empty plasmid, PCR reaction solution (PCR Master Mix Buffer main component is harmless Taq archaeal dna polymerases, salt and water composition in), and above to human body, there is no safety issues.

Description of the drawings

The present invention is further elaborated with specific example below in conjunction with the accompanying drawings.

Fig. 1 is the production technological process of the present invention；

Fig. 2 is that the amplification curve of the present invention is followed successively by from top to bottom：HPV-18, HPV-16, HPV-52, HPV-58 amplification knot Fruit is schemed;

Fig. 3 is that the amplification curve of the present invention is followed successively by from top to bottom：HPV-31, HPV-33, HPV-35 amplification figures；

Fig. 4 is that the amplification curve of the present invention is followed successively by from top to bottom：HPV-31, HPV-39, HPV-33 amplification figure；

Fig. 5 is that the amplification curve of the present invention is followed successively by from top to bottom：HPV-31, HPV-33, HPV-45 amplification figure；

Fig. 6 is that the amplification curve of the present invention is followed successively by from top to bottom：HPV-31, HPV-33, HPV-35 amplification figure；

Fig. 7 is that the amplification curve of the present invention is followed successively by from top to bottom：HPV-31, HPV-33, HPV-56 amplification figure；

Fig. 8 is that the amplification curve of the present invention is followed successively by from top to bottom：HPV-31, HPV-33, HPV-59 amplification figure；

Fig. 9 is that the amplification curve of the present invention is followed successively by from top to bottom：HPV-31, HPV-68, HPV-33 amplification figure；

Figure 10 is the HPV16 canonical plottings of the present invention;

Figure 11 is the HPV18 canonical plottings of the present invention；

Figure 12 is the HPV52 canonical plottings of the present invention；

Figure 13 is the HPV58 canonical plottings of the present invention；

Figure 14 is the HPV31 canonical plottings of the present invention；

Figure 15 is the HPV33 canonical plottings of the present invention.

Specific implementation mode

Embodiment

A kind of fluorescent PCR kit of detection 13 kinds of hypotypes of human papilloma virus, including following each component：PCR is mixed Liquid；PCR reaction solution；Negative reference product；Internal reference product；Positive criteria product.

1. primer and probe

Pertinent literature is consulted, according to specific conservative's gene of 13 kinds of hypotypes of the Access to publication reported, logs in GenBank Correlated series are obtained, all sequences are compared using DNAMAN softwares and are analyzed, most conservative gene order Primer is selected Premier5.0 software Design primers log in NCBI and compare analysis by BLAST, and it is most strong that each hypotype filters out a pair of of specificity Primer and corresponding specific probe.The primer of each hypotype is closed with probe by Sangon Biotech (Shanghai) Co., Ltd. At, to ensure specificity and the sensitivity of multiple fluorescence quantitative PCR, all primer and probes with the amplification in GenBank Target sequence carries out BLAST, and the Tm values of all primer and probes are almost consistent.In order to increase target sequence is expanded with corresponding type HPV The specificity of hybridization, every probe and non-specific hybridization sequence at least 9 bases not complementary pairing.Using Bioer (can detect 6 kinds of fluorescence simultaneously) carries out multiple fluorescence PCR experiment in LineGene9600 fluorescence quantitative PCR instruments.

2, the identification and preservation of plasmid

The recombinant plasmid of all hypotypes is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.

The preservation of plasmid：The plasmid of extracting is placed in -20 DEG C of preservations.Qualitative reference product use same store method, will Plasmid after quantitative is diluted to 1 × 10 with distilled water⁰Copy/l-1 × 10 μ⁹Copy/μ l, -20 DEG C of preservations.

The positive criteria product is the standard plasmid molecule DNA of the highly conserved sequence of different subtype containing HPV, the internal reference The standard plasmid molecule DNA that product are the highly conserved sequence containing human body gene is examined, the negative reference product are the distilled water of sterilizing.

3, the extraction of DNA profiling

(1) sample is taken out, oscillation mixing (is thawed) as should be first set room temperature to freeze sample.1ml samples are drawn in band screw socket Centrifuge tube in, 12000rpm is centrifuged 5 minutes, and careful inhale abandons supernatant, retains sediment；

(2) it is added in 50 μ l cell pyrolysis liquids to sample sediment, is blown and beaten repeatedly with pipette tips, mixing, 95-100 DEG C of boiling water Bath/dry bath 10 minutes (avoiding booster)；

(3) sample 12000rpm is centrifuged 5 minutes, retains supernatant, takes 2 μ l for fluorescence quantitative PCR detection (if sample is split It is not used on the day of solution product, it is proposed that be stored in -20 DEG C of C).All experimental implementations should use the pipette tips and centrifuge tube of sterilizing, institute There is the preparation of sample to be carried out in sample process area.

4, quantitative fluorescent PCR condition optimizing

By the combination of the primer and probe of each concentration within the scope of 100-400nM, it is anti-to form different quantitative fluorescent PCRs System is answered, final PCR reaction systems are determined according to reaction efficiency and curve.

5, the specificity of quantitative fluorescent PCR, sensitivity tests

Specificity：Use the viral DNAs of known HPV types as detection sample, according to the difference of PCR mixed liquors, sample Addition is divided into two kinds of addition manners of 3 weights and 4 weights：When mixed liquor is first pipe, HPV-16,18,52,58 DNA sample are taken respectively Each 1 μ l;When mixed liquor is second pipe, HPV-31 is taken, 33 DNA sample and HPV-35, one in 39,45,51,56,59,68 Each 1 μ l of DNA sample, separately constitute 7 kind 3 same, reaction system and reaction condition are same as above.

Sensitivity experiments：The other HPV recombinant plasmids of four kinds of single types are subjected to 10 times of doubling dilutions at 1.0 × 10⁰Copy/ μl-1.0×10⁹Copy/μ l, compares as the template of multiple fluorescence quantitative PCR, while with aseptic double-distilled water, to different dilute The template for degree of releasing carries out fluorescent PCR detection according to fixed reaction system and amplification condition, and each sample that detects at least repeats Twice, the software analysis result carried with instrument after the completion of amplification establishes the minimum concentration of this method detection, assesses this method The detection sensitivity of HPV quantitative fluorescent PCRs.

6. the preparation of multiple fluorescence quantitative PCR standard curve

Using fixed multiple fluorescence PCR reaction system, HPV-16,18,52,58 4 kind of hypotype purpose amplification will be contained The recombinant plasmid of the known copy number of segment is uniformly mixed, and will contain HPV-31,33 two kinds of hypotype purpose amplified fragments it is known The recombinant plasmid of copy number is equally uniformly mixed, and continuous 10 times of doubling dilutions are divided to two pipes to be added to quantitative fluorescent PCR main reaction liquid In, each item data that composite analyser provides sets rational threshold value (Threshold) and baseline (Baseline), carries out Interpretation of result prepares standard curve；

8. result judgement

(1) analysis condition is set：Result is automatically analyzed according to instrument institute band analysis software.

(2) quality control standard：Blank control occurs without amplification curve and Ct values, the Ct values of the Ct values and standard items of internal reference control It is corresponding, and there is typical amplification curve, it is invalid otherwise to regard experiment.

(3) result and judgement：

It is negative：Without Ct values, no amplification curve.

Positive and its judgement：Value≤35 Ct, and typical amplification curve occur and be determined as the positive, certainly according to standard curve It is dynamic to obtain quantitative values.

Effective principle：If value >=35 Ct, sample amounts testing result is unreliable, need to reform experiment；If no Ct values are the moon Property.

As a result

1. the above-mentioned mixed liquor being made of probe and upstream and downstream primer is dissolved with following 14 kinds of probes：For detecting human milk head The probe A of 16 hypotype of shape tumor virus；Probe B for detecting HPV type 18 hypotype；For detecting people's papilloma The probe C of 2 hypotype of virus 5;Probe D for detecting 58 hypotype of human papilloma virus；For detecting human papilloma virus The probe E of 31 hypotypes;Probe F for detecting 33 hypotype of human papilloma virus；It is sub- for detecting human papilloma virus 35 The probe G of type；Probe H for detecting 39 hypotype of human papilloma virus；For detecting 45 hypotype of human papilloma virus Probe I；Probe J for detecting 51 hypotype of human papilloma virus；Probe for detecting 56 hypotype of human papilloma virus K；Probe L for detecting 59 hypotype of human papilloma virus；Probe M for detecting 68 hypotype of human papilloma virus；With In the probe N of the internal reference plasmid of control.

The mixed liquor of the probe and upstream and downstream primer composition is sub-packed in first and second liang of pipes, and 0.5-1.5 μ l are contained in first pipe Probe A and its each 1.5-2.5 μ l of upstream and downstream primer；Each 1.5-2.5 μ l of probe B and its upstream and downstream primer of 0.5-1.5 μ l； Each 1.5-2.5 μ l of probe C and its upstream and downstream primer of 1.0-2.0 μ l；The probe D and its upstream and downstream primer of 0.7-1.7 μ l is each 1.5-2.5μl;And sterilizing ultra-pure water is supplemented to 100 μ l.Probe E containing 0.4-1.4 μ l in second pipe and its upstream and downstream primer Each 1.5-2.5 μ l；Each 1.5-2.5 μ l of probe F and its upstream and downstream primer of 0.62-1.62 μ l；The probe G of 0.5-1.5 μ l and its Each 1.5-2.5 μ l of upstream and downstream primer；Each 1.5-2.5 μ l of probe H and its upstream and downstream primer of 0.5-1.5 μ l;0.3-l.3 μ l's Probe I and its each 1.5-2.5 μ l of upstream and downstream primer；Each 1.5-2.5 μ l of probe J and its upstream and downstream primer of 0.5-1.5 μ l； Each 1.5-2.5 μ l of probe K and its upstream and downstream primer of 0.3-1.3 μ l；The probe L and its upstream and downstream primer of 0.7-1.7 μ l is each 1.5-2.5μl；Each 1.5-2.5 μ l of probe M and its upstream and downstream primer of 0.5-1.5 μ l；The probe N of 0.1-1.1 μ l and thereon, Each 0.7-1.7 μ l of downstream primer and supplement sterilizing ultra-pure water are to 100 μ l.

The PCR reaction solution is stored in the third pipe, by the Taq archaeal dna polymerases of 6-8 μ l, Buffer, 80- of 60-100 μ l The MgCl of 100 μ l₂, 15-25 μ l dNTPs composition.

Reaction condition, which is arranged, is：The first step, reacts 5min by 37 DEG C；Second step, reacts 5min by 94 DEG C；Third walks, 94 DEG C DEG C, react 15s；4th step, reacts 40s by 50 DEG C;Third walks and the 4th step recycles 40 times, and fluorescence is read in the 4th step.

2. the specificity of multiple fluorescence quantitative PCR, sensitivity tests result：The specificity of each hypotype primer and probes of HPV It is 100%, sees Fig. 2-9.HPV-16,18,52,58,31,33 detection sensitivity are respectively：10⁴Copy/μ l, 10⁴Copy/μ l、10⁵Copy/μ l, 10⁴Copy/μ l, 10⁶Copy/μ l, 10⁵Copy/μ l.

3. the standard curve of each hypotype prepares result：PCR reaction systems after each hypotype HPV standard curves are optimized carry out It prepares, HPV-16, the r of 18,52,58,31,33 standard curve²Respectively 0.970,0.995,0.970,0.986,0.982, 0.983, see Figure 10-15.

One of the above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, Any technical person familiar with the field within the technical scope disclosed by the invention, the change that can be expected without creative work Change or replace, should be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with claims Defined by subject to protection domain.

Claims

1. detecting the PCR kit for fluorescence quantitative of 13 kinds of hypotypes of human papilloma virus, kit includes following each component：PCR Reaction solution, PCR mixed liquors, positive criteria product, internal reference product and negative reference product；The PCR reaction solution includes Taq DNA poly- Synthase, Buffer, MgCl₂And dNTPs, the positive criteria product are containing 6 common high-risk HPV hypotypes：HPV-16,18,31, The standard plasmid molecule DNA of 33,52,58 highly conserved sequence, the internal reference product are containing the highly conserved sequence of human body gene The standard plasmid molecule DNA of row, the negative reference product are the ultra-pure water of sterilizing, and the PCR mixed liquors expand respectively including 13 sets Increase the primer and probe of the probe and 1 set of detection human body gene of HPV13 hypotype of primer and detection of HPV13 hypotype, respectively It is described below：

Forward primer sequence for detecting 16 hypotype of human papilloma virus is SEQ ID NO：1, reverse primer sequences SEQ ID NO：2, fluorescence probe sequence is SEQ ID NO：3, wherein 5 ' end mark fluorescent reporter group FAM, 3 ' mark fluorescents are quenched Group BHQ；It is SEQ ID NO for detecting HPV type 18 hypotype forward primer sequence：4, reverse primer sequences are SEQ ID NO：5, fluorescence probe sequence is SEQ ID NO：6, wherein 5 ' end mark fluorescent reporter group HEX, 3 ' mark fluorescents Quenching group BHQ；Forward primer sequence for detecting 31 hypotype of human papilloma virus is SEQ ID NO：7, reverse primer Sequence is SEQ ID NO：8, fluorescence probe sequence is SEQ ID NO：9, wherein 5 ' end mark fluorescent reporter group HEX, 3 ' marks Remember fluorescent quenching group BHQ；Forward primer sequence for detecting 33 hypotype of human papilloma virus is SEQ ID NO：10, instead It is SEQ ID NO to primer sequence：11, fluorescence probe sequence is SEQ ID NO：12, wherein 5 ' end mark fluorescent reporter groups ROX, 3 ' mark fluorescent quenching group BHQ；Forward primer sequence for detecting 35 hypotype of human papilloma virus is SEQ ID NO：13, reverse primer sequences are SEQ ID NO：14, fluorescence probe sequence is SEQ ID NO：15, wherein 5 ' end mark fluorescents Reporter group FAM, 3 ' mark fluorescent quenching group BHQ；Forward primer sequence for detecting 39 hypotype of human papilloma virus For SEQ ID NO：16, reverse primer sequences are SEQ ID NO：17, fluorescence probe sequence is SEQ ID NO：18, wherein 5 ' ends Mark fluorescent reporter group FAM, 3 ' mark fluorescent quenching group BHQ；Forward direction for detecting 45 hypotype of human papilloma virus Primer sequence is SEQ ID NO：19, reverse primer sequences are SEQ ID NO：20, fluorescence probe sequence is SEQ ID NO：21, Wherein 5 ' end mark fluorescent reporter group FAM, 3 ' mark fluorescent quenching group BHQ；It is sub- for detecting human papilloma virus 51 The forward primer sequence of type is SEQ ID NO：22, reverse primer sequences are SEQ ID NO：23, fluorescence probe sequence is SEQ ID NO：24, wherein 5 ' end mark fluorescent reporter group FAM, 3 ' mark fluorescent quenching group BHQ；For detecting people's papilloma The forward primer sequence of 2 hypotype of virus 5 is SEQ ID NO：25, reverse primer sequences are SEQ ID NO：26, fluorescence probe sequence It is classified as SEQ ID NO：27, wherein 5 ' end mark fluorescent reporter group Cy5,3 ' mark fluorescent quenching group BHQ；For detecting people The forward primer sequence of 56 hypotype of papillomavirus is SEQ ID NO：28, reverse primer sequences are SEQ ID NO：29, it is glimmering Light probe sequence is SEQ ID NO：30, wherein 5 ' end mark fluorescent reporter group FAM, 3 ' mark fluorescent quenching group BHQ；With It is SEQ ID NO in detecting the forward primer sequence of 58 hypotype of human papilloma virus：31, reverse primer sequences are SEQ ID NO：32, fluorescence probe sequence is SEQ ID NO：33, wherein base is quenched in 5 ' end mark fluorescent reporter group ROX, 3 ' mark fluorescents Group BHQ；Forward primer sequence for detecting 59 hypotype of human papilloma virus is SEQ ID NO：34, reverse primer sequences are SEQ ID NO：35, fluorescence probe sequence is SEQ ID NO：36, wherein 5 ' end mark fluorescent reporter group FAM, 3 ' labels are glimmering Optical quenching group BHQ；Forward primer sequence for detecting 68 hypotype of human papilloma virus is SEQ ID NO：37, reversely draw Object sequence is SEQ ID NO：38, fluorescence probe sequence is SEQ ID NO：39, wherein 5 ' end mark fluorescent reporter group FAM, 3 ' mark fluorescent quenching group BHQ；The forward primer sequence of detection human body gene for internal reference is SEQ ID NO：40, reversely Primer sequence is SEQ ID NO：41, fluorescence probe sequence is SEQ ID NO：42, wherein 5 ' end mark fluorescent reporter groups Cy5,3 ' mark fluorescent quenching group BHQ.

2. the fluorescent PCR kit of detection 13 kinds of hypotypes of human papilloma virus according to claim 1, feature exist In：The mixed liquor of the probe and primer composition is sub-packed in two pipe of first, second；

The probe and its each 1.5-2.5 μ l of upstream and downstream primer of HPV16 are detected in first pipe containing 0.5-1.5 μ l；Detect HPV18's Each 1.5-2.5 μ l of probe and its upstream and downstream primer of 0.5-1.5 μ l；Detect the probe of the 1.0-2.0 μ l of HPV52 and its upper and lower Swim each 1.5-2.5 μ l of primer；Detect the probe and its each 1.5-2.5 μ l of upstream and downstream primer of the 0.7-1.7 μ l of HPV58；And it mends Ultra-pure water sterilize to 100 μ l；

0.4-1.4 μ l probes and its each 1.5-2.5 μ l of upstream and downstream primer containing detection HPV31 in second pipe；Detect HPV33's 0.62-1.62 μ l probes and its each 1.5-2.5 μ l of upstream and downstream primer；Detect 0.5-1.5 μ l probes and its upstream and downstream of HPV35 Each 1.5-2.5 μ l of primer；Detect the 0.5-1.5 μ l probes and its each 1.5-2.5 μ l of upstream and downstream primer of HPV39；Detect HPV45 0.3-1.3 μ l probes and its each 1.5-2.5 μ l of upstream and downstream primer；Detect 0.5-1.5 μ l probes and its upstream and downstream of HPV51 Each 1.5-2.5 μ l of primer；Detect the 0.3-1.3 μ l probes and its each 0.5-1.5 μ l of upstream and downstream primer of HPV56；Detect HPV59 0.7-1.7 μ l probes and its each 1.5-2.5 μ l of upstream and downstream primer；Detect 0.5-1.5 μ l probes and its upstream and downstream of HPV68 Each 1.5-2.5 μ l of primer；The probe and its each 0.7-1.7 μ l of upstream and downstream primer of 0.1-1.1 μ l detection human body genes；And it mends Ultra-pure water sterilize to 100 μ l.

3. the fluorescent PCR kit of detection 13 kinds of hypotypes of human papilloma virus according to claim 1, feature exist In：The PCR reaction solution is stored in the third pipe, by the Taq archaeal dna polymerases of 6-10 μ l, PCR Buffer, the 80- of 60-100 μ l The MgCl of 100 μ l₂, 15-25 μ l dNTPs composition.