CN104450954A - Fluorescent PCR (polymerase chain reaction) kit and method for detecting 13 subtypes of human papillomavirus - Google Patents

Abstract

The invention discloses a fluorescent PCR (polymerase chain reaction) kit and a method for detecting 13 subtypes of a human papillomavirus. The kit comprises a PCR liquid, a PCR mixed liquid, a positive standard substance, an internal reference substance and a negative reference substance. According to the fluorescent PCR kit disclosed by the invention, a unique quadruple fluorescent quantitative PCR technology is adopted, PCR amplification primers and detection probes corresponding to the 13 subtypes of the human papillomavirus are respectively designed, six high-risk subtypes of the human papillomavirus are quantitatively detected in two tubes by one step in a typing mode, and unusual seven high-risk subtypes in the Chinese population which are detected in the typing mode are combined.

Description

Detect fluorescent PCR kit and the method thereof of human papillomavirus 13 kinds of hypotypes

Technical field

The present invention relates to a kind of fluorescent PCR kit and the method thereof that detect human papillomavirus 13 kinds of hypotypes, belong to the detection field of human papillomavirus hypotype.The present invention relates to application multiple real time fluorescence quantifying PCR technology to shape rapidly in two PCR reaction tubess the human papillomavirus (HPV-16 of detection by quantitative 6 kinds of high-risk hypotypes simultaneously, 18,31,33,52,58) and merge sizing detection 7 kinds of Chinese uncommon high-risk hypotype human papillomavirus (HPV-35,39,45,51,56,59,68) method.

Background technology

Human papillomavirus (Human Papilloma Virus, HPV)

Being a kind of double-stranded DNA virus, belonging to Papillomavirus, is the virus of a kind of specific infection skin or mucous membrane stratified epithelium.At present, the hypotype that HPV has been found has close to 200 kinds, and wherein major part does not show obvious symptom after infecting the mankind, and minority part has pathological manifestations, some may cause mankind's wart, as mankind verruca vulgaris, the pointed condyloma of growth near genitals skin and mucous membrane.Some high-risk hypotypes can cause the tumour of the mankind, cause the generation of cervical cancer, carcinoma of vagina, anus cancer, penile cancer etc.Wherein contacted by property and cause the pathology of genital region more than the propagation of 30 kinds of HPV hypotypes.

It is the direct cause of disease of cervical cancer that HPV infects, and has latent period slowly from the generation infected to final cervical cancer to Most patients.Therefore, regularly in time the early screening of detection in cervical cancer of HPV, auxiliary diagnosis are carried out to cervical exfoliated cell and more seems particularly important in the link such as later evaluation.Cervical cancer early symptom is not obvious, and examination early warning, to early discovery cervical cancer, reduces mortality ratio very important.The diagnosis of current HPV places one's entire reliance upon the detection of HPV DNA in clinical samples.HPV somatotype and clinical binding have confirmed that different HPV genotype and different virus loads have different carinogenicity, and thus the somatotype detection by quantitative of HPV DNA has great importance to forecasting before the nosetiology of cervical cancer and female genital tract tumor and cancer.

Because HPV still can not cultivate so far in vitro, again without suitable laboratory animal, thus Morphological Identification and molecular Biological Detection technology are depended on to its detection.Current HPV detection method mainly contains histiocytosis's detection of science, dot blotting, fluorescence in situ hybridization, Southern hybrid method, polymerase chainreaction method (Polymerase Chain Reaction, PCR) and hybrid capture etc.Cytology method susceptibility and specific degree lower, dot blotting has radioactivity.The method that susceptibility is higher has in situ hybridization and normal PCR method, but ribozyme trace hybridization in situ is complicated, and normal PCR method specificity is very low, and false positive rate is higher, and unsuitable larger scale clinical uses.

Current HC-II be one by Food and Drug Administration (FDA) ratify can the technology of detection HPVDNA of Clinical practice, but it can not carry out concrete somatotype to HPV, and there is the problem of cross hybridization.

Multiple fluorescence quantitative PCR technology not only can detect concrete type can also detect all types, is a method having large development prospect.For existing methodical shortcoming and defect, various detection method is organically combined by continuous effort, is learnt from other's strong points to offset one's weaknesses by we, develop simple, fast and easy, inexpensive, and the very high HPV detection method of susceptibility, specificity being applied in the examination of cervical cancer.

Real-time fluorescence PCR technology based on fluorescence labeling probe, is most widely used in clinical diagnosis domestic at present.In the real-time fluorescence PCR reaction system adopting probe method, according to the kind of detected pathogenic agent, substance or multiple real time fluorescence round pcr can also be divided into, one or more pairs of Specific PCR primers and probe is often comprised in reaction system, groped by the condition in R&D process, probe and primer only with corresponding template specificity combine, its binding site is between two primers.5 ' end of probe is marked with reporter group (Reporter, R), and as FAM, VIC, ROX etc., 3 ' end is marked with fluorescent quenching group (Quencher, Q), as BHQ1, BHQ2, TAMRA etc.When probe is complete time, the fluorescent energy that reporter group is launched is quenched group absorptions, and instrument can't detect signal.Along with the carrying out of PCR, Taq archaeal dna polymerase runs into the probe be combined with template in chain extension process, and probe will cut off by its 5 ' → 3 ' exonuclease activity, and reporter group is away from quenching group, and its energy can not be absorbed, and namely produces fluorescent signal.So often through a PCR circulation, fluorescent signal is also the same with object fragment, has the process that a sync index increases.Adopt the key of real-time fluorescence PCR technology for detection HPV each hypotype be need to design can be special, sensitive the PCR primer detecting each hypotype and probe.

Two class HPV diagnostic kit one classes are had to be produce HPV diagnostic kit with PCR fluorescent method and single fluorescence dye in the market, because probe molecules all in product all uses identical fluorescence dye, this product can not tell HPV hypotype classification, also cannot detect the amount of HPV different subtype.Another kind of product gene chips is produced, and this product can tell various different HPV hypotype classification, but can not detect their amount.In addition the test kit of gene chips, complicated operation, the test duration is long, cost is high, not easily promote.

Summary of the invention

Main purpose of the present invention is to provide one can disposable detection human papillomavirus 13 kinds of hypotype fluorescent PCR kits and method thereof.This invention is used for carrying out hypotype resolution and quantitative assay to kind of the humam papillomavirus genotype that high-risk Chinese are common of 6 in genitourinary tract secretory product and cervical exfoliated cell, carries out merging qualitative detection to 7 kinds of high-risk uncommon virus subtypes.

The fluorescence PCR detecting method of human papillomavirus 13 kinds of hypotypes comprises the steps:

(1) design of primers: the human papillomavirus retrieving acquisition 13 kinds of hypotypes in gene pool, i.e. HPV-16,18,31,33,35,39,45,51,52,56,58,59,68 specific conservative genes, by online sequence alignment, determine the highly conserved sequence district of each hypotype, as target gene specific nucleotide sequence design special primer to be checked;

(2) design of fluorescent probe: according to the target gene specific nucleotide sequence design fluorescent probe to be detected of 13 kinds of hypotypes in step (1);

(3) structure of standard molecule: according to the primer in step (1), utilizes gene clone technology to build 6 kinds respectively containing HPV-16,18,31,33,52, the standard plasmid molecule in 58 highly conserved sequence districts;

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

(5) optimization of multiple fluorescence PCR reaction system and foundation: by regulating the primer of different concns and the combination of probe, forming different multiple fluorescence quantitative PCR reaction systems, determining final PCR reaction system according to reaction efficiency and curve;

(6) typical curve preparation: use the multiple fluorescence PCR reaction system in step (5), by the recombinant plasmid containing object amplified fragments of known copy number continuously by the doubling dilution of 10 times, join in quantitative fluorescent PCR reaction as detected sample, every data that composite analyser provides, set rational threshold value (Threshold) and baseline (Baseline), carry out interpretation of result, preparation standard curve;

(7) clinical samples detects: the viral DNA extracted with the clinical samples of known HPV genotype and virus load, as template, uses (4) in claim and (5) method and condition to carry out multiple fluorescence quantitative PCR amplification further.

Described specific conservative gene refers to capsid protein coding region (L1) gene.

Described gene specific nucleotide sequence to be detected refers to for a fragment gene sequence specific to each subtype virus gene.

Described specificity fluorescent quantification PCR primer refers to the oligonucleotide chain that length is 20-30bp, identical or complementary with target gene specific nucleotide sequence to be detected.

Described specificity fluorescent quantitative PCR fluorescent probe, referring to length is that the oligonucleotide chain of 20-30bp is identical or complementary with target gene specific nucleotide sequence to be detected, and 5 ' end of all probes and 3 ' end all use fluorescence mark.

Step (1), primer described in (2) and specific quantification PCR fluorescent probe sequence thereof are as follows:

HPV-16：

Forward primer sequence is:

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

Reverse primer sequences is:

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

Fluorescent 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 is:

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

Fluorescent 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 is:

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

Fluorescent 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 is:

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

Fluorescent 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 is:

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

Fluorescent 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 is:

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

Fluorescent 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 is:

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

Fluorescent 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 is:

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

Fluorescent 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 is:

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

Fluorescent 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 is:

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

Fluorescent 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 is:

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

Fluorescent 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 is:

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

Fluorescent 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 is:

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

Fluorescent probe sequence M is:

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

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

Forward primer sequence is:

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

Forward primer sequence is:

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

Fluorescent probe sequence N is: (5 '-CY5-TCTGTACCATCAGACAGCGACCGGTA-BHQ-3 ')

CY5, FAM, HEX, ROX are the 5 ' fluorescent reporter group holding mark four kinds different, 3 ' mark BHQ fluorescent quenching group.

Described multiple fluorescence quantitative PCR reaction system, refers to the quadruple quantitative fluorescent PCR system be made up of four groups of primers, four kinds of probes and PCR reaction solution; And the nine heavy quantitative fluorescent PCR reaction systems to be made up of nine groups of primers, nine kinds of probes and PCR reaction solution.

Described standard molecule, refers to the artificial recombination plasmid utilizing gene clone technology to build, and often kind of plasmid is only recombinated a kind of special target-gene sequence to be checked.

Described multiple fluorescence quantitative PCR reaction normal curve refers to: HPV-16,18,31,33,52, the standard plasmid of 58 hypotypes different extent of dilution quantitative fluorescent PCR reaction system detects gained, the r of typical curve ²all be greater than 0.97, meet the requirement of quantitative fluorescent PCR typical curve.

The present invention is according to the conserved genetic sequences of 13 of HPV virus kinds of hypotypes, log in GenBank and obtain correlated series, application DNAMAN software is to all sequences compare of analysis, select the most conservative gene order Primer Premier5.0 software design primer, log in NCBI by BLAST compare of analysis, the HPV of often kind of hypotype filters out the strongest primer of a pair specificity and corresponding specific probe, in Bioer LineGene9600 quantitative real time PCR Instrument, (6 kinds of fluorescence can be detected) simultaneously carry out substance and multiple fluorescence PCR experiment, optimize multiple fluorescence quantitative PCR reaction system, determine the sensitivity of the method, specificity, repeatability etc.

The main purpose of invention is achieved through the following technical solutions: a kind of fluorescent PCR kit detecting human papillomavirus 13 kinds of hypotypes, comprises following component: PCR reaction solution; PCR mixed solution; Negative reference product; Positive criteria product; Internal reference product.Described PCR mixed solution contains 14 kinds of probes: for detecting the probe A of human papillomavirus 16 hypotype; For detecting the probe B of HPV type 18 hypotype; For detecting the probe C of human papillomavirus 52 hypotype; For detecting the probe D of human papillomavirus 58 hypotype; For detecting the probe E of human papillomavirus 31 hypotype; For detecting the probe F of human papillomavirus 33 hypotype; For detecting the probe G of human papillomavirus 35 hypotype; For detecting the probe H of human papillomavirus 39 hypotype; For detecting the probe I of human papillomavirus 45 hypotype; For detecting the probe J of human papillomavirus 51 hypotype; For detecting the probe K of human papillomavirus 56 hypotype; For detecting the probe L of human papillomavirus 59 hypotype; For detecting the probe M of human papillomavirus 68 hypotype; The probe N of internal reference product.Described PCR reaction solution comprises Taq archaeal dna polymerase, Buffer, MgCl ₂, dNTPs; Described internal reference product are the standard plasmid DNA of human body gene, and described negative reference product are sterilized water, and positive criteria product are the HPV plasmid DNA of known copy number.

The mixed solution of described probe and upstream and downstream primer composition is sub-packed in first and second liang of pipes, probe A wherein containing 0.5-1.5 μ l in first pipe and upstream and downstream primer each 1.5-2.5 μ l thereof, the probe B of 0.5-1.5 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof, the probe C of 1.0-2.0 μ l and the probe D of upstream and downstream primer each 1.5-2.5 μ l, 0.7-1.7 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof thereof and benefit sterilizing ultrapure water to 100 μ l, in second pipe containing 0.4-1.4 μ l probe E and on, downstream primer each 1.5-2.5 μ l, the probe F of 0.62-1.62 μ l and on, downstream primer each 1.5-2.5 μ l, the probe G of 0.5-1.5 μ l and on, downstream primer each 1.5-2.5 μ l, the probe H of 0.5-1.5 μ l and on, downstream primer each 1.5-2.5 μ l, the probe I of 0.3-1.3 μ l and on, downstream primer each 1.5-2.5 μ l, the probe J of 0.5-1.5 μ l and on, downstream primer each 1.5-2.5 μ l, the probe K of 0.3-1.3 μ l and on, downstream primer each 1.5-2.5 μ l, the probe L of 0.7-1.7 μ l and on, downstream primer each 1.5-2.5 μ l, the probe M of 0.5-1.5 μ l and on, downstream primer each 1.5-2.5 μ l, the probe N of 0.1-1.1 μ l and on, downstream primer each 0.7-1.7 μ l and benefit sterilizing ultrapure water to 100 μ l.

As each composition optimized in reaction solution, described PCR reaction solution is stored in the third pipe, by the MgCl of the Taq archaeal dna polymerase of 6-10 μ l, Buffer, 80-100 μ l of 60-100 μ l ₂, 15-25 μ l dNTPs composition.

For detecting the detection method of the fluorescent PCR kit of human papillomavirus 13 kinds of hypotypes, step is as follows:

(1) prepare pcr amplification solution: the mixed solution 5 μ l pipetting probe A-D and upstream and downstream primer composition thereof from first pipe, pipette PCR reaction solution 5 μ l from the third pipe, testing sample 2 μ l, the distilled water 8 μ l of sterilizing puts into PCR pipe (1); Do negative reference with 2 μ l distilled waters simultaneously, be designated as PCR pipe (2).From second pipe, pipette the mixed solution 5 μ l of probe E-N and upstream and downstream primer composition thereof, pipette PCR reaction solution 5 μ l from the third pipe, and testing sample 2 μ l, sterilizing distilled water 8 μ l put into PCR pipe (3); Do negative reference with 2ul distilled water simultaneously, be designated as PCR pipe (4).

(2) fluoroscopic examination: prepared PCR pipe is placed on real-time fluorescence PCR instrument, arranging reaction conditions is: the first step, 37 DEG C, reaction 5min; Second step, 94 DEG C, reaction 5min; 3rd step, 94 DEG C, reaction 15s; 4th step, 50 DEG C, reaction 40s; 3rd step and the circulation of the 4th step carry out 42 times, finally read fluorescence.

Main innovation of the present invention is: the present invention has multiple Taq Man probe, and for each HPV hypotype, the probe designed contains the fluorescence dye of a uniqueness corresponding thereto, therefore can tell various HPV hypotype.Be different from like product in the market, our pcr amplification is Multiplex real-time PCR amplification, and the intensity of various fluorescence dye reflects the pcr amplification amount of the HPV hypotype of its correspondence in real time, thus energy quantitative analysis goes out the content of various hypotype.What this test kit was exclusive have employed quadruple fluorescent quantitative PCR technique, the correspondent probe of difference designer papillomavirus 13 kinds of hypotypes and upper and lower primer, be divided into 2 pipes can disposable detection 13 kinds of hypotype human papillomaviruses, often pipe be divided into FAM, HEX, ROX, CY5 tetra-passages to distinguish hypotypes.

Evaluation of the present invention: (1) accuracy in detection is high.The present invention be a kind of can in early days the clinical modal 6 kinds of high-risk hypotypes of quick, easy, effective detection HPV virus and 7 kinds belong to high-risk hypotype but the multiple real time fluorescence quantifying PCR detection method of the uncommon HPV virus of Chinese.HPV-16,18,31,33,52,58 adopts somatotype quantitative analyses, and HPV-35,39,45,51,59,68 adopts and merges sizing and analyze, and different subtype detects and do not interfere with each other, and proves that its specificity is better; Random detection 50 clinical samples, contrast with the result of triumphant general like product detection kit, degree of conformity reaches 98.5%, and consistent with sequencing result; By same sample, (virus load is 10 ⁴copy/μ l) carry out 10 pcr amplifications, result is the positive, owing to have employed fluorescein-labelled probe, its detection sensitivity is improved greatly, detection time shortens greatly, easy and simple to handle, and specially can carry out large batch of sample analysis detection, more existing substance fluorescent quantitative PCR technique is greatly improved in detected energy, compared with HC-II technology detection sensitivity, consuming time, quantitative etc. in all improve a lot and improvement.(2) safety in utilization is good.The present invention is external diagnosis reagent, not containing any objectionable constituent, with person under inspection without directly contacting, safe and reliable.The internal reference product adopted in test kit are plasmid DNA, negative reference product are empty plasmid, in PCR reaction solution (PCR Master Mix buffer), main component is harmless Taq archaeal dna polymerase, salt and water composition, and above do not exist safety issue to human body.

Accompanying drawing explanation

Below in conjunction with accompanying drawing and specific examples, the present invention is further elaborated.

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

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

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

Fig. 4 is that 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 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 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 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 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 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 HPV16 canonical plotting of the present invention;

Figure 11 is HPV18 canonical plotting of the present invention;

Figure 12 is HPV52 canonical plotting of the present invention;

Figure 13 is HPV58 canonical plotting of the present invention;

Figure 14 is HPV31 canonical plotting of the present invention;

Figure 15 is HPV33 canonical plotting of the present invention.

Embodiment

Embodiment

Detect a fluorescent PCR kit for human papillomavirus 13 kinds of hypotypes, comprise following component: PCR mixed solution; PCR reaction solution; Negative reference product; Internal reference product; Positive criteria product.

1. primer and probe

Consult pertinent literature, according to specific conservative's gene of reported Access to publication 13 kinds of hypotypes, log in GenBank and obtain correlated series, application DNAMAN software is to all sequences compare of analysis, select the most conservative gene order Primer Premier5.0 software design primer, log in NCBI by BLAST compare of analysis, often kind of hypotype filters out the strongest primer of a pair specificity and corresponding specific probe.Primer and the probe of each hypotype are synthesized by Sangon Biotech (Shanghai) Co., Ltd., for guaranteeing specificity and the sensitivity of multiple fluorescence quantitative PCR, all primers and probe all carry out BLAST with the amplified target sequence in GenBank, and all primers are almost consistent with the Tm value of probe.In order to increase the specificity with corresponding type HPV amplified target sequence hybridization, every bar probe and non-specific hybridization sequence have 9 bases not complementary pairing at least.Adopt (6 kinds of fluorescence can be detected) in Bioer LineGene9600 quantitative real time PCR Instrument to carry out multiple fluorescence PCR experiment simultaneously.

2, the qualification of plasmid and preservation

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 adopt same store method, and the plasmid distilled water is quantitatively diluted to 1 × 10 ⁰copy/μ l-1 × 10 ⁹copy/μ l ,-20 DEG C of preservations.

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

3, the extraction of DNA profiling

(1) sample is taken out, vibration mixing (thawing as should be first put room temperature for frozen sample).Draw 1ml sample in the centrifuge tube of band screw socket, centrifugal 5 minutes of 12000rpm, careful suction abandons supernatant, retains throw out;

(2) add 50 μ l cell pyrolysis liquids in sample throw out, repeatedly blow and beat with rifle head, mix, 95-100 DEG C of boiling water bath/dry bath 10 minutes (avoiding booster);

(3) centrifugal 5 minutes of sample 12000rpm, retains supernatant, gets 2 μ l for fluorescence quantitative PCR detection (if sample split product did not use the same day, suggestion was kept at-20 DEG C of C).All experimental implementation should adopt rifle head and the centrifuge tube of sterilizing, and the preparation of all samples is all carried out in sample process district.

4, quantitative fluorescent PCR condition optimizing

By the primer of each concentration within the scope of 100-400nM and the combination of probe, form different quantitative fluorescent PCR reaction systems, determine final PCR reaction system according to reaction efficiency and curve.

5, the specificity of quantitative fluorescent PCR, sensitivity test

Specificity: with the viral DNA of known HPV type as detection sample, according to the difference of PCR mixed solution, the interpolation of sample is divided into 3 heavy and 4 heavy two kinds of addition manners: when mixed solution is first pipe, get HPV-16 respectively, 18,52, each 1 μ l of DNA sample of 58; When mixed solution is second pipe, get HPV-31, the DNA sample of 33 and HPV-35,39,45,51,56,59, each 1 μ l of a DNA sample in 68, respectively form 7 kind 3 same, reaction system and reaction conditions the same.

Sensitivity experiments: other HPV recombinant plasmid of four kinds of single types is carried out 10 times of doubling dilutions and becomes 1.0 × 10 ⁰copy/μ l-1.0 × 10 ⁹copy/μ l, as the template of multiple fluorescence quantitative PCR, contrast with aseptic double-distilled water simultaneously, according to fixed reaction system and amplification condition, fluorescent PCR detection is carried out to the dilution template of difference, each detection sample at least repeats twice, the software analysis result that the rear instrument that increased carries, establishes the minimum concentration that the method detects, the detection sensitivity of the HPV quantitative fluorescent PCR of assessment the method.

6. the preparation of multiple fluorescence quantitative PCR typical curve

Use fixed multiple fluorescence PCR reaction system, by containing HPV-16,18,52, the recombinant plasmid of the known copy number of 58 4 kind of hypotype object amplified fragments mixes, will containing HPV-31, the recombinant plasmid of the known copy number of 33 two kinds of hypotype object amplified fragments mixes equally, continuous 10 times of doubling dilutions, two pipes are divided to join in quantitative fluorescent PCR main reaction liquid, every data that composite analyser provides, set rational threshold value (Threshold) and baseline (Baseline), carry out interpretation of result, preparation standard curve;

8. result judges

(1) analysis condition setting: be with analysis software automatic analysis result according to instrument.

(2) quality control standard: blank occurs without amplification curve and Ct value, the Ct value of internal reference contrast is corresponding with the Ct value of standard substance, and occurs typical amplification curve, otherwise it is invalid to look experiment.

(3) result and judgement:

Negative: without Ct value, without amplification curve.

The positive and judgement thereof: Ct value≤35, and occur that namely typical amplification curve is judged to be the positive, according to typical curve automatic acquisition quantitative values.

Effective principle: if Ct value >=35, then sample amounts detected result is unreliable, experiment of need reforming; If be negative without Ct value.

Result

1. the above-mentioned mixed solution be made up of probe and upstream and downstream primer is dissolved with following 14 kinds of probes: for detecting the probe A of human papillomavirus 16 hypotype; For detecting the probe B of HPV type 18 hypotype; For detecting the probe C of human papillomavirus 52 hypotype; For detecting the probe D of human papillomavirus 58 hypotype; For detecting the probe E of human papillomavirus 31 hypotype; For detecting the probe F of human papillomavirus 33 hypotype; For detecting the probe G of human papillomavirus 35 hypotype; For detecting the probe H of human papillomavirus 39 hypotype; For detecting the probe I of human papillomavirus 45 hypotype; For detecting the probe J of human papillomavirus 51 hypotype; For detecting the probe K of human papillomavirus 56 hypotype; For detecting the probe L of human papillomavirus 59 hypotype; For detecting the probe M of human papillomavirus 68 hypotype; For the probe N of internal reference plasmid contrasted.

The mixed solution that described probe and upstream and downstream primer form is sub-packed in first and second liang of pipes, the probe A containing 0.5-1.5 μ l in first pipe and upstream and downstream primer each 1.5-2.5 μ l thereof; The probe B of 0.5-1.5 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof; The probe C of 1.0-2.0 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof; The probe D of 0.7-1.7 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof; And supplementary sterilizing ultrapure water to 100 μ l.Probe E containing 0.4-1.4 μ l in second pipe and upstream and downstream primer each 1.5-2.5 μ l thereof; The probe F of 0.62-1.62 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof; The probe G of 0.5-1.5 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof; The probe H of 0.5-1.5 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof; The probe I of 0.3-l.3 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof; The probe J of 0.5-1.5 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof; The probe K of 0.3-1.3 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof; The probe L of 0.7-1.7 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof; The probe M of 0.5-1.5 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof; The probe N of 0.1-1.1 μ l and upstream and downstream primer each 0.7-1.7 μ l thereof and supplementary sterilizing ultrapure water to 100 μ l.

Described PCR reaction solution is stored in the third pipe, by the MgCl of the Taq archaeal dna polymerase of 6-8 μ l, Buffer, 80-100 μ l of 60-100 μ l ₂, 15-25 μ l dNTPs composition.

Arranging reaction conditions is: the first step, 37 DEG C, reaction 5min; Second step, 94 DEG C, reaction 5min; 3rd step, 94 DEG C DEG C, reaction 15s; 4th step, 50 DEG C, reaction 40s; 3rd step and the 4th step circulate 40 times, read fluorescence in the 4th step.

2. the specificity of the specificity of multiple fluorescence quantitative PCR, each hypotype primer of sensitivity test result: HPV and probe is 100%, sees Fig. 2-9.HPV-16,18,52,58,31, the detection sensitivity of 33 is respectively: 10 ⁴copy/μ l, 10 ⁴copy/μ l, 10 ⁵copy/μ l, 10 ⁴copy/μ l, 10 ⁶copy/μ l, 10 ⁵copy/μ l.

3. the typical curve of each hypotype prepares result: the PCR reaction system of each hypotype HPV typical curve after optimizing is prepared, HPV-16, the r of the typical curve of 18,52,58,31,33 ²be respectively 0.970,0.995,0.970,0.986,0.982,0.983, see Figure 10-15.

The above; be only one of the specific embodiment of the present invention; but protection scope of the present invention is not limited thereto; any those of ordinary skill in the art are in the technical scope disclosed by the present invention; the change can expected without creative work or replacement, all should be encompassed within protection scope of the present invention.Therefore, the protection domain that protection scope of the present invention should limit with claims is as the criterion.

Claims (4)

1. detect fluorescent PCR kit and the method thereof of human papillomavirus 13 kinds of hypotypes, test kit comprises following component: PCR reaction solution, PCR mixed solution, positive criteria product, internal reference product and negative reference product; Described PCR reaction solution comprises Taq archaeal dna polymerase, Buffer, MgCl ₂and dNTPs, described positive criteria product are the standard plasmid molecule DNA containing HPV different subtype highly conserved sequence, described internal reference product are the standard plasmid molecule DNA containing human body gene highly conserved sequence, described negative reference product are the ultrapure water of sterilizing, described PCR mixed solution comprises increase the respectively primer of HPV13 hypotype and the probe and 1 that detects HPV13 hypotype of 13 covers and overlaps primer and the probe of human body gene, is described below respectively:

Be SEQ ID NO:1 for detecting the forward primer sequence of human papillomavirus 16 hypotype, reverse primer sequences is SEQ ID NO:2, fluorescent probe sequence is SEQ ID NO:3, wherein 5 ' end mark fluorescent reporter group FAM, 3 ' mark fluorescent quenching group BHQ; Be SEQ IDNO:4 for detecting HPV type 18 hypotype forward primer sequence, reverse primer sequences is SEQ IDNO:5, fluorescent probe sequence is SEQ ID NO:6, wherein 5 ' end mark fluorescent reporter group HEX, 3 ' mark fluorescent quenching group BHQ; Be SEQ IDNO:7 for detecting the forward primer sequence of human papillomavirus 31 hypotype; reverse primer sequences is SEQ IDNO:8; fluorescent probe sequence is SEQ IDNO:9, wherein 5 ' end mark fluorescent reporter group HEX, 3 ' mark fluorescent quenching group BHQ; Be SEQ ID NO:10 for detecting the forward primer sequence of human papillomavirus 33 hypotype; reverse primer sequences is SEQ ID NO:11; fluorescent probe sequence is SEQ ID NO:12, wherein 5 ' end mark fluorescent reporter group ROX, 3 ' mark fluorescent quenching group BHQ; Be SEQ ID NO:13 for detecting the forward primer sequence of human papillomavirus 35 hypotype, reverse primer sequences is SEQ ID NO:14, fluorescent probe sequence is SEQ ID NO:15, wherein 5 ' end mark fluorescent reporter group FAM, 3 ' mark fluorescent quenching group BHQ; Be SEQ ID NO:16 for detecting the forward primer sequence of human papillomavirus 39 hypotype, reverse primer sequences is SEQ ID NO:17, fluorescent probe sequence is SEQ ID NO:18, wherein 5 ' end mark fluorescent reporter group FAM, 3 ' mark fluorescent quenching group BHQ; Be SEQID NO:19 for detecting the forward primer sequence of human papillomavirus 45 hypotype; reverse primer sequences is SEQ ID NO:20; fluorescent probe sequence is SEQ ID NO:21, wherein 5 ' end mark fluorescent reporter group FAM, 3 ' mark fluorescent quenching group BHQ; Be SEQ ID NO:22 for detecting the forward primer sequence of human papillomavirus 51 hypotype; reverse primer sequences is SEQ ID NO:23; fluorescent probe sequence is SEQ ID NO:24, wherein 5 ' end mark fluorescent reporter group FAM, 3 ' mark fluorescent quenching group BHQ; Be SEQ IDNO:25 for detecting the forward primer sequence of human papillomavirus 52 hypotype; reverse primer sequences is SEQ ID NO:26; fluorescent probe sequence is SEQ ID NO:27, wherein 5 ' end mark fluorescent reporter group CY5,3 ' mark fluorescent quenching group BHQ; Be SEQ ID NO:28 for detecting the forward primer sequence of human papillomavirus 56 hypotype, reverse primer sequences is SEQ ID NO:29, fluorescent probe sequence is SEQ ID NO:30, wherein 5 ' end mark fluorescent reporter group CY5,3 ' mark fluorescent quenching group BHQ; Be SEQ ID NO:31 for detecting the forward primer sequence of human papillomavirus 58 hypotype; reverse primer sequences is SEQ ID NO:32; fluorescent probe sequence is SEQ ID NO:33, wherein 5 ' end mark fluorescent reporter group ROX, 3 ' mark fluorescent quenching group BHQ; Be SEQIDNO:34 for detecting the forward primer sequence of human papillomavirus 59 hypotype; reverse primer sequences is SEQ IDNO:35; fluorescent probe sequence is SEQ ID NO:36, wherein 5 ' end mark fluorescent reporter group FAM, 3 ' mark fluorescent quenching group BHQ; Be SEQ IDNO:37 for detecting the forward primer sequence of human papillomavirus 68 hypotype, reverse primer sequences is SEQ IDNO:38, fluorescent probe sequence is SEQ ID NO:39, wherein 5 ' end mark fluorescent reporter group FAM, 3 ' mark fluorescent quenching group BHQ; Forward primer sequence for the human body gene of internal reference is SEQ ID NO:40, reverse primer sequences is SEQ ID NO:41, fluorescent probe sequence is SEQ ID NO:42, wherein 5 ' end mark fluorescent reporter group CY5,3 ' mark fluorescent quenching group BHQ.

2. the fluorescent PCR kit of detection human papillomavirus according to claim 1 13 kinds of hypotypes and method thereof, is characterized in that: the mixed solution that described probe and primer form is sub-packed in first, second two pipe;

Containing the probe A of 0.5-1.5 μ l and the probe B of upstream and downstream primer each 1.5-2.5 μ l0.5-1.5 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof thereof in first pipe; The probe C of 1.0-2.0 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof; The probe D of 0.7-1.7 μ l and upstream and downstream primer each 1.5-2.5 μ l thereof; And mend sterilizing ultrapure water to 100 μ l;

Containing 0.4-1.4 μ l probe E and upstream and downstream primer each 1.5-2.5 μ l thereof in second pipe; 0.62-1.62 μ l probe F and upstream and downstream primer each 1.5-2.5 μ l thereof; 0.5-1.5 μ l probe G and upstream and downstream primer each 1.5-2.5 μ l thereof; 0.5-1.5 μ l probe H and upstream and downstream primer each 1.5-2.5 μ l thereof; 0.3-1.3 μ l probe I and upstream and downstream primer each 1.5-2.5 μ l thereof; 0.5-1.5 μ l probe J and upstream and downstream primer each 1.5-2.5 μ l thereof; 0.3-1.3 μ l probe K and upstream and downstream primer each 0.5-1.5 μ l thereof; 0.7-1.7 μ l probe L and upstream and downstream primer each 1.5-2.5 μ l thereof; 0.5-1.5 μ l probe M and upstream and downstream primer each 1.5-2.5 μ l thereof; 0.1-1.1 μ l probe N and upstream and downstream primer each 0.7-1.7 μ l thereof; And mend sterilizing ultrapure water to 100 μ l.

3. one kind adopts fluorescent PCR kit and the method thereof of detection human papillomavirus according to claim 1 13 kinds of hypotypes, it is characterized in that: described PCR reaction solution is stored in the third pipe, by Taq archaeal dna polymerase, the PCR Buffer of 60-100 μ l, the MgCl of 80-100 μ l of 6-10 μ l ₂, 15-25 μ l dNTPs composition.

4. the using method of the PCR kit for fluorescence quantitative of detection human papillomavirus according to claim 1 13 kinds of hypotypes, is characterized in that comprising the steps:

(1) solution of each reaction tubes is prepared: comprise PCR mixed solution 5 μ l, PCR reaction solution 5 μ l, the ultrapure water 8 μ l of sterilizing, testing sample 2 μ l, totally 20 μ l; Wherein testing sample replaces with sterilized water as negative control.

(2) prepared PCR pipe be placed on Bior LineGene9600 real-time fluorescence PCR instrument, arranging reaction conditions is: the first step, and 37 DEG C of C react 5min; Second step, 94 DEG C of C react 5min; 3rd step, 94 DEG C of DEG C of reaction 15s; 4th step, 50 DEG C of reaction 40s; 3rd step and the 4th step circulate 42 times, read fluorescence in the 4th step;

(3) making of typical curve: by known copy number respectively containing other HPV-16 of single type, 18,31,33,52, the recombinant plasmid of 58 object amplified fragments, during use continuous 10 doubling dilutions to concentration 1.0 × 10 ⁰copy/μ l-1.0 × 10 ⁹between copy/μ l, as the standard substance of multiple fluorescence quantitative PCR; Get the template of standard substance 2 μ l as quadruple quantitative fluorescent PCR of different concns, use sterilizing ultrapure water experiment product in contrast simultaneously, fluorescent quantitative PCR detection is carried out according to the reaction system of establishing and amplification condition to each weaker concn standard substance template simultaneously, all standard substance samples at least detect twice, the software automatic analysis result that the rear instrument that increased carries, thus determine the detection limit of the method, the detection sensitivity of assessment HPV quantitative fluorescent PCR, and production standard product calibration curve;

(4) clinical samples detects: with the DNA extracted in clinical HPV sample as template, carries out multiple fluorescence quantitative PCR amplification by the method for above-mentioned (1) and (2) and condition.