CN101251469A - Fluorescence PCR method for diagnosis of human papilloma viral infection - Google Patents

Fluorescence PCR method for diagnosis of human papilloma viral infection Download PDF

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CN101251469A
CN101251469A CNA2007100765840A CN200710076584A CN101251469A CN 101251469 A CN101251469 A CN 101251469A CN A2007100765840 A CNA2007100765840 A CN A2007100765840A CN 200710076584 A CN200710076584 A CN 200710076584A CN 101251469 A CN101251469 A CN 101251469A
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hpv
sequence
probe
pcr
dna
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杨梦甦
黄鹤
黄明辉
柏干荣
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Genetel Pharmaceuticals Shenzhen Co Ltd
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Genetel Pharmaceuticals Shenzhen Co Ltd
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Abstract

The present invention relates to a fluorescent PCR (polymerase chain reaction) detection method for diagnosing low risk type human papilloma virus (HPV) infection, belonging to the in vitro nucleic acid diagnosis field. The present invention comprises a multiple polymerase chain reaction (PCR) system based on fluorescent PCR technique, includes a forward primer and a reverse primer which aim at a plurality of HPV types, as well as a fluorescent probe, and can simultaneously detect DNA comprising six HPV types at most, namely HPV6, 11, 40, 42, 43 and 44, in one reaction tube under proper PCR conditions. The detection method can conveniently and rapidly diagnoss HPV infection in clinical samples, is high in specificity, and has great clinical value for preventing and controlling condyloma acuminatum in an early stage, blocking infection sources and reducing HPV infection.

Description

The fluorescence PCR method of diagnosis of human papilloma viral infection
Technical field
The invention belongs to external diagnostic nucleic acid field, is a kind of one or more human papillomavirus (HPV) infection fluorescent polyase chain reaction (PCR) method that detects simultaneously in clinical sample.
Background technology
Condyloma acuminatum (Condyloma acuminata; Condyloma acuminatum; Genital warts) is human infection Human infectious warts virus (HPV) and a kind of epidermoma sample hyperplasia of causing.Virus is duplicated on the basalis of infected skin and mucosal tissue, and induces epitheliosis, destroys the adjusting and controlling growth of cell, causes that the local proliferation venereal disease becomes.
Papillomavirus belongs to the Papillomavirus of papovaviridae, it comprise the papillomavirus of multiple animal and Human infectious warts virus (Human Papillomavirus, HPV).Wherein Human infectious warts virus (HPV) is the common dna virus of a kind of obligate infected person epidermis and mucous membrane scaly epithelium, can pass through direct or indirect contact stain article or sex track transmission, cause the multiple optimum papilloma or the wart of human skin and mucous membrane, a carcinogenicity is also had in the infection of some type.According to its carcinogenic danger degree, HPV is divided into low risk and high-risk-type, low risk mainly causes condyloma acuminatum, comprise types such as HPV6/11/40/42/43/44 (G M Clifford, et al, Worldwide distribution of humanpapillomavirus types in cytologically normal women in the Intemational Agency for Research on CancerHPV prevalence surveys a pooled analysis, Lancet 2005; 366:991～98); High-risk-type then comprises (MV Jacobs, et al, A general primer GP5 such as HPV16/18/31/33/35/58 +/ GP6 +-mediated PCR-enzymeimmunoassay method for rapid detection of 14 high-risk and 6 low-risk human papillomavirusgenotypes in cervical scrapings J.Clin.Microbiol., Mar 1997; 35:791～795.), its persistent infection can finally cause cervical carcinoma.
The condyloma acuminatum case increases day by day in recent years, occupy first place (the Strand A of sexually transmitted disease in American-European countries, RylanderE, Wilander E, et al, HPV infection in male partners of women with squamous intraepithelial neoplasia and/or high-riskHPV.Acta Derm Venereol, 1995,75:312～316), according to China's venereal disease monitoring point report, genitals HPV recall rate is 30.69/10 ten thousand (Chen Mingliang etc., the application of FQ-PCR detection in HPV6, the diagnosis of 11 type condyloma acuminatums.China's leprosy skin disease magazine, 2004,20 (2): 189～190), second (the Frega A that is only second to gonorrhoea row China sexually transmitted disease, et al, Giant condyloma acuminatumor buschke Lowenstein tumor:review of the literature and report of three cases treated by CO 2Lasersurgery.A long-term follow-up.Anticancer Res.2002,22:1201～1204).
Present diagnosis for condyloma acuminatum, main dependence is not bought sexual intercourse history, vulva neoplasm form and acetic acid and is tested (A Wikstrom in vain, et al, The acetic acid test in evaluation of subclinical genital papillomavirus infection:a comparative study onpenoscopy, histopathology, virology and scanning electron microscopy findings, Sex.Transm.Inf., Apr1992; 68:90～99.), lack quick and precisely the also method of early diagnosis.And should disease latent period grow (average 3 months), subclinical infection is common, skin disfigurement attitude is various, very easily causes omission.Fluorescent PCR sensitivity height, specificity is good, in real time the monitoring reaction process, reaction time can be controlled in two and one-half-hours, and was the stopped pipe operation, need not last handling process, can avoid reaction product to pollute to greatest extent, be expected to replace the early diagnosis that classic method is carried out HPV DNA.But the fluorescent PCR diagnostic products that detects the HPV low risk in the market only can detect two kinds of types (nearly 6 kinds of HPV6/11, common type), omission most probably clinically.
Fluorescent PCR technology (Fluoresence PCR) nineteen ninety-five takes the lead in succeeding in developing (M.J.Espy et al by U.S. PE company, Real-TimePCR in Clinical Microbiology:Applications for Routine Laboratory Testing.Clinical Microbiology Reviews, Jan.2006, Vol.19, No.1.165-256), it has the high sensitivity of PCR, the specificity of DNA hybridization and the advantage of spectral technique accurate quantification concurrently, visual result can directly be monitored the variation in the PCR process.Compare with regular-PCR, but its result's Real Time Observation, product does not need detected through gel electrophoresis, and stopped pipe is operated fully, has reduced the risk of PCR product pollution effectively.
The fluorescence probe of fluorescent PCR technology has now developed the formation polytype, as TaqMan probe (M.Krause et al, Comparative, Collaborative, and On-Site Validation of a TaqMen PCR Method as a Tool for Certified Production ofFresh, Campylobacter-Free Chickens.Applied and Environmental Microbiology, Aug.2006, Vol.72, No.8,5463-5468.), Molecular Beacon probe (Aneta J.at al, Molecular-Beacon Multiplex Real-Time PCRAssay for Detection of Vibrio cholerae.Applied and Environmental Microbiology, Sept.2006, Vol.72, No.9,6424-6428.), FRET probe etc., what this method related to is the TaqMan probe technique, the TaqMan probe is the specific oligonucleotide strand of a two ends mark fluorescent group, 5 ' end mark fluorescent reporter group, 3 ' end mark fluorescent quenching group.Its principle of work is: 1) polymerization: primer and probe combine with the target sequence specificity, and the Taq enzyme begins to synthesize to 3 ' end from 5 ' end along template, and this moment, probe was kept perfectly, and the fluorescence of fluorescence report group is not produced fluorescence signal by the cancellation of fluorescent quenching group.2) shear: when the Taq enzyme is blended into when running into probe along template, with probe 5 ' end report fluorophor shearing, the fluorescence report group that gets off that dissociates can send fluorescence.3) synthetic finishing: intensity of fluorescence strengthens with the PCR cycle index, and proportional with the quantity of PCR product.The fluoroscopic examination instrument sees through wavelength and the concentration change that PCR pipe lid/tube wall can directly detect fluorescence signal, so the fluorescent PCR detection technique can realize that stopped pipe monitors the variation of PCR overall process in real time, and the PCR product need not subsequent detection and processing.
The TaqMan probe is divided into two kinds according to the difference of the fluorescent quenching group of its 3 ' end mark: common TaqMan probe and TaqMan MGB probe.The quenching group of MGB probe is non-fluorescent quenching group (Non-Fluorescent Quencher), and itself does not produce fluorescence, can reduce the intensity of background signal greatly, improves signal to noise ratio (S/N ratio).Also be connected with MGB (Minor Groove Binder) modification group (Igor V.Kutyavin et al on the probe simultaneously, 3 '-Minor groove binder-DNA probes increase sequence specificity at PCR extensiontemperatures Nucleic Acids Research, 2000, Vol.28, No.2,655-661.), the Tm value of probe can be improved about 10 ℃.Therefore in order to obtain same Tm value, the MGB probe can get shorter than general T aqMan probe design, and the space length of reporter group and quenching group is nearer, can obtain better cancellation effect.
The method that the present invention relates to can be in a PCR reaction tube detects nearly 6 kinds of HPV low risks (HPV6/11/40/42/43/44) simultaneously, is applicable to the early prevention of condyloma acuminatum and provides guidance for course of disease monitoring.
Summary of the invention
The present invention be more particularly directed to a kind of multiple fluorescence PCR technology of utilizing and in a PCR reaction tube, detect the genotypic diagnostic method of multiple HPV fast, abreast simultaneously.
General PCR only adopts a pair of primer, produces a nucleic acid fragment by pcr amplification.The present invention then adopts multiplex PCR method (MultiplexPCR, the multiple nucleic acid TRAP), promptly in same PCR reaction system, add the primer more than two pairs, each reacts (Philip S.Bemard, Real-Time PCR Technology for Cancer Diagnostics.ClinicalChemistry.2002 to primer to special separately target sequence while amplification PCR; 48; 1178-1185.).Its reaction principle, reaction reagent and operating process are identical with general PCR.(TaqMan MGB probe can design shortlyer with respect to general T aqMan probe, has not only reduced synthetic cost effectively, also makes the success ratio of design greatly improve to comprise 6 TaqMan MGB probes in the reaction system of the present invention.In addition, TaqMan MGB probe improves the difference of Tm value between pairing and non-matching sequence, helps carrying out more multiple PCR) and 12 upstream primers, 12 downstream primers are respectively at 6 kinds of DNA target sequences that hang down danger HPV types.Can in same reaction tube, detect 6 kinds low danger HPV types simultaneously, but big time saver reduce cost, provide more diagnostic messages more accurately for clinical, more press close to the clinical examination demand.
6 kinds of different HPV target sequences owing to need increase simultaneously, a primer and probe surplus needing in the system to add ten, the complicacy of system (causing the generation of a large amount of primer dimers thus) will reduce the efficient of PCR greatly, thereby need be in the complexity that guarantees to reduce as far as possible under the specific prerequisite system.Concrete grammar is as follows: use software Primer Express 2.0, (studies show that between the various genomic L1 region sequence of HPV, to have certain homology sequence and polymorphic sequence at the 6 kinds low L1 districts that endanger the HPV types.Its sequence difference also is the foundation that different HPV types are divided), search TaqMan MGB probe, screening is in the probe of conservative section, (probe of selecting the same area as far as possible is to reduce the complicacy of system) be stationary probe then, by the corresponding supporting primer of Primer Express 2.0 search, determine that finally the framework of system is: design 6 probes at 6 types; Downstream primer derives from high conserved region section (reduction complicacy); Upstream primer derives from polymorphism section (guaranteeing enough specificitys).
Description of drawings
The initial copy number that the fluorescent PCR amplification curve of the quantitative reference material of Fig. 1 .HPV, curve are from left to right corresponding is respectively (1.0 * 10 7, 1.0 * 10 6, 1.0 * 10 5, 1.0 * 10 4, 1.0 * 10 3Copy/microlitre).
The typical curve that the quantitative reference material of Fig. 2 .HPV detects, coefficient R 2=0.995, illustrate that linear relationship is very good, can be used for the quantitative test of HPV DNA copy number.
The amplification curve diagram that Fig. 3 .HPV clinical sample fluorescent PCR detects.
Embodiment
1. primer and probe design (see Table 1 and table 2) are with synthetic
Primer and probe all entrust specialized company synthetic, and wherein primer is the PAGE purifying, and probe is the HPLC purifying.Probe 5 ' end flag F AM fluorophor, 3 ' end is in conjunction with MGB and NFQ quenching group.
Table 1. detects primer and the sequence table of TaqMan-MGB probe oligonucleotides of HPV
Sequence numbering The sequence title Oligonucleotide sequence (5 '-3 ') Base length (bp)
SEQ ID NO.1 The HPV6 probe ATATCGCCAT CCTGTATAAC A 21
SEQ ID NO.2 The HPV11 probe TGTCCCCATC CTGTATAA 18
SEQ ID NO.3 The HPV40 probe TrCAGGATGG CGACATG 17
SEQ ID NO.4 The HPV42 probe ATATCCCCAT CCTGAATAA 19
SEQ ID NO.5 The HPV43 probe CATGTCACCA TCCTGTATAA 20
SEQ ID NO.6 The HPV44 probe CACCATCCTC AATTACACTA G 21
SEQ ID NO.7 The HPV6 forward primer GGTAAAGGTA AACAGTGTAC TAATACACCT GTA 33
SEQ ID NO.8 The HPV6 reverse primer ATAGCACCAA AGCCTGTGTC AA 22
SEQ ID NO.9 The HPV11 forward primer GGGTACACAA TGTTCAAATA CCTCTGTA 28
SEQ ID NO.10 The HPV11 reverse primer CATAGCACCA AAGCCTGTAT CAAC 24
SEQ ID NO.11 The HPV40 forward primer TGTCCTGTAT TAGAATTAAA AACTGAG 27
SEQ ID NO.12 The HPV40 reverse primer GATAGCACCA AAGCCAGTAT CCA 23
SEQ ID NO.13 The HPV42 forward primer TAAAGGTACT GCCTGTACAC CACAGT 26
SEQ ID NO.14 The HPV42 reverse primer GCCCCAAACC CTACATCCA 19
SEQ ID NO.15 The HPV43 forward primer TCAGGGTGTG CCTTGCAAC 19
SEQ ID NO.16 The HPV43 reverse primer CCTGTAGGGA AGCAAAATCC ATT 23
SEQ ID NO.17 The HPV44 forward primer GGTAAAGGCA AGCAGTGTAA TAATGTT 27
SEQ ID NO.18 The HPV44 reverse primer TCATGGCTCC AAAACCAGTG T 21
The target sequence gene pool of corresponding HPV amplified production numbering and position in table 2. table 1
The HPV genotype Gene pool numbering (GI No.) The target sequence position Amplified production length (bp)
HPV6 6002612 6285-6400 116
HPV11 333026 6274-6385 112
HPV40 397014 6325-6396 72
HPV42 9627156 6343-6447 105
HPV43 41057205 6260-6393 134
HPV44 1020242 6192-6309 118
2.HPV the preparation of plasmid reference material
Use primer amplified HPV L1 district target sequence, purified PCR product is connected on the pGEM-T easy carrier ( PGEM-T easy vector system connects kit), be converted in the escherichia coli jm109 competent cell then, by blue hickie screening, make up 6 kinds of HPV low risks (HPV6,11,40,42,43,44) recombinant plasmid dna respectively as the sensitivity reference material, and 13 kinds of HPV high-risk-types (HPV16,18,31,33,35,39,45,51,52,56,58,59,68) recombinant plasmid dna is as the specificity reference material, and the HPV recombinant plasmid of structure is identified through two-way dna sequencing.
3. sample collection and extraction
I. genitals or crissum there are excipuliform body hyperplasia, suspect to be the condyloma acuminatum person. gather excipuliform surface cast-off cells.The cotton swab that soaks into physiological saline is the tissue surface of wiping excipuliform back and forth 3 times firmly, obtains cast-off cells.Cotton swab after the sampling is put into the sample tube that has the 1ml stroke-physiological saline solution.Fully rinsing.
Ii. to other suspicious the infecteds, before the collection,, change cotton swab with cotton swab secretion that uterine neck mouth or urethral orifice is too much wiped clean gently, soak into cotton swab with physiological saline and be close to uterine neck mouth or urethral orifice mucous membrane. slightly firmly rotated for 2 weeks, to obtain secretion and cast-off cells.Cotton swab after the sampling is put into the sample tube that has the 1ml stroke-physiological saline solution, fully rinsing.
Iii. skin is decreased piece of tissue, get piece of tissue and smash to pieces and place the PCR sample processing tube.Centrifugal again after 20 minutes with the immersion of 1ml physiological saline, later step is identical with the secretion sample preparation.
Iv. use the commercial goods kit to extract HPV DNA.
4. reference substance is selected
The negative contrast of end user's genomic DNA (30 μ g/ml): HPV6 recombinant plasmid (2.0 * 10 5Copies/ μ l) is the strong positive contrast; HPV6 recombinant plasmid (4.0 * 10 3Copies/ μ l) be critical positive control.
5.PCR reaction is formed
According to the form below (table 3) preparation PCR reactant liquor also adds HPV dna profiling (each test must comprise feminine gender, the critical positive and strong positive contrast).Preparation finishes and does mixing and the upward machine reaction of centrifugal back slightly.
Table 3.PCR reaction is formed
Material name Final concentration
PCR Buffer (0.8-2)×
MgCl 2 2-5mM
dNTP 0.1-0.5mM
The HPV primer Each 0.01-0.50 μ M
The HPV probe Each 0.01-0.50 μ M
UNG 0.05-1U
The Taq enzyme 0.5-3U
H 2O In right amount
Dna profiling 2μl
Final volume 40μl
6. response procedures is set
The fluorescence detection channel (it is Reporter that FAM is set, and None is Quencher) of collecting the FAM fluorescence signal is set, reaction tube is put into the fluorescent PCR instrument begin amplification, response procedures following (is example with ABI 7000):
Table 4.PCR program setting
7. the result judges
To ABI 7000, the baseline scope is 6-15 circulation or selects (Auto baseline) automatically by software that setting threshold (threshold) makes it to surpass the mxm. of random amplification curve.
8. quality control standard (table 5)
Table 5. quality-control product standard testing result (is example with ABI 7000)
Figure A20071007658400072
9. result's report (table 6)
Table 6. report pattern detection result (is example with ABI 7000)
Figure A20071007658400073
10. limitation
This method uses a kind of fluorescence signal (FAM) to detect 6 kinds of HPV types (HPV6,11,40,42,43,44), does not specifically distinguish the HPV type, but does not influence the auxiliary diagnosis that in the clinical use HPV is infected.
11. this method mainly is applicable to:
A) detect the main virulence factor that low risk HPV6,11,40,42,43 and 44 etc. causes condyloma acuminatum.
B) the condyloma acuminatum patient's follows up a case by regular visits to.
Testing result should be used in combination with the clinical information of other diagnostics method acquisition and suitable physical examination.
LR HPV_ST25.txt
SEQUENCE LISTING
＜110〉Genetel Pharmaceuticals (Shenzhen) Co., Ltd.
＜120〉fluorescence PCR method of diagnosis of human papilloma viral (HPV) infection
<130>
<160>18
<170>PatentIn version 3.4
<210>1
<211>21
<212>DNA
<213>Human papillomavirus type 6
<400>l
atatcgccat cctgtataac a 21
<210>2
<211>18
<212>DNA
<213>Human papillomavirus type 11
<400>2
tgtccccatc ctgtataa 18
<210>3
<211>17
<212>DNA
<213>Human papillomavirus type 40
<400>3
ttcaggatgg cgacatg 17
<210>4
<211>19
<212>DNA
<213>Human papillomavirus type 42
<400>4
atatccccat cctgaataa 19
<210>5
<211>20
<212>DNA
<213>Human papillomavirus type 43
<400>5
catgtcacca tcctgtataa 20
<210>6
<211>21
<212>DNA
<213>Human papillomavirus type 44
<400>6
caccatcctc aattacacta g 21
<210>7
<211>33
<212>DNA
<213>Human papillomavirus type 6
<400>7
ggtaaaggta aacagtgtac taatacacct gta 33
LR HPV_ST25.txt
<210>8
<211>22
<212>DNA
<213>Human papillomavirus type 6
<400>8
atagcaccaa agcctgtgtc aa 22
<210>9
<211>28
<212>DNA
<213>Human papillomavirus type 11
<400>9
gggtacacaa tgttcaaata cctctgta 28
<210>10
<211>24
<212>DNA
<213>Human papillomavirus type 11
<400>10
catagcacca aagcctgtat caac 24
<210>11
<211>27
<212>DNA
<213>Human papillomavirus type 40
<400>11
tgtcctgtat tagaattaaa aactgag 27
<210>12
<211)23
<212>DNA
<213>Human papillomavirus type 40
<400>12
catagcacca aagccagtat cca 23
<210>13
<211>26
<212>DNA
<213>Human papillomavirus type 42
<400>13
taaaggtact gcctgtacac cacagt 26
<210>14
<211>19
<212>DNA
<213>Human papillomavirus type 42
<400>14
gccccaaacc ctacatcca 19
<210>15
<211>19
<212>DNA
<213>Human papillomavirus type 43
<400>15
tcagggtgtg ccttgcaac 19
<210>16
<211>23
<212>DNA
LR HPV_ST25.txt
<213>Human papillomavirus type 43
<400>16
cctgtaggga agcaaaatcc att 23
<210>17
<211>27
<212>DNA
<213>Human papillomavirus type 44
<400>17
ggtaaaggca agcagtgtaa taatgtt 27
<210>18
<211>21
<212>DNA
<213>Human papillomavirus type 44
<400>18
tcatggctcc aaaaccagtg t 21

Claims (3)

1. the fluorescence PCR method that infects of a diagnosis of human papilloma viral (HPV) comprises with the lower part:
(1) oligonucleotide sequence with nearly 6 kinds of HPV DNA complementation is used for the primer that the pcr amplification clinical sample obtains DNA; Here, the primer sequence of each type comprises:
(a) forward primer of energy and a kind of HPV type gene pairs first site hybridization of target sequence upstream of answering;
(b) reverse primer of second the site hybridization in energy and a kind of HPV type gene pairs target sequence downstream of answering;
(c) forward primer and reverse primer are not limited to the sequence that the sequence table of this instructions is listed.
(2) be used to detect the one or more oligonucleotide sequence probes of fluorescence labeling of HPV, this probe can and in target sequence the HPV sequence hybridization between the hybridization of above-mentioned first and second sites.Probe sequence can be sequence or its complementary series of SEQ ID No.1-6, also can comprise derive out by listed sequence or its complementary series, difference is not more than the oligonucleotide sequence of 8 bases.
(3) sample of obtaining is carried out pcr amplification, it is characterized in that comprising one or more pairs of primers and probe, as template, one or more HPV DNA target sequence fragments increase in the reaction system with HPV DNA.By detecting the variation of one or more different fluorescence signals in the PCR reaction system, determine whether infect HPV in the sample, and the content of HPV.
2. fluoroscopic examination according to claim 1, it is characterized in that the described fluorescence probe that is used for compound detection is the TaqMan probe, what label probe 5 ' was held (includes but not limited to following several: FAM for a kind of fluorescence report group, TET, JOE, VIC, HEX, ROX, TAMRA, CY3, CY3.5, CY5, CY5.5, Oregon Green, CAL Red, Red 640, Texas Red), 3 ' end (includes but not limited to following several: TAMRA for a kind of fluorescent quenching group, DABCYL, ECLIPSE, NFQ), 3 ' end of probe can be to have DNA ditch compound (MGB, Minor Groove Binder) to modify.
3. require can use one or more fluorescence probes in the described reaction system according to right 2, the fluorescence report group of institute's mark can be one or more.
CNA2007100765840A 2007-08-24 2007-08-24 Fluorescence PCR method for diagnosis of human papilloma viral infection Pending CN101251469A (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
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CN101487063B (en) * 2009-02-25 2011-07-20 潮州凯普生物化学有限公司 Human papilloma virus infection gene amplification fluorescent detection kit
CN102341696A (en) * 2009-03-03 2012-02-01 万迈医疗仪器有限公司 Detection system and method for high sensitivity fluorescent assays
CN101613764B (en) * 2009-08-03 2012-05-23 广州锐达生物科技有限公司 High-risk type HPV fluorescence PCR screening method and primer, probe and kit thereof
CN103173565A (en) * 2013-01-10 2013-06-26 湖南圣湘生物科技有限公司 Low-risk human papillomavirus (HPV)6/11 detection kit
CN104561385A (en) * 2015-01-24 2015-04-29 中国农业科学院兰州兽医研究所 PCR kit for detecting papilloma virus HPV6
CN106929602A (en) * 2015-12-30 2017-07-07 上海星耀医学科技发展有限公司 A kind of low risk HPV kit for detecting nucleic acid

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101487063B (en) * 2009-02-25 2011-07-20 潮州凯普生物化学有限公司 Human papilloma virus infection gene amplification fluorescent detection kit
CN102341696A (en) * 2009-03-03 2012-02-01 万迈医疗仪器有限公司 Detection system and method for high sensitivity fluorescent assays
CN101613764B (en) * 2009-08-03 2012-05-23 广州锐达生物科技有限公司 High-risk type HPV fluorescence PCR screening method and primer, probe and kit thereof
CN103173565A (en) * 2013-01-10 2013-06-26 湖南圣湘生物科技有限公司 Low-risk human papillomavirus (HPV)6/11 detection kit
CN104561385A (en) * 2015-01-24 2015-04-29 中国农业科学院兰州兽医研究所 PCR kit for detecting papilloma virus HPV6
CN106929602A (en) * 2015-12-30 2017-07-07 上海星耀医学科技发展有限公司 A kind of low risk HPV kit for detecting nucleic acid
CN106929602B (en) * 2015-12-30 2021-06-25 上海星耀医学科技发展有限公司 Low-risk human papilloma virus nucleic acid detection kit

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