CN104818342A - Detection kit, detection system and detection method for 19 high-risk human papilloma viruses (HPVs) - Google Patents
Detection kit, detection system and detection method for 19 high-risk human papilloma viruses (HPVs) Download PDFInfo
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Abstract
The invention discloses a detection kit, detection system and detection method for 19 high-risk HPVs. The 19 high-risk HPVs comprise HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82. According to the invention, a plurality of general primers are employed for amplification of target fragments, and specific fluorescence probes are used for real-time detection of the amplified fragments; since the general primers are employed, the primer amount of the system and complexity of the system are reduced, cost is saved, and all the high risk types are detected through combined action of 10 primers; and 20 specific fluorescence probes are used to distinguish different types. With the detection kit, detection system and detection method provided by the invention, the 19 high-risk HPVs can be detected in a reaction tube; high sensitivity and good specificity are obtained; operation is simple and fast; and for patients, screening cost is saved.
Description
Technical field
The invention belongs to biological technical field, relating to a kind of primer, probe, test kit and method thereof for detecting 19 kinds of high-risk human mammilla papillomavirus particularly.
Background technology
Human papillomavirus (HPV) is that a kind of papilloma vacuolating virus A belonging to papovaviridae belongs to, and be spherical DNA virus, the tesselated epithelium of human body skin mucous membrane can be caused to breed, and the mankind are its unique hosts.Determined that HPV type has exceeded 190 kinds at present, about more than 50 plant the directed stratified squamous epithelium infecting human body reproductive tract skin and mucous membrane.Clinically, according to HPV different subtype virulence large I, HPV is divided into high-risk-type and the large class of low risk two.High-risk-type is except can causing genitalia wart, the more important thing is and cause genitalia cancer, cervical cancer and high-grade cervical intraepithelial neoplasia (cin), high-risk-type is the main arch-criminal causing cervical cancer, and the cervical cancer of about 99.7% is caused by the lasting of high-risk human mammilla papillomavirus or repeated infection; And low risk human papillomavirus (HPV) mainly causes the benign lesions such as the pointed condyloma of men and women's genitalia to comprise low pathology in epithelium of cervix uteri (CIN I), if benign lesion is diagnosed not in time and treated the possibility also having and develop into canceration.Therefore, targetedly complete detection is carried out to high-risk HPV, the early diagnosis and therapy of cervical cancer is had great importance.
Traditional cytology detection method only can detect the change of cytology aspect, and its sensitivity is low, poor specificity, false negative rate and false positive rate are high, and can not carry out somatotype to HPV.
Summary of the invention
The object of the present invention is to provide the primer for detecting 19 kinds of high-risk HPV s, probe, 19 kinds of high-risk HPV s comprise HPV16,18,26,31,33,35,39,45,51,52,53,56,58,59,66,68,70,73,82; Its primer sequence is as shown in table 1, and probe sequence is as shown in table 2, and interior label primer probe is as shown in table 3.
Table 1 primer and sequence
Table 2 probe and sequence
| HPV type | Probe title | Probe sequence | Sequence number |
| HPV16 | HPV16-P1-C | CGCAGTACAAATATGTCATTATGTGCTGCC | SEQ ID NO:11 |
| HPV18 | HPV18-P3-C | TTAACAATATGTGCTTCTACACAGTCTCCTGTACC | SEQ ID NO:12 |
| HPV26 | HPV26-P1-F | GTTGATACCACCCGCAGTACTAACCTTACC | SEQ ID NO:13 |
| HPV31 | HPV31-P1-F | CCAATATGTCTGTGTGTGCTGCAATTGC | SEQ ID NO:14 |
| HPV33 | HPV33-P1 | CCACTCGCAGTACTAATATGACTTTATGCACAC | SEQ ID NO:15 |
| HPV35 | HPV35-P1-F | GTCTGTGTGTTCTGCTGTGTCTTCTAGTGACAG | SEQ ID NO:16 |
| HPV39 | HPV39-P1-F | CCTGTCTACCTCTATAGAGTCTTCCATACCTTCTAC | SEQ ID NO:17 |
| HPV45 | HPV45-P1 | TTATGTGCCTCTACACAAAATCCTGTGCC | SEQ ID NO:18 |
| HPV51 | HPV51-P1 | CTATTAGCACTGCCACTGCTGCAGTTTCC | SEQ ID NO:19 |
| HPV52 | HPV52-P1-F | CAGTTGTAGATACCACTCGTAGCACTAACATGACTTTA | SEQ ID NO:20 |
| HPV53 | HPV53-P1-F | CATGACTCTTTCTGCAACCACACAGTCTATG | SEQ ID NO:21 |
| HPV56 | HPV56-P1 | CTGCTACAGAACAGTTAAGTAAATATGATGCACG | SEQ ID NO:22 |
| HPV58 | HPV58-P1 | GATACCACTCGTAGCACTAATATGACATTATGCAC | SEQ ID NO:23 |
| HPV59 | HPV59-P1 | CAGCACCAATCTTTCTGTGTGTGCTTC | SEQ ID NO:24 |
| HPV66 | HPV66-P1-F | CTACCAGAAGTACCAACATGACTATTAATGCAGC | SEQ ID NO:25 |
| HPV68 | HPV68-A-P2-F | CGTTGTGGATACAACGCGCAGTACTAATT | SEQ ID NO:26 |
| HPV68 | HPV68-B-P2-F | TATTTCTTACTGTTGTGGATACCACTCGCAGTACC | SEQ ID NO:27 |
| HPV70 | HPV70-P1-F | ATTGTCTGCCTGCACCGAAACGG | SEQ ID NO:28 |
| HPV73 | HPV73-P1-F | GTGTAGGTACACAGGCTAGTAGCTCTACTACAACG | SEQ ID NO:29 |
| HPV82 | HPV82-P3-F | CACTGCTGTTACTCCATCTGTTGCACAA | SEQ ID NO:30 |
Table 3 interior label primer probe and sequence
| Primed probe title | Sequence | Sequence number |
| E-Ex4-F1 | TCAGCAACAACCCTGCCCTG | SEQ ID NO:31 |
| E-Ex4-R1 | GAAGGTCTTGGTGGACCCGTC | SEQ ID NO:32 |
| E-Ex4-P1 | TGGGACGGGACACGTTGCACCTCT | SEQ ID NO:33 |
Another object of the present invention is to the test kit that detection 19 kinds of high-risk HPVs are provided, it is characterized in that, described test kit comprises the primer of sequence as shown in SEQ ID NO:1 ~ SEQ ID NO:10, and sequence is as one or more of the specific probe of SEQ ID NO:11 ~ SEQ ID NO:30, and the interior label primer of sequence as shown in SEQ ID NO:31 ~ SEQ ID NO:33 and probe;
Described test kit adopts noncompetitive interior mark detection system, choose one section of conservative region of human genome as interior mark amplification object fragment, probe mark VIC fluorescence, has without exception with or without correctly adding with pcr amplification reaction process for monitoring sample DNA as interior mark reagent.For the probe mark CY5 fluorophor of 16/18 high-risk HPV DNA, the probe mark FAM fluorophor of other 17 kinds of high-risk HPV DNA, interior mark probe mark VIC fluorophor.
Described test kit composition is as shown in table 4.
Interior mark detected result can reflected sample DNA quality truly and experimental implementation situation, and can be used as the Quality Control to reagent, DNA quality and experimental implementation, the amplification region of selection is the section that human EGFR gene is guarded relatively, and target sequence length is about 100bp.
Table 4 test kit forms
The present invention provides a kind of reaction system for detecting 19HPVs on the other hand, specific as follows:
The using method of test kit of the present invention comprises the following steps:
1. sample collection: applicable specimen types has urogenital tract secretion cotton swab, reproductive tract scraping blade, biopsy sample etc.;
2. extract sample DNA;
3. extracted DNA is mixed with mixed enzyme according to corresponding ratio, and join in PCR reaction tubes; And HPV19 positive control and HPV19 negative control are added respectively in corresponding PCR reaction tubes;
4. judge detected result according to the Ct value of fluorescent PCR amplification instrument display;
5. the explanation of detected result, as shown in table 5:
1. HEX (or VIC) passage should have obviously or have the amplification curve of " S " type trend to rise, and Ct value≤29.If Ct value >29 or HEX passage rise without obvious amplification curve, illustrate that the DNA added contains PCR inhibitor or DNA add-on is very few, need again to extract or increase DNA applied sample amount and test again; In such cases, if FAM and the CY5 passage of sample wherein one or two rise and Ct value≤27, then this sample is without the need to again detecting, result be judged as high-risk HPV DNA the positive.
2. the amplification curve of CY5 passage is typical " S " type and Ct value≤27, then result is judged as that HPV 16/18 type is positive.
1. the amplification curve of FAM passage is typical " S " type and Ct value≤27, then result is judged as that other high-risk-types of HPV are positive.
2. a sample may be that HPV 16/18 type and other high-risk-types are simultaneously positive.
Table 5 sample results judges
The invention has the beneficial effects as follows:
Molecular diagnostic techniques then can improve sensitivity and the specificity of HPV detection.Cytology detection method and molecular diagnosis method are combined the diagnostic level that can improve cervical cancer and the change of other anogenital cancers better.The invention provides a kind of highly sensitive and high specific for detecting the primer of human papillomavirus 19 somatotype, probe and detection system.This detection kit adopts many universal primers to increase to object fragment, utilizes special fluorescent probe to detect in real time amplified fragments, simple and efficient to handle, provides a kind of fast and reliable detection method.The method can realize the disposable detection of same reaction tubes 19 kinds of human papillomaviruss (HPV), substantially reduces time and the examination expense of detection, for clinical early diagnosis provides important evidence.
The present invention can detect 19 kinds of high-risk HPVs in same reaction tubes, namely the present invention adopts general primer and for other Idiotype probe various, by adding the difference of the probe of Idiotype, can form and detect one or more, 19 kinds of high-risk HPVs can be detected at most.The amplified fragments selected is positioned at the genomic L1 gene regions of HPV, many universal primers are adopted to increase to object fragment, special fluorescent probe is utilized to detect in real time amplified fragments, adopt noncompetitive interior mark detection system, choose one section of conservative region of human genome as interior mark amplification object fragment, probe mark VIC fluorescence, has without exception with or without correctly adding with pcr amplification reaction process for monitoring sample DNA as interior mark reagent.For the probe mark CY5 fluorophor of 16/18 high-risk HPV DNA, the probe mark FAM fluorophor of other 17 kinds of high-risk HPV DNA, interior mark probe mark VIC fluorophor.Finally judge according to pcr amplification curve the high-risk HPV DNA that whether there is test kit sensing range in the middle of system.Adopt universal primer, decrease the complicacy of system primer consumption and system, saved cost; Article 10, all high-risk-types detect by primer acting in conjunction; Article 19, specific probe is distinguished each type.Pcr amplification reaction system of the present invention contains UNG enzyme simultaneously, can selectivity rupture containing the uridylic glycosidic link in the PCR fragment of dU, effectively reduce the false positive because of PCR primer pollution generation.
The present invention's highly sensitive (reach 5 copies/reaction, improve more than 2 orders of magnitude than existing certain methods), high specificity, simple and efficient to handle, meanwhile, for patients, save examination expense.
Accompanying drawing explanation
Fig. 1 is HPV16 and HPV18 sensitivity analysis figure.
Fig. 2 is 17 kinds of HPV sensitivity analysis figure.
Fig. 3 is that national full-length genome somatotype reference material (HPV16 and HPV18) detects figure.
Fig. 4 is that national full-length genome somatotype reference material (HPV26/31/33/35/45/56) detects figure.
Fig. 5 is that national full-length genome somatotype reference material (HPV58/59/66/6/73/82) detects figure.
Fig. 6 is national full-length genome somatotype reference material negative reference product N1-N5 detection figure.
Fig. 7 is that national full-length genome somatotype reference material (HPV6/11/61/67/69/71/81) detects figure.
Fig. 8 is clinical negative sample detected result figure.
Fig. 9 is HPV16/18 positive clinical sample amplification figure.
Figure 10 is other high-risk-type positive clinical pattern detection result figure.
Embodiment
Below in conjunction with specific embodiment, the present invention is set forth further.Should be understood that these embodiments are only not used in for the present invention to limit the scope of the invention.Unless otherwise defined or described herein, the scientific and technical term described in this patent and those of ordinary skill in the art understand there is identical implication.
Embodiment 1
The present embodiment for template, utilizes test kit of the present invention to carry out real-time PCR detection with 19 kinds of high-risk human mammilla papillomavirus DNA plasmids in full-length genome somatotype reference material:
(1) PCR reaction system is set up
Apply probe and the special primer of above-mentioned design, get 5 μ L reference material DNA as reaction template, adopt following PCR reaction system to increase, pcr amplification reaction system is:
The PCR reaction tubes having added template is put into real-time PCR detection instrument, is undertaken by following amplification program: the first stage: 50 DEG C of 2min, 95 DEG C of 5min, 1 circulation; Subordinate phase: 95 DEG C of 5s, 40 DEG C of 30s, 72 DEG C of 30s, 10 circulations; Phase III: 95 DEG C of 5s, 60 DEG C of 35s, 72 DEG C of 30s, 35 circulations; Phosphor collection: collect FAM, HEX (or VIC) and CY5 signal during the phase III 60 DEG C.
(2) detected result is judged according to the Ct value of fluorescent PCR amplification instrument display, as shown in table 5.
1. HEX (or VIC) passage should have obviously or have the amplification curve of " S " type trend to rise, and Ct value≤29.If Ct value >29 or HEX passage rise without obvious amplification curve, illustrate that the DNA added contains PCR inhibitor or DNA add-on is very few, need again to extract or increase DNA applied sample amount and test again; In such cases, if FAM and the CY5 passage of sample wherein one or two rise and Ct value≤27, then this sample is without the need to again detecting, result be judged as high-risk HPV DNA the positive.
2. the amplification curve of CY5 passage is typical " S " type and Ct value≤27, then result is judged as that HPV 16/18 type is positive.
3. the amplification curve of FAM passage is typical " S " type and Ct value≤27, then result is judged as that other high-risk-types of HPV are positive.
4. a sample may be that HPV 16/18 type and other high-risk-types are simultaneously positive.
Sensitivity analysis: getting concentration is quantitatively the reference material DNA of 500 copies, carries out 10 times of gradient dilutions, do 3 dilution gradients altogether, every secondary response adds 5 μ L, and 8 parallel carries out amplified reaction.Result shows, sensitivity of the present invention can detect 5 copies/μ L.
Detected result shows, detection system of the present invention accurately can detect 19 kinds of high-risk HPVs, and the sensitivity of detection can reach 5 copies/μ L.
Embodiment 2
Reaction system is as embodiment 1, test kit of the present invention is adopted to detect each component in human papillomavirus country full-length genome somatotype reference material, the reaction system that investigation is finally determined is to the detection case of human papillomavirus full-length genome somatotype National reference, and test-results is as Fig. 3-7.
Test-results shows, this test kit detects the negative reference product N1-N5 in human papillomavirus full-length genome somatotype National reference and is feminine gender, to 7 kinds of low risk (HPV6 in human papillomavirus full-length genome somatotype National reference, 11, 61, 67, 69, 71, 81) positive reference material is all detected as feminine gender, to 13 kinds of high-risk-type (HPV16 in human papillomavirus full-length genome somatotype National reference, 18, 26, 31, 33, 35, 45, 56, 58, 59, 66, 73, 82) positive reference material detects and is the positive, show that the detection coincidence rate of reaction system to human papillomavirus full-length genome somatotype National reference finally determined is 100%.
Embodiment 3
The present invention is used to detect clinical sample, detect the sample that 80 examples are clinical altogether, and with the human papillomavirus kit for detecting nucleic acid of Yaneng Biotechnology (Shenzhen) Co., Ltd. (PCR-fluorescence probe method) (17 type) in contrast.Real-time fluorescence PCR system of the present invention is utilized to detect the step of 80 routine clinical samples as follows.
(1) detect sample DNA to extract
Detect sample and comprise urogenital tract secretion cotton swab, reproductive tract scraping blade, biopsy sample etc.
The sample transfer 1mL gathered in a clean centrifuge tube, centrifugal 1 minute of 12,000rpm; Carefully siphon away 700 μ L supernatant liquors, in residue precipitated liquid, add 350 μ L Buffer VDL12 (adding dehydrated alcohol); Concussion mixing 10s, with hand held centrifuge 5 ~ 10s, is placed in 80 DEG C, brooder digestion 3min; Concussion mixing 10s, hand held centrifuge 5 ~ 10s, move to whole liquid rotating in CB3DNA adsorption column, the centrifugal 30s of 12000rpm; Outwell the liquid in collection tube, in DNA adsorption column, add 400 μ L Buffer RTW, the centrifugal 3min of 12000rpm (not empty get rid of); Abandon collection tube, adsorption column is carefully transferred in clean 1.5mL centrifuge tube, drip 80 μ L Buffer BDE toward DNA adsorption film center; Room temperature leaves standstill 2min, the centrifugal 1min of 12000rpm, collects sample DNA and preserves.
(2) PCR reaction system is set up
Apply probe and the special primer of above-mentioned design, get 5 μ L reference material DNA as reaction template, adopt following PCR reaction system to increase, pcr amplification reaction system is:
The PCR reaction tubes having added template is put into real-time PCR detection instrument, is undertaken by following amplification program: the first stage: 50 DEG C of 2min, 95 DEG C of 5min, 1 circulation; Subordinate phase: 95 DEG C of 5s, 40 DEG C of 30s, 72 DEG C of 30s, 10 circulations; Phase III: 95 DEG C of 5s, 60 DEG C of 35s, 72 DEG C of 30s, 35 circulations; Phosphor collection: collect FAM, HEX (or VIC) and CY5 signal during the phase III 60 DEG C.
(3) detected result is judged according to the Ct value of fluorescent PCR amplification instrument display, as shown in table 5.
1. HEX (or VIC) passage should have obviously or have the amplification curve of " S " type trend to rise, and Ct value≤29.If Ct value >29 or HEX passage rise without obvious amplification curve, illustrate that the DNA added contains PCR inhibitor or DNA add-on is very few, need again to extract or increase DNA applied sample amount and test again; In such cases, if FAM and the CY5 passage of sample wherein one or two rise and Ct value≤27, then this sample is without the need to again detecting, result be judged as high-risk HPV DNA the positive.
2. the amplification curve of CY5 passage is typical " S " type and Ct value≤27, then result is judged as that HPV 16/18 type is positive.
3. the amplification curve of FAM passage is typical " S " type and Ct value≤27, then result is judged as that other high-risk-types of HPV are positive.
4. a sample may be that HPV 16/18 type and other high-risk-types are simultaneously positive.
Result shows, the 80 routine clinical samples detected altogether, and contrast agents box detects positive sample 39 example, negative sample 41 example, and this test kit detects positive sample 43 example, negative sample 37 example; Compare with contrast agents box, the positive coincidence rate of this test kit reaches 92.3%, and negative match-rate reaches 97.6%, and total coincidence rate reaches 95%, and test kit has been ratified in test-results visualizingre agent box and market good coincidence rate.As Fig. 8-10.
Claims (5)
1., for detecting a test kit for 19 kinds of high-risk human mammilla papillomavirus (HPV), it is characterized in that, comprise:
1) primer
General HPV-F1TGCCTGGGGYAATCAGTTATTTGTTAC SEQ ID NO:1
General HPV-R3CCTCTGCCATGICKAATATATTSCTTAA SEQ ID NO:2
General HPV-R5CCTCTGCCATGYCKAATATAYTSCTTAA SEQ ID NO:3
General HPV-R6CCTCTGCCATGYCKAATATACTSCTTAA SEQ ID NO:4
HPV39 HPV-390-F1TGCCTGGGGCAATCAGTTATTTGTTAC SEQ ID NO:5
HPV53 HPV-530-R1CCTCTGCCATGTCTAAGGTATTCCTTAA SEQ ID NO:6
HPV51 HPV-510-R CCTCTGCCATGACTAACCTATTCCTTAA SEQ ID NO:7
HPV16 HPV-160-R CCTCTGCCATGYCTAATATAYTSCTTAA SEQ ID NO:8
HPV18 HPV-180-F TGCCTGGCATAATCAATTATTTGTTAC SEQ ID NO:9
HPV52 HPV-520-R CCTCTGCCATGTCTAATATATTCCTTAA SEQ ID NO:10；
2) probe
HPV16 HPV16-P1-C CGCAGTACAAATATGTCATTATGTGCTGCC SEQ IDNO:11
HPV18 HPV18-P3-C TTAACAATATGTGCTTCTACACAGTCTCCTGTACCSEQ ID NO:12
HPV26 HPV26-P1-F GTTGATACCACCCGCAGTACTAACCTTACC SEQ IDNO:13
HPV31 HPV31-P1-F CCAATATGTCTGTGTGTGCTGCAATTGC SEQ ID NO:14
HPV33 HPV33-P1 CCACTCGCAGTACTAATATGACTTTATGCACAC SEQ IDNO:15
HPV35 HPV35-P1-F GTCTGTGTGTTCTGCTGTGTCTTCTAGTGACAG SEQ IDNO:16
HPV39 HPV39-P1-F CCTGTCTACCTCTATAGAGTCTTCCATACCTTCTACSEQ ID NO:17
HPV45 HPV45-P1 TTATGTGCCTCTACACAAAATCCTGTGCC SEQ ID NO:18
HPV51 HPV51-P1 CTATTAGCACTGCCACTGCTGCAGTTTCC SEQ ID NO:19
HPV52 HPV52-P1-F CAGTTGTAGATACCACTCGTAGCACTAACATGACTTTASEQ ID NO:20
HPV53 HPV53-P1-F CATGACTCTTTCTGCAACCACACAGTCTATG SEQ IDNO:21
HPV56 HPV56-P1 CTGCTACAGAACAGTTAAGTAAATATGATGCACG SEQ IDNO:22
HPV58 HPV58-P1 GATACCACTCGTAGCACTAATATGACATTATGCAC SEQ IDNO:23
HPV59 HPV59-P1 CAGCACCAATCTTTCTGTGTGTGCTTC SEQ ID NO:24
HPV66 HPV66-P1-F CTACCAGAAGTACCAACATGACTATTAATGCAGC SEQ IDNO:25
HPV68 HPV68-A-P2-F CGTTGTGGATACAACGCGCAGTACTAATT SEQ IDNO:26
HPV68 HPV68-B-P2-F TATTTCTTACTGTTGTGGATACCACTCGCAGTACCSEQ ID NO:27
HPV70 HPV70-P1-F ATTGTCTGCCTGCACCGAAACGG SEQ ID NO:28
HPV73 HPV73-P1-F GTGTAGGTACACAGGCTAGTAGCTCTACTACAACGSEQ ID NO:29
HPV82 HPV82-P3-F CACTGCTGTTACTCCATCTGTTGCACAA SEQ ID NO:30
3) mark in
E-Ex4-F1 TCAGCAACAACCCTGCCCTG SEQ ID NO:31
E-Ex4-R1 GAAGGTCTTGGTGGACCCGTC SEQ ID NO:32
E-Ex4-P1 TGGGACGGGACACGTTGCACCTCT SEQ ID NO:33。
2. one as claimed in claim 1 is for detecting the test kit of 19 kinds of high-risk human mammilla papillomavirus (HPV), it is characterized in that,
Also comprise:
HPV19 mixed enzyme: comprise Taq archaeal dna polymerase, UNG enzyme 1 pipe 27 μ L
HPV19 positive control: comprise HPV 18/26 type plasmid 1 pipe 50 μ L.
3. for detecting a reaction system of 19 kinds of HPVs, specific as follows:
DEPC H2O
10 × PCR damping fluid
MgCl2 20.0-50.0mmol
Each couple of primer 50-200 μm ol of claim 1
Each couple of probe 50-200 μm ol of claim 1
dNTP 20.0-30.0mmol
Mixed enzyme 0.5-1.0 μ L
Cumulative volume 30-50 μ L.
4. simultaneously detecting and a method of somatotype 19 kinds of HPV in biological sample for non-diseases diagnostic purpose, said method comprising the steps of:
1) sample collection;
2) sample DNA is extracted;
3) extracted DNA is mixed with mixed enzyme according to corresponding ratio, and join in PCR reaction tubes; And HPV19 positive control and HPV19 negative control are added respectively in corresponding PCR reaction tubes;
4) detected result is judged according to the Ct value of fluorescent PCR amplification instrument display;
5) explanation of detected result, as shown in the table:
1. HEX (or VIC) passage should have obviously or have the amplification curve of " S " type trend to rise, and Ct value≤29: if Ct value >29 or HEX passage rise without obvious amplification curve, illustrate that the DNA added contains PCR inhibitor or DNA add-on is very few, need again to extract or increase DNA applied sample amount and test again; In such cases, if FAM and the CY5 passage of sample wherein one or two rise and Ct value≤27, then this sample is without the need to again detecting, result be judged as high-risk HPV DNA the positive;
2. the amplification curve of CY5 passage is typical " S " type and Ct value≤27, then result is judged as that HPV 16/18 type is positive;
1. the amplification curve of FAM passage is typical " S " type and Ct value≤27, then result is judged as that other high-risk-types of HPV are positive;
2. a sample may be that HPV 16/18 type and other high-risk-types are simultaneously positive.
5. a kind of simultaneously detecting and the method for somatotype 19 kinds of HPV in biological sample for non-diseases diagnostic purpose as claimed in claim 4, is characterized in that: described sample comprises urogenital tract secretion cotton swab, reproductive tract scraping blade, biopsy sample.
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Cited By (8)
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| CN105603121A (en) * | 2016-01-15 | 2016-05-25 | 艾康生物技术(杭州)有限公司 | Method, oligonucleotide and kit for detecting high-risk HPV (human papilloma viruses) |
| CN105950788A (en) * | 2016-06-15 | 2016-09-21 | 亚能生物技术（深圳）有限公司 | Primers, probes and kit for detecting 18 high-risk type HPV nucleic acids |
| CN107267663A (en) * | 2017-05-08 | 2017-10-20 | 常州金麦格生物技术有限公司 | HPV detection methods and kit |
| CN108660254A (en) * | 2018-05-30 | 2018-10-16 | 杭州千基生物科技有限公司 | Genital tract causal agent detection of nucleic acids primer, probe, kit and detection method |
| CN110938712A (en) * | 2019-12-27 | 2020-03-31 | 苏州药明检测检验有限责任公司 | Primer, probe, kit and method for detecting human papilloma virus based on real-time fluorescent quantitative PCR technology |
| CN111455108A (en) * | 2020-04-14 | 2020-07-28 | 天津普瑞赛斯分子诊断技术有限责任公司 | Kit and detection method for detecting 14 high-risk HPV (human papilloma Virus) types |
| CN114214463A (en) * | 2021-12-28 | 2022-03-22 | 广州安必平医药科技股份有限公司 | Primer probe composition for detecting HPV (human papillomavirus) and application thereof |
| WO2024187843A1 (en) * | 2023-03-14 | 2024-09-19 | 广州达安基因股份有限公司 | Primer, primer-probe composition and kit for detecting high-risk human papillomavirus |
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| CN105950788A (en) * | 2016-06-15 | 2016-09-21 | 亚能生物技术（深圳）有限公司 | Primers, probes and kit for detecting 18 high-risk type HPV nucleic acids |
| CN105950788B (en) * | 2016-06-15 | 2019-07-30 | 亚能生物技术（深圳）有限公司 | Detect the primer, probe and kit of 18 kinds of high-risk HPV nucleic acid |
| CN107267663A (en) * | 2017-05-08 | 2017-10-20 | 常州金麦格生物技术有限公司 | HPV detection methods and kit |
| CN108660254A (en) * | 2018-05-30 | 2018-10-16 | 杭州千基生物科技有限公司 | Genital tract causal agent detection of nucleic acids primer, probe, kit and detection method |
| CN110938712A (en) * | 2019-12-27 | 2020-03-31 | 苏州药明检测检验有限责任公司 | Primer, probe, kit and method for detecting human papilloma virus based on real-time fluorescent quantitative PCR technology |
| CN111455108A (en) * | 2020-04-14 | 2020-07-28 | 天津普瑞赛斯分子诊断技术有限责任公司 | Kit and detection method for detecting 14 high-risk HPV (human papilloma Virus) types |
| CN111455108B (en) * | 2020-04-14 | 2022-09-13 | 天津普瑞赛斯分子诊断技术有限责任公司 | Kit and detection method for detecting 14 high-risk HPV (human papilloma Virus) types |
| CN114214463A (en) * | 2021-12-28 | 2022-03-22 | 广州安必平医药科技股份有限公司 | Primer probe composition for detecting HPV (human papillomavirus) and application thereof |
| WO2024187843A1 (en) * | 2023-03-14 | 2024-09-19 | 广州达安基因股份有限公司 | Primer, primer-probe composition and kit for detecting high-risk human papillomavirus |
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