CN102952894A - Color based loop-mediated isothermal amplification technology for detecting HPV6, HPV11, HPV16, HPV18, HPV52 and HPV58 - Google Patents
Color based loop-mediated isothermal amplification technology for detecting HPV6, HPV11, HPV16, HPV18, HPV52 and HPV58 Download PDFInfo
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Abstract
The invention belongs to the biotechnological application field, and relates to the detection and the monitoring of Chinese-common six HPVs of cervical scraping smear specimens of suspected HPV (human papilloma virus) infected patients with genital damage (comprising genital warts and cervical epithelial neoplasia) as main symptoms in disease prevention control mechanisms at all levels, papilloma virus detection network laboratories, sentinel point hospitals and the like. The conserved sequences of E6, E7 or L1 genes of six HPV subtypes are analyzed by bioinformatics professional software through applying gene color based loop-mediated isothermal amplification (LAMP) technology, six primers of each subtype, which completely couple with eight binding domains in each target sequence, are designed, single reverse transcription and nested PCR secondary application steps are omitted, an HNB (hydroxynaphthol blue) dye is added before the reaction, the amplification in a 63DEG C isothermal environment for 1h can be completed only through one step, and a detection result can be determined through the color change observed with naked eyes and the white precipitate at the bottom of a tube. Compared with traditional PCR methods, the technology provided in the invention has the advantages of safety, specificity, sensitivity, and convenience.
Description
Invention field
The present invention uses the ring mediated reverse transcription isothermal amplification technique based on the HNB color to detect common 6 kinds of papilloma viruss (HPV) E6, E7 or the L1 gene of Chinese.Be used for Disease Prevention and Control Institutions at different levels, HPV Sampling network laboratory, Sentinel point hospital is to detection and the monitoring of 6 kinds of common HPV of Chinese.
Background of invention
Cervical cancer occupies second in world's woman cancer, the annual newly-increased case 49.3 ten thousand in the whole world, the women about 270,000 who dies from cervical cancer every year.Wherein the generation of 99% cervical cancer is relevant with parillomarvirus infections.HPV is divided into low risk and high-risk-type.The LR-HPV infection can cause human innocent tumour and Genital warts, and it then is the major cause that causes cervical cancer that HR-HPV infects.The common low risk of Chinese is 6 types and 11 types, and common high-risk-type is 16 types, 18 types, 52 types and 58 types.
Be the quality of Effective Raise China uterine neck generaI investigation and the sickness rate of controlling China's cervical cancer, must enlarge the coverage of monitoring network, expand network laboratories to whole districts and cities one-level.Target is to make more than 300 districts and cities in the whole nation all possess the ability that can carry out the uterine neck examination, conscientiously accomplishes early discovery, report morning, early treatment.This problem is planned to build the method for the common HPV of visual LAMP detection Chinese of vertical color-based, have fast, the characteristics of sensitivity and simple operations, need not the PCR instrument, need not electrophoresis equipment, to for improving China disease surveillance network laboratories and Sentinel point hospital to the monitoring capability of the common HPV of Chinese, realize providing strong technical guarantee to HPV the infected's rapid screening.
The LAMP technology of color-based should have the incomparable advantage of traditional gene diagnosis method in the context of detection of dna virus, compare with some conventional gene test means, this technology has larger advantage aspect operability and detection time and the equipment requirements.This is mainly reflected in the following aspects.At first, can shorten to its detection time in one hour, sensitivity can reach and detect the above viral nucleic acid molecule of ten copies.Except using some polysaccharases commonly used, substantially without particular requirement, just can finish this testing at common biology laboratory to hardware device.In test is implemented, only need to finish experimental implementation once going on foot, reduced opportunities for contamination and made things convenient for the Quality Control work in the experimentation.The second, this technology has been omitted the secondary amplification step of independent reverse transcription and nest-type PRC, and amplified reaction carries out under 63 ℃ of isothermal conditions in addition, does not have the change renaturation process of nucleic acid, has not only saved a large amount of time to have reduced again the opportunities for contamination of amplification of nucleic acid.The 3rd, amplified reaction only could carry out in the situation of eight land couplings in the complete target sequence with identifying of six primers smoothly, and all these characteristics have all reduced the background of amplified reaction to a great extent, thereby detection specificity is improved.Can form visual magnesium pyrophosphate white precipitate in the amplified reaction of the 4th, LAMP, the HNB dyestuff adds before amplification, so naked eyes also can be observed the variation of color in white casse thing in the positive reaction pipe and the reaction tubes, can directly judge detected result.
Summary of the invention
The present invention be at present to the improvement of the common 6 kinds of HPV detection technique means of Chinese with replenish, it has safe, special, sensitive, advantage easily.This is mainly reflected in the following aspects.At first, amplified reaction only could carry out in the situation of eight land couplings in the complete target sequence with identifying of six primers smoothly, and all these characteristics have all reduced the background of amplified reaction to a great extent, thereby detection specificity is improved.The second, this technology has been omitted the secondary amplification step of independent reverse transcription and nest-type PRC, only needs can finish experimental implementation once going on foot, and does not have the change renaturation process of nucleic acid, has reduced the opportunities for contamination of amplification of nucleic acid, has improved the susceptibility and the security that detect.The 3rd, all amplification procedures all can be finished under 63 ℃ of equalities of temperature, need not the PCR instrument, have reduced the requirement to Experimental Hardware, because the HNB dyestuff adds before amplification, can directly judge detected result by naked eyes, need not electrophoresis.The 4th, shortened the required time of detecting than PCR.
2. select respectively HPV6, HPV11, HPV16, HPV18, HPV52, the E6 of HPV58 type, E6, E7, L1, E6, the corresponding LAMP primer of the relative conserved regions design of L1 gene, comprising two outer primers (F3, B3) and two inner primers (FIP, BIP), two loops close primer (LF, LB).The concrete sequence of all primers sees Table 1.
Table 1:LAMP detects the nucleotide primer sequence table of 6 kinds of common HPV hypotypes in the Chinese population
| Primer names | Sequences(5’-3’) |
| HPV6E6-F3 | CAAATGCCTCCACGTCTG |
| HPV6E6-B3 | AAATTCTAGGCAGCACGC |
| HPV6E6-FIP | ACACAATTAATTTGCAACGTATGCACAACGACCATAGACCAGT |
| HPV6E6-BIP | GCACTGACCACTGCAGAGATCTGCATATGGATAGCCGC |
| HPV6E6-LF | GATAGATTAAACGTCTTGCAC |
| HPV6E6-LB | AACAGCTAAAGGTCCTGTTTCG |
| HPV11E6-F3 | GTAAAGATGCCTCCACGT |
| HPV11E6-B3 | TGCAGTTCTAAGCAACAGG |
| HPV11E6-FIP | ACTGAATTTGCAGAGTGTGCAAAGGCAACATCCATAGACCAGT |
| HPV11E6-BIP | TGCAGGAATGCACTGACCACTTTTGGAAAGTTGTCTCGCCAC |
| HPV11E6-LF | AAGATTAAACGTCTTGCAC |
| HPV11E6-LB | CGCAGAGATATATGCATATGCCT |
| HPV16E7-F3 | AGACAACTGATCTCTACTGTT |
| HPV16E7-B3 | CTTCCAAAGTACGAATGTCTAC |
| HPV16E7-FIP | TTCTGCTTGTCCAGCTGGACGCAATTAAATGACAGCTCAGAG |
| HPV16E7-BIP | CCGGACAGAGCCCATTACAATGTGTGTGCTTTGTACGCA |
| HPV16E7-LF | CATCTATTTCATCCTCCTC |
| HPV16E7-LB | TGCAAGTGTGACTCTACGCT |
| HPV18L1-F3 | CGCGTCCTTTATCACAGG |
| HPV18L1-B3 | TGGAATCCCCATAAGGATC |
| HPV18L1-FIP | GGCACCATATCCAGTATCTACCATAATTGCCCCCCTTTAGAACT |
| HPV18L1-BIP | TGCAAGATACTAAATGTGAGGTACCGCAGACATTTGTAAATAA TCAGGAT |
| HPV18L1-LF | TCACCATCTTCCAAAACTG |
| HPV18L1-LB | ATTGGATATTTGTCAGTCT |
| HPV52E6-F3 | TTGAGGATCCAGCAACAC |
| HPV52E6-B3 | GCGTAGGCACATAATACACA |
| HPV52E6-FIP | GCACACACTGCAGCCTTATTTCTTTTGACCCTGCACGAATTGTG |
| HPV52E6-BIP | AAGAGCTACAACGAAGAGAGGTCGCCATATGGATTATTGTCTC |
| HPV52E6-LF | CACCGATTCTTCCAGCACC |
| HPV52E6-LB | GTTTCTATTTACAGATTTACG |
| HPV58L1-F3 | ACAGGGAATGCTTATCTATGG |
| HPV58L1-B3 | TGTACCAAAGTCCATGCA |
| HPV58L1-FIP | GCATTATTGTTACAGGCAACACCTTGTTTAATTGGCTGTAAACCTC |
| HPV58L1-BIP | GCTGCTACTGATTGTCCTCCATTCCAAACCCTGTATCTACCAT |
| HPV58L1-LF | ACCCCAATGCTCACCAGTG |
| HPV58L1-LB | TTCTATTATTGAGGATGG |
3. set up following testing process, seen for details as follows:
(1) synthetic primer: invitrogen company is synthetic and through the HPLC purifying.
(2) viral DNA of sample to be measured being pressed Qiagen company extracts the test kit step process, obtains the DNA of sample.
(3) LAMP reaction.
(4) positive reaction result with the naked eye can be observed white precipitate and color becomes sky blue from purple, and the light absorption value of also available 650nm is judged
Embodiment
Embodiment 1: the LAMP of color-based detects human papillomavirus HPV6,11,16,18,52, the specificity of 58 types
Press the viral DNA in the Qiagen test kit explanation extraction cervical smear sample.Set up following amplification reaction system: 2.5 μ l Bst dna polymerase buffer liquid (10 *), 2.5 μ l dNTP (10mmol/L), article six, (primer concentration is F3 and B3:5pmol/ μ L to each 1 μ l of primer, BIP and FIP:40pmol/ μ L, Loop-1 and Loop-2:20pmol/ μ L), 8 μ l dH2O, 1 μ l Betaine (250mmol/L), 1 μ lMgSO4 (150mmol/L), 1 μ l HNB (3mmol/L), 1 μ l Bst archaeal dna polymerase (8U/ μ L), 2 μ l DNA samples.Mixing, 63 ℃, water-bath 1h, 80 ℃, the 5min termination reaction,, observe colour-change and white precipitate.Only have the HPV hypotype corresponding with primer that specific amplification is arranged, other papilloma viruss (HPV6,11,16,18,31,33,39,45,51,52,56,58,59,68) are without amplification.
Embodiment 2: the LAMP of color-based detects human papillomavirus HPV6,11,16,18,52, the sensitivity of 58 types
Utilize F3 and B3 increase corresponding HPV hypotype, then carrier construction pMD-18T-HPVDNA; Carrier is transformed into competent cell TOP10 enlarged culturing; Extract plasmid and survey OD260 with the OMEGA plasmid extraction kit; Calculate plasmid copy number; With the plasmid gradient dilution.The system that LAMP detects is with embodiment 1.The sensitivity of 6 kinds of HPV hypotype detections all can reach the levels of 100 copies.
Embodiment 3: the result that the LAMP of color-based detects 6 kinds of common HPV can with the naked eye (observe colour-change and white precipitate) or the colourimetric number judgement
After the LAMP reaction is finished, the variation of the color that detects by an unaided eye and the white precipitate at the pipe end, also desirable 20 μ l measure the absorption value of 650nm.The effective naked eyes of positive reaction can be observed white precipitate at the pipe end, and color has become reacted sky blue, the light absorption value of 650nm 〉=0.30 from reacting front purple in the pipe.
Claims (5)
1. pathogenic agent human papillomavirus (HPV) E6 who is used for people's neoplasmata genitalis and Genital warts, the loop-mediated isothermal amplification technique based on the HNB color of E7 or L1 gene test (LAMP), comprising: 36 Oligonucleolide primers that are used for that 6 kinds of common HPV viruses of Chinese population detect and HNB (hydroxynaphthol blue) dyestuff.
2. common 6 kinds of HPV virus E6 in the claim 1 described Chinese population, the primer of the loop-mediated isothermal amplification technique (LAMP) of E7 or L1 gene test comprise gene order that table 1 is listed and complementary sequence or the variant of every kind of sequence thereof.
3. common 6 kinds of HPV virus E6 in the claim 1 described Chinese population, the loop-mediated isothermal amplification technique (LAMP) of E7 or L1 gene test comprises 6 kinds of HPV viruses and variant thereof.
4. claim 3 described ranges of application of the present invention comprise Disease Prevention and Control Institutions at different levels, papilloma virus Sampling network laboratory, and Sentinel point hospital is used for 6 kinds of common HPV Detectings of Chinese population and monitoring.
5. common 6 kinds of HPV virus E6 in the claim 1 described Chinese population, E7 or; Response procedures and the trace routine of the loop-mediated isothermal amplification technique based on the HNB color of L1 gene test (LAMP).
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104076150A (en) * | 2013-03-28 | 2014-10-01 | 沈萍萍 | Detection method of HPV-E6 proteins |
| CN106148571A (en) * | 2016-09-06 | 2016-11-23 | 清华大学 | The reagent set of detection HPV HPV16 and HPV18 |
| CN106834547A (en) * | 2017-03-27 | 2017-06-13 | 杭州迪安生物技术有限公司 | A kind of kit for human papilloma virus E6/E7 genetic tests and its application based on real-time isothermal duplication |
| CN107557492A (en) * | 2017-09-14 | 2018-01-09 | 温州美众医学检验所 | LAMP detections are combined with primer and its reaction system |
| CN109055611A (en) * | 2018-08-24 | 2018-12-21 | 深圳市芯思微生物科技有限公司 | For detecting the kit of HPV |
| CN109694927A (en) * | 2019-03-01 | 2019-04-30 | 纽奥维特(成都)生物科技有限公司 | Primer system of HPV (human papillomavirus) as well as detection method and application thereof |
| CN114317828A (en) * | 2022-01-14 | 2022-04-12 | 广州朗坤生物科技有限公司 | LAMP primer combination, reaction system and method for detecting HPV types 52 and 58 |
| CN114480728A (en) * | 2021-11-25 | 2022-05-13 | 四川大学华西第二医院 | LAMP primer group and kit for bedside rapid detection of HPV (human papilloma Virus) and using method thereof |
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2011
- 2011-08-23 CN CN 201110242077 patent/CN102952894A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104076150A (en) * | 2013-03-28 | 2014-10-01 | 沈萍萍 | Detection method of HPV-E6 proteins |
| CN106148571A (en) * | 2016-09-06 | 2016-11-23 | 清华大学 | The reagent set of detection HPV HPV16 and HPV18 |
| CN106834547A (en) * | 2017-03-27 | 2017-06-13 | 杭州迪安生物技术有限公司 | A kind of kit for human papilloma virus E6/E7 genetic tests and its application based on real-time isothermal duplication |
| CN106834547B (en) * | 2017-03-27 | 2021-01-05 | 杭州迪安生物技术有限公司 | Kit for human papilloma virus E6/E7 gene detection based on real-time isothermal amplification and application thereof |
| CN107557492A (en) * | 2017-09-14 | 2018-01-09 | 温州美众医学检验所 | LAMP detections are combined with primer and its reaction system |
| CN109055611A (en) * | 2018-08-24 | 2018-12-21 | 深圳市芯思微生物科技有限公司 | For detecting the kit of HPV |
| CN109694927A (en) * | 2019-03-01 | 2019-04-30 | 纽奥维特(成都)生物科技有限公司 | Primer system of HPV (human papillomavirus) as well as detection method and application thereof |
| CN114480728A (en) * | 2021-11-25 | 2022-05-13 | 四川大学华西第二医院 | LAMP primer group and kit for bedside rapid detection of HPV (human papilloma Virus) and using method thereof |
| CN114317828A (en) * | 2022-01-14 | 2022-04-12 | 广州朗坤生物科技有限公司 | LAMP primer combination, reaction system and method for detecting HPV types 52 and 58 |
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