CN101487063A - Human papilloma virus infection gene amplification fluorescent detection kit - Google Patents
Human papilloma virus infection gene amplification fluorescent detection kit Download PDFInfo
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- CN101487063A CN101487063A CNA2009100374526A CN200910037452A CN101487063A CN 101487063 A CN101487063 A CN 101487063A CN A2009100374526 A CNA2009100374526 A CN A2009100374526A CN 200910037452 A CN200910037452 A CN 200910037452A CN 101487063 A CN101487063 A CN 101487063A
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Abstract
The invention discloses a probe and a primer which have nucleotide sequence complementary with HPV DNA, wherein, the probe is selected from SEQ ID Nos: 1 to 13; the primer is selected from SEQ ID SEQ ID Nos: 14 to 39; the invention also discloses a fluorescence identification kit comprising the probe and the primer and used for indentifying nucleic acid amplification infected by high-risk human papillomavirus, which contains 13 varieties of high-risk human papillomavirus subtypes and can finish extracting and detecting 80 to 100 portions of clinical samples DNA in 3 hours. The invention has the advantages of high sensitivity and specificity and low price, thus being easy to be accepted by patients and easy for popularizing and spreading.
Description
Technical field
The present invention relates to a kind ofly have with probe, the primer of human papillomavirus HPV DNA complementary nucleotide sequence and contain the human papilloma virus infection gene amplification fluorescent detection kit of this probe, primer.
Background technology
Human papillomavirus (HPV) and cervical cancer are in close relations, and HPV can be divided into two kinds of high-risk-type and low risks, and the cases of cervical cancer of clinical proof 95～99.7% is relevant with the high-risk HPV virus infection, and HPV infects and makes the relative risk of suffering from cervical cancer increase by 250 times.The existence of HPV is just indicating the existence of disease, diagnoses HPV to infect ahead of time, to removing the persistent infection of female genital tract, prevents that it from having great significance to the cervical cancer differentiation,
Traditionally, it is the conventional means of population screening that cervical smear detects, but since this method only to observe as diagnostic base, drawn materials, smear is made quality, read chip technology influences, its accuracy rate is low, the false positive height, poor repeatability, result who obtains and analysis rate of missed diagnosis can reach 30%.Molecular diagnostic techniques can roughly be divided into two kinds, and a kind of is not need the DNA amplification molecule, directly utilizes nucleic acid probe to detect to target molecule by hybridization arrest, and the shortcoming of this method is that sensitivity is low, generally will use radioelement
32The P mark just can reach testing goal; Another kind is to increase to molecule, can be further divided into two classes again: (1) molecules of interest (target molecule) amplification, promptly to the check (as the PCR method) of increasing of molecules of interest (target molecule) gene; (2) amplification of signal is generally caught viral DNA as probe with hybridization with nucleic acid, carries out amplification of signal again and detects.
Smear the sheet inspection with traditional cell and compare, the advantage that PCR detects is apparent, and it is highly sensitive, can detect the HPV DNA of denier.Developed a kind of novel PCR detection method in recent years again, i.e. real-time fluorescence PCR detection method.Though it is quick, easy to adopt conventional round pcr to detect the HPV infection in the past, has two bigger defectives:,, then easily pollute and cause false positive as careless manipulation 1. amplified production being carried out in the process of aftertreatment.2. can only be qualitative, can not be quantitative, can not observe the relation of HPV load and cervical lesions, thereby limit its application in clinical position.The real-time fluorescence PCR technology has been added a mark on the conventional PCR basis fluorescence double-tagging probe of 2 fluorogenes.One is marked at 5 of probe ' end, is called the fluorescence report group, and another is marked at 3 of probe ' end, is called fluorescence and suppresses group.Both constitute the transmission ofenergy structure, and promptly 5 ' end fluorescence that fluorophor sent can be absorbed by 3 ' end fluorophor or suppress.The real-time fluorescence PCR technology combines gene amplification, molecular hybridization and photochemistry together, the whole process of pcr amplification and product analysis is all carried out under the single tube sealing condition, and, realized pcr amplification product is carried out Real-time and Dynamic Detection and automatic analytical results by microcomputer control.This method detects cervical HPV infection, has advantages such as quick, easy, highly sensitive, high specificity, both has been applicable to that extensive examination also is applicable in the clinical routine work to adopt.
Summary of the invention
The object of the present invention is to provide a kind of probe, have and HPV DNA complementary nucleotide sequence, its middle probe is selected from SEQ ID Nos:1～13, and any 15～28 bases in the nucleotide sequence scope of each probe all can be used as probe sequence.
| Sequence number | The HPV type | |
| SEQ NO1 | ||
| 16 | 5′-gac aac tga tct cta ctg tta tga gca att-3′ | |
| |
18 | 5′-tat tct ttg cag caa ggg aac atg gca tac-3′ |
| |
31 | 5′-att gaa cta caa atg atg ttg gaa aca tta-3′ |
| |
33 | 5′-gtt agc atc aaa gac caa agc att tca agt-3′ |
| |
35 | 5′-gga acc cga ggc aac tga cct ata ctg tta-3′ |
| SEQ NO6 | 39 | 5′-atg gca cta gaa agt gtt gca caa act gaa-3′ |
| SEQ NO7 | 45 | 5′-tgg aaa atg caa tac tat tta cag caa ggg-3′ |
| SEQ NO8 | 51 | 5′-cca gaa aga cgg gct gga cag gct acg tgt-3′ |
| SEQ NO9 | 52 | 5′-gcc tgc caa gct att gaa cta caattg gca-3′ |
| SEQ NO10 | 56 | 5′-gga tga agt aga cca ttt gca gga gcg gcc-3′ |
| SEQ NO11 | 58 | 5′-taa agc gtt tca agt aat tga act gca aat-3′ |
| SEQ NO12 | 59 | 5′-gtc aaa ccg tac ttc cac tgt aat gcc ctg-3′ |
| SEQ NO13 | 68 | 5′-att atg cag cac gag aac gtg gta tgc ata-3′ |
Another object of the present invention is to provide a kind of primer, have and HPV DNA complementary nucleotide sequence, wherein primer is selected from SEQ ID Nos:14～39, and any 20～25 bases in the nucleotide sequence scope of every primer all can be used as primer sequence.The nucleotidesequence of each primer is as follows:
3 of probe of the present invention ' employing fluorescent mark, fluorescent mark can be: FAM, VIC, JOE, HEX, TET, Cy3.
5 of probe of the present invention ' employing fluorescent mark, fluorescent mark can be: TAMARA, MGB.
Main purpose of the present invention is to provide a kind of nucleic acid amplification fluorescent identification kit of high-risk human papilloma virus infection, comprising:
(1) nucleotide probe of different HPV types, its middle probe are one at least and are selected from SEQ IDNos:1～13;
(2) various primers, the dna sequence dna of the clinical sample that is used for increasing, this primer is selected from SEQID Nos:14～39;
(3) Chang Gui amplifing reagent;
(4) Chang Gui dna cleavage reagent;
(5) quality control reference substance.
Existing multiple HPV detection kit appears on the market both at home and abroad up to now, domestic used HPV detection kit major part is to utilize PCR detection method (comprising real time fluorescent PCR method), after Auele Specific Primer special by HPV or its each somatotype carries out pcr amplification, agarose electrophoresis or fluorescent quantitation obtain net result, and many of these test kits are used for high-risk HPV-16,18, low risk HPV6,11 HPV virus detects, detection for other types HPV virus does not relate to, and nucleic acid amplification (PCR) the fluorescence identification kit that high-risk human papillomavirus of the present invention (HPV) infects comprises 13 kinds of high-risk human papillomaviruss (HPV) hypotype, can in 3 hours, finish 80～100 parts of clinical sample DNA of extracting and detection simultaneously, sensitivity and specificity height, cheap, accepted by extensive patients easily, therefore be easy to penetration and promotion.
Description of drawings
Below be the description of the drawings, be convenient to understand the purpose and the concrete feature of foregoing invention.
Figure one is the detected results (200 copy/types/reaction) of 13 kinds of high-risk HPV templates on ABI7000, and red arrow 1～13 is represented the amplification curve of HPV16,18,31,33,35,39,45,51,52,56,58,59,68 hypotypes respectively from top to bottom.
Figure two be HPV58, HPV56, HPV52, HPV18, HPV16 through pcr amplification rear electrophoresis detection figure, wherein M, N, 1,2,3,4,5 represent molecular weight marker, negative control, HPV58,56,52,18 and 16 hypotypes respectively.
Figure three is HPV45, HPV68 electrophoresis detection figure behind pcr amplification, and wherein M, 1,2 represents molecular weight marker, HPV45 and HPV68 hypotype respectively.
Figure four is HPV31, HPV59, HPV51 electrophoresis detection figure behind pcr amplification, wherein 1,2,3, M represents HPV31, HPV59, HPV51 and molecular weight marker thing respectively.
Figure five is HPV33, and HPV35, HPV39 detect figure through the pcr amplification rear electrophoresis, and wherein M, N, 1,2,3 represent molecular weight marker, negative control, HPV33,35 and 39 respectively.
Embodiment
The present invention will be described in detail by the following examples.
The inventor is by analyzing the DNA of clinical sample, preparation has and the probe of SEQ ID Nos:1～13 of the DNA complementary nucleotide sequence of 13 kinds of high-risk HPVs (16,18,31,33,35,39,45,51,52,56,58,59,68) and the primer of SEQ ID Nos:14～39 respectively, detects HPV and infects and identify the HPV high-risk-type.
Nucleic acid amplification (PCR) the fluorescence identification kit that high-risk human papillomavirus provided by the invention (HPV) infects comprises above-mentioned probe and primer, archaeal dna polymerase, also comprise PCR MIX, the latter comprises that all PCR beyond the removing template react required reagent, and is used for the HPV53 plasmid of positive control and the ultrapure water of negative control.
Test kit of the present invention uses step as follows:
[sample extraction]
(1) gather specimen types:
Women's uterine neck mouth cast-off cells
(2) acquisition method:
Before the sampling with cotton swab with uterine neck many secretory product wiped clean gently of making a slip of the tongue, change cotton swab then, be close to the uterine neck oral mucous membrane with cotton swab or hairbrush that cell-preservation liquid soaks into, slightly firmly rotate several weeks, to obtain cervical exfoliated cell, cotton swab after the sampling or hairbrush are put into the sample hose that has 1 ml cells preservation liquid, fully after the rinsing, cotton swab or adherent the extracting of hairbrush are abandoned.
(3) store method:
The sample of gathering should not surpass 24 hours 2～8 ℃ of preservations, and-20 ℃ of preservations are no more than three months, but-70 ℃ of prolonged preservation are avoided the multigelation sample.
[operation steps]
1 sample preparation (carrying out) in the sample preparation district
1.1 take out sample to be tested, HPV53 plasmid and ultrapure water, place room temperature to thaw, and the vibration mixing.
1.2 get the 500ul sample to be tested, centrifugal 10 minutes of 13000rpm, the careful suction abandoned supernatant, keeps precipitation.
1.3 in throw out, add 50ul dna cleavage liquid, the vibration mixing.
1.4 100 ℃ of boiling water bath/dried baths 10 minutes, centrifugal 10 minutes of 13000rpm keeps supernatant standby (DNA for discharging in the supernatant is not if used the same day, in-20 ℃ of preservations).
Annotate: positive control need not to handle, and can be directly used in pcr amplification.
2 amplifing reagents are prepared (area in preparation is carried out before PCR)
2.1 from test kit, take out HR-HPV PCR MIX, archaeal dna polymerase, after room temperature is melted and is mixed, centrifugal 10 seconds of 8000rpm.
2.2 prepare amplifing reagent according to following table:
Everyone part reaction cumulative volume is: the sample DNA that 18ul HR-HPV PCR MIX+2ul handles well; Each reaction HR-HPV PCR MIX cumulative volume=18 (HR-HPV PCR MIX) * PCR reaction tubes pipes are counted n (n=sample number+1 pipe negative control+1 pipe positive control).
2.3 with the abundant mixing of the amplifing reagent for preparing, 8, centrifugal 10 seconds of 000rpm adds 18ul respectively in n the PCR reaction tubes of setting, transfer to the sample preparation district.
3 application of samples (carrying out) in the sample process district
3.1 add sample DNA, negative control, positive control that 2ul handles well in the PCR of correspondence reaction tubes respectively, the tight pipe lid of lid is done centrifugal slightly.
3.2 transfer to detection zone, put into corresponding fluorescent PCR detector, the record sample is put order.
(4) pcr amplification (carrying out) at detection zone
4.1 cycling condition setting
1 circulation of 95 ℃ of 10min
94℃ 10sec
55℃ 30sec
94℃ 10sec
58℃ 30sec
Cycling program is provided with as follows when using rich day Line-Gene quantitative real time PCR Instrument:
Reaction system is made as 20ul.
4.2 instrument detecting channel selecting
It is report fluorescence that selected report fluorescence, ABI7000 select FAM, and cancellation fluorescence is None, and Passive Reference selects none, and the report fluorescence of rich day Line-Gene quantitative real time PCR Instrument is first channel.
[result's judgement]
The setting of 1 threshold value
1.1 on ABI7000, carry out being defined as of interpretation of result base line: get 3～10 round-robin fluorescent signals; The threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve (random noise line), and Ct value=0.0 is as the criterion.
1.2 zero adjustment is when carrying out interpretation of result on rich day quantitative real time PCR Instrument: get 3～10 round-robin fluorescent signals, definite threshold line of baseline and threshold value (0.1＜threshold line＜1) is just above the vertex of normal negative control product amplification curve (random noise line), and Ct value=0.0 is as the criterion.Yield value is 70.
2 Quality Controls contrast
2.1 negative control CT value all should be shown as Undet.
2.2 positive control CT value≤26.
3 results judge
3.1 the CT value of sample is shown as Undet and is judged as feminine gender.
3.2 CT value≤33 of sample are judged as the positive; If the sample of 33＜CT value＜35 suggestion is reformed, reform that CT value＜35 are positive as a result, otherwise negative.
[test kit diagnostic result statistics of the present invention]
Table one: the distribution of clinical examination test case
Table two: the consistence of test kit detected result of the present invention and coincidence rate
Table three: test kit detected result statistics of the present invention
Table four: test kit detected result Performance Evaluation of the present invention
The foregoing description operating process only needs about 3 hours, and is simple to operate, and can finish many parts of clinical sample DNA and detection simultaneously.From above-mentioned diagnostic result cartogram, sensitivity and specificity height that test kit of the present invention detects reach 94% and 87% respectively, and effect has fast and accurately been played in the diagnosis of disease, satisfy requirements for clinical application fully.
Sequence table
＜110〉Chaozhou Kaipu Biochemistry Co., Ltd.
＜120〉human papilloma virus infection gene amplification fluorescent detection kit
<160>39
<210>1
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV16 probe
<400>1
<210>2
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV18 probe
<400>2
<210>3
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV31 probe
<400>3
<210>4
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV33 probe
<400>4
<210>5
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV35 probe
<400>5
<210>6
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV39 probe
<400>6
<210>7
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV45 probe
<400>7
<210>8
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV51 probe
<400>8
<210>9
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV52 probe
<400>9
<210>10
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV56 probe
<400>10
<210>11
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV58 probe
<400>11
<210>12
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV59 probe
<400>12
<210>13
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV68 probe
<400>13
<210>14
<211>43
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the HPV16 primer 1
<400>14
<210>15
<211>39
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV16 primer 2
<400>15
<210>16
<211>46
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the HPV18 primer 1
<400>16
<210>17
<211>42
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV18 primer 2
<400>17
<210>18
<211>46
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the HPV31 primer 1
<400>18
<210>19
<211>44
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV31 primer 2
<400>19
<210>20
<211>44
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the HPV33 primer 1
<400>20
<210>21
<211>43
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV33 primer 2
<400>21
<210>22
<211>40
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the HPV35 primer 1
<400>22
<210>23
<211>40
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV35 primer 2
<400>23
<210>24
<211>41
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the HPV39 primer 1
<400>24
<210>25
<211>45
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV39 primer 2
<400>25
<210>26
<211>46
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the HPV45 primer 1
<400>26
<210>27
<211>46
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV45 primer 2
<400>27
<210>28
<211>41
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the HPV51 primer 1
<400>28
<210>29
<211>44
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV51 primer 2
<400>29
<210>30
<211>44
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the HPV52 primer 1
<400>30
<210>31
<211>40
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV52 primer 2
<400>31
<210>32
<211>44
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the HPV56 primer 1
<400>32
<210>33
<211>42
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV56 primer 2
<400>33
<210>34
<211>44
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the HPV58 primer 1
<400>34
<210>35
<211>44
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV58 primer 2
<400>35
<210>36
<211>45
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the HPV59 primer 1
<400>36
<210>37
<211>44
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV59 primer 2
<400>37
<210>38
<211>40
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the HPV68 primer 1
<400>38
<210>39
<211>40
<212>DNA
＜213〉artificial sequence
<220>
＜223〉HPV68 primer 2
<400>39
Claims (5)
1, a kind of probe that has with HPV DNA complementary nucleotide sequence, its middle probe is selected from SEQ ID Nos:1～13, and any 15～28 bases in the nucleotide sequence scope of each probe all can be used as probe sequence.
2, a kind of primer that has with HPV DNA complementary nucleotide sequence, wherein primer is selected from SEQ ID Nos:14～39, and any 20～25 bases in the nucleotide sequence scope of every primer all can be used as primer sequence.
3, a kind of nucleic acid amplification fluorescent identification kit of high-risk human papilloma virus infection comprises:
(1) nucleotide probe of different HPV types, its middle probe are one at least and are selected from SEQ ID Nos:1～13;
(2) various primers, the dna sequence dna of the clinical sample that is used for increasing, this primer is selected from SEQ ID Nos:14～39;
(3) Chang Gui amplifing reagent;
(4) Chang Gui dna cleavage reagent;
(5) quality control reference substance.
4, the described probe of claim 1 is characterized in that, 3 of probe ' employing fluorescent mark, and fluorescent mark is FAM, VIC, JOE, HEX, TET or Cy3.
5, the described probe of claim 1 is characterized in that, 5 of probe ' employing fluorescent mark, and fluorescent mark is TAMARA or MGB.
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| CN2009100374526A CN101487063B (en) | 2009-02-25 | 2009-02-25 | Human papilloma virus infection gene amplification fluorescent detection kit |
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| CN101487063A true CN101487063A (en) | 2009-07-22 |
| CN101487063B CN101487063B (en) | 2011-07-20 |
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| CN102229930A (en) * | 2011-06-10 | 2011-11-02 | 亚能生物技术（深圳）有限公司 | PCR group, method and kit for detecting human papilloma virus (HPV) |
| CN102367487A (en) * | 2011-09-09 | 2012-03-07 | 中国医学科学院病原生物学研究所 | High accuracy detection method of human papilloma virus genotype |
| CN102994646A (en) * | 2011-09-19 | 2013-03-27 | 潘振华 | Fluorescent nucleic acid amplification detection method of human papilloma virus |
| CN110885906A (en) * | 2012-10-11 | 2020-03-17 | 简·探针公司 | Compositions and methods for detecting human papillomavirus nucleic acids |
| CN103114033A (en) * | 2012-11-08 | 2013-05-22 | 杭州腾越生物科技有限公司 | Array type multiplex microfluidic isothermal amplification chip for HPV (human papillomavirus) detection |
| CN103276107A (en) * | 2013-05-13 | 2013-09-04 | 中国医学科学院病原生物学研究所 | Method for detecting and identifying human polyomavirus with high sensitivity |
| CN103276107B (en) * | 2013-05-13 | 2014-11-19 | 中国医学科学院病原生物学研究所 | Method for detecting and identifying human polyomavirus with high sensitivity |
| CN105506173A (en) * | 2014-09-25 | 2016-04-20 | 苏州新波生物技术有限公司 | Nucleic acid detection kit for human papilloma virus, use method and application thereof |
| WO2018059581A1 (en) * | 2016-09-30 | 2018-04-05 | 广州易活生物科技有限公司 | Probe for hpv virus genotyping detection based on efirm technique, kit and use |
| CN109554505A (en) * | 2018-12-26 | 2019-04-02 | 广东和信健康科技有限公司 | The probe and its detection kit of one group of detection HPV high-risk-type and middle danger type nucleic acid and application |
| CN109554505B (en) * | 2018-12-26 | 2022-02-18 | 广东和信健康科技有限公司 | Group of probes for detecting HPV high-risk and medium-risk nucleic acids, detection kit and application thereof |
| CN112143832A (en) * | 2020-09-30 | 2020-12-29 | 东南大学 | Fluorescent sensor for detecting HPV-18 virus infection |
| CN112143832B (en) * | 2020-09-30 | 2024-04-09 | 东南大学 | Fluorescent sensor for detecting HPV-18 virus infection |
| CN114592087A (en) * | 2021-12-16 | 2022-06-07 | 山东博弘基因科技有限公司 | High-risk human papilloma virus nucleic acid detection kit |
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