CN103966356A - Human immunodeficiency virus type 1 one-step fluorescence quantitative RT-PCR detection kit - Google Patents
Human immunodeficiency virus type 1 one-step fluorescence quantitative RT-PCR detection kit Download PDFInfo
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Abstract
The present invention provides a human immunodeficiency virus type 1 one-step fluorescence quantitative RT-PCR detection kit, which comprises a RT-PCR reaction solution, a RT-PCR enzyme mixture, a primer probe, a negative control, a strong positive control, a weak positive control, and calibration substances No.1-5. According to the present invention, a one-step fluorescence quantitative reaction can be directly performed on the extracted HIV-1RNA, the virus loading of the HIV-1RNA in a sample can be detected, the reference gene is adopted as the internal control, and the UNG enzyme is adopted to prevent pollution; and the kit has characteristics of simple one-step amplification method, short procedure, easy operation, pollution prevention, strong detection result specificity, high sensitivity, clear result and high result reliability, and can be used for detection of the virus loading of the human immunodeficiency virus type 1 (HIV-1) RNA in blood serum.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of Measurement for Biotechnique, relate in particular to a kind of for human immunodeficiency virus type 1 (HIV-1) RNA fluorescence quantitative detection kit.
Background technology
Human immunodeficiency virus (Human Immunodeficiency Virus, HIV) is a kind of slow virus (Lentivirus) that infects human immunity system cells, belongs to the one of retrovirus (Retrovirus).This virus is destroyed the immunologic function of human body, cause the immune resistibility that loses, thereby cause various diseases pathogenic agent to be able to survive in human body, develop into last, causing acquired immune deficiency syndrome (AIDS) (is acquired immune deficiency syndrome (AIDS), Acquired Immunodeficiency Syndrome, AIDS), for a kind of so far without the effective mortality transmissible disease of therapy.From 1981 since the U.S. finds first and confirms, acquired immune deficiency syndrome (AIDS) has been captured the life that exceedes 3,000 ten thousand people, make it become one of global prevailing disease of tool destructive force in history, approximately have in the world in by the end of May, 2,011 6,400 ten thousand people's aids infection poison by, on average have 7000 new cases every day.At present, South Asia and South East Asia become the second severely afflicated area after Sub-Saharan Africa.China CDC estimates, ends to the end of the year 2011 China survival HIV carriers and AIDS patient approximately 780,000 people, annual new the infected 4.8 ten thousand people, dead 2.8 ten thousand people.Epidemic situation has covered national all provinces, autonomous regions and municipalities, and at present China faces acquired immune deficiency syndrome (AIDS) morbidity and dead peak period, and has been started to population to spread by high risk population such as drug abuse, unlicensed prostitutes, and anti-Chinese mugwort, the situation is tense for anti-Chinese mugwort.
After HIV infects, the variation of virus load and the process of disease have close dependency, the detection of HIV virus load can predictive disease generation, development and prognosis, therefore, the detection of HIV virus load can monitoring disease evolution, and provide foundation for formulating corresponding treatment plan and strategy; And the higher pregnant woman of virus load causes the danger of mother-to-baby transmission larger, can determine whether pregnant woman needs to take antiviral and reduce the mother-to-baby transmission of HIV according to virus load.The effect of anti HIV-1 virus medicine is mainly to suppress copying of virus, reduces virus load.Therefore, the curative effect of monitoring, Prevention of MTCT and the observation antiviral of the detection of virus load to disease all has great importance.
According to gene difference, HIV can be divided into HIV-1 type and HIV-2 type.Generally, HIV-1 virus is the pathogenic agent that most HIV infect, and HIV-2 virus mainly appears at Africa, especially in area, West Africa.The circulation way of HIV-2 is identical with HIV-1, all can generator opportunistic infections and AIDS, but immune deficiency due to HIV-2 develops comparatively slowly and gentle.The hypotype difference that different areas are popular, also there is some difference for the epidemic status of same hypotype in different areas.At present, the popular HIV-1 type that is mainly in world wide.In the U.S., because HIV-2 is comparatively rare, HIV-2 is not listed in conventional sense.Popular HIV is mainly M group HIV-1 type and (there is not yet the report of HIV-1 N group and O group at present within the border within Chinese territory, the report of HIV-2 type cases of infection is few), its genotype is mainly B/B ', BC recombinant type (comprising CRF 07_BC recombinant type and CRF 08_BC recombinant type) and AE recombinant type (CRF 01_AE recombinant type).And HIV-1 genotype has certain areal variation, the popular HIV genotype in different areas is also not quite similar, and as CRF 07_BC recombinant type is popular in the ground such as Sichuan, Xinjiang, CRF 08_BC recombinant type is popular in the ground such as Guangxi.
At present, the detection side of HIV totally can be divided into antibody test and virus detection two large classes.Virus detects and comprises that cell cultures (virus separates), p24 Detection of antigen and viral nucleic acid detect.Early stage is mainly the antibody that detects anti-HIV by serological test to the diagnosis of HIV, indirectly diagnoses HIV to infect.In recent years, molecular biology method was constantly applied in the detection of HIV, and the laboratory diagnostic method of HIV makes great progress, and detection of nucleic acids has become the developing direction of HIV laboratory diagnosis.
Traditional nucleic acid detection method is to adopt nucleic acid hybridization technique to detect object nucleic acid, although adopt radiolabeled probe can improve the susceptibility of detection, but still on the low side, can not meet clinical needs.Nucleic acid detection technique development recent years rapidly, is mainly to adopt various amplification amplifying techniques to improve the sensitivity detecting.Along with the maturation of amplification technique, the target sequence of low copy can be become logarithm level to amplify amplification, adopt the on-radiation detection system that specific activity probe is sensitiveer (as electrochemical luminescence system etc.) simultaneously, the sensitivity that obviously improves detection of nucleic acids, and reduce the pollution of radioactive substance.The method that detects HIV-1 RNA viruses carrying capacity mainly contains branched DNA and detects (bDNA), reverse transcription polymerase chain reaction (RT-PCR) and amplification of nucleic acid sequences detection (NASBA) etc., wherein, the fluorescent quantitative RT-PCR method based on TaqMan probe technique has developed into maturation, has commonly used and effective detection means.
TaqMan probe technique is the fluorescent quantitative PCR technique of high special.TaqMan probe is a kind of oligonucleotide probe, and fluorophor is connected to 5 ' of probe-end, and quenching group is at probe 3 '-end.In the time that probe is complete or match with target sequence, the fluorescence of fluorophor transmitting, because approaching and be quenched with the quenching group of 3 '-end, produces without fluorescent signal.In the time carrying out PCR reaction extension process,
taq5' → 3' exonuclease activity of archaeal dna polymerase is degraded probe, and fluorophor is separated with quenching group, produces fluorescent signal.PCR reaction cycle of every experience, just has the amplified production of a part to generate, and is accompanied by the generation of the fluorescent signal of a part.Along with the increase of amplification cycles number, the fluorophor signal discharging constantly accumulates, and is the process that an Exponential Synchronization increases, and the intensity of fluorescent signal has just represented the copy number of amplified production.This technology has high specificity, level of automation high, efficiently solves the problems such as PCR pollution.HIV-1 fluorescent quantitation detects just based on this principle design, for the diagnosis of HIV-1 early infection, extensive examination, curative effect performance analysis, new drug development etc. provide good technical support.At present, HIV-1 virus load reagent mainly contains following several in the world: COBAS AmpliPrep/COBASTaqMan HIV-1 Test (Roche), Abbott RealTime HIV-1 Amplification Kit (Abbott), Versant HIV-1 RNA 3.0 (bDNA, Simens), Roche Amplicor HIV-1 Monitor Test (Roche), NucliSens HIV-1 QT (NASBA) and NucliSens HIV-1 EasyQ (EasyQ) etc.Da'an Gene Company, Zhongshan University of China and Qiagen Bioengineering (Shenzhen) Co., Ltd. have also developed HIV-1 nucleic acid fluorescent quantitative diagnostic reagent, have obtained national production permit.
For avoiding the false negative of reaction result, in reaction system, add reference gene and internal reference probe simultaneously.Reference gene is to contain and the different slow virus RNA of probe sequence sequence in amplification gene, it with the region of HIV-1 amplification gene aligning primer specific combination.Reference gene is by HIV-1 amplification gene upstream and downstream Auele Specific Primer in conjunction with the amplified production that also can produce equal length (137 bp), and its based composition is consistent with HIV-1 amplification gene sequence.
Utilize the antipollution characteristic of UNG enzyme simultaneously, in pcr amplification reaction, replace dTTP with dUTP, so only in amplified fragments, contain dUTP; And dUTP makes the amplicon polluting be easy to the enzyme liberating by UNG before amplification target DNA: the PCR product of this dUTP of containing is hatched together with UNG enzyme, because of the N-glycosyl bond between UDG enzyme cleavable uridylic base and sugared phosphoric acid skeleton, can from strand or double-stranded DNA, eliminate dUTP and stop
taqthe extension of archaeal dna polymerase, thus the ability being increased again lost.UNG enzyme loses activity above at 50 DEG C, can not destroy patient HIV-1 RNA like this through thermal cycling.UNG enzyme has no effect to the template that does not contain dUTP, and therefore, the HIV-1 RNA only extracting from HIV-1 positive patients sample serum in reaction system is not because directly being carried out RT-PCR reaction by UNG enzyme liberating.
This technology is by analyzing the each genotype sequence alignment of HIV-1, obtain, universal primer special for the each genotype of HIV-1 and fluorescent probe, by detecting various HIV-1 standard serums and clinical serum, determine the various detection indexs of this test kit, determine the minimum copy number of specificity, susceptibility and detection that this test kit detects, for the making a definite diagnosis of HIV-1, Large-scale Screening, severity extent judgement, therapeutic evaluation, new drug development etc. provide a convenience, cheap detection means.Its gordian technique is to obtain for special, responsive universal primer and the general probe of various HIV-1 hypotypes and strain isolated, prepares high specific, does susceptibility, reproducible HIV-1 fluorescent quantificationally PCR detecting kit.
Summary of the invention
The object of the invention is to: a kind of accurately easy method for human immunodeficiency virus type 1 (HIV-1) RNA detection by quantitative is provided; Another object is to provide a kind of test kit for the method.
Technical scheme of the present invention is: a kind of human immunodeficiency virus type 1 fluorescence quantitative detecting method and test kit are provided, adopt single stage method fluorescent quantitative PCR technique to detect human immunodeficiency virus type 1 (HIV-1) RNA.Starfish credit medical science and technology Development Co., Ltd post method purified reagent (pellosil absorption method) is carried out after nucleic acid extraction HIV-1 positive serum sample in the use, directly preparation reaction system increases, reaction system preparation is convenient, amplification program step is easy, proliferation time is short, without recirculation repeatedly, prevent product pollution.The inventive method is easy and simple to handle, susceptibility is high, result is distinct, reliability is high.
Test kit provided by the invention comprises: RT-PCR reaction solution, RT-PCR enzyme mixture, primer probe, negative control, strong positive contrast, weak positive control, calibration object No. 1 ~ No. 5.The nucleic acid that test kit provided by the invention detects need be by using Shanghai Xingyao Medical Technology Development Co., Ltd.'s post method purified reagent (pellosil absorption method) to carry out nucleic acid extraction to HIV-1 positive serum sample, the HIV-1 RNA extracting is directly carried out to fluorescence quantitative RT-RCR detection, and wherein said RT-PCR reaction solution is Tris-HCl (pH8.3) 20 mM, KCl 100 mM, gelatin 0.2 mg/ml, dATP, dGTP, each 0.4 mM of dCTP, dUTP, MgCl
2the mixed solution of 6 mM; Described enzyme mixture be reversed transcriptive enzyme,
taqthe mixture of archaeal dna polymerase and UNG enzyme (reversed transcriptive enzyme 3 U/ul,
taqarchaeal dna polymerase 2 U/ul, UNG enzyme 1 U/ul); Described primer probe is the mixed solution (each 6.25 uM of primer, HIV-1 specific probe 2.5 uM, internal reference probe 2.5 uM) of a pair of HIV-1 Auele Specific Primer, a HIV-1 specific probe and an internal reference probe; Described negative control is the normal human serum without HIV-1 RNA; The human serum that described strong positive contrast is the slow virus that contains HIV-1 gene fragment of high density; The human serum of the slow virus that contains HIV-1 gene fragment that described weak positive control is lower concentration; The human serum of the slow virus that contains HIV-1 gene fragment that described calibration object is concentration known gradient No. 1 ~ No. 5.The HIV-1 gene-specific primer that detects use divides upstream primer and downstream primer:
Upstream primer sequence < SEQ ID No.3 > is: 5 '-GGCTGTTGGAAATGTGG-3 ';
Downstream primer sequence < SEQ ID No.4 > is: 5 '-GCTGTTGGCTCTGGTCTG-3 ';
HIV-1 specific probe sequence < SEQ ID No.5 > is: 5 '-FAM-AATTCCCCGGCCTTCCTTTGTTG-TAMRA-3 ';
Internal reference specific probe sequence < SEQ ID No.6 > is: 5 '-JOE-CCACCAATTGGTTCCTCTCTGTG-TAMRA-3 '.
Reagent provided by the invention is stored in-20 DEG C, reduces multigelation as far as possible.
Test kit using method of the present invention:
Each detection all should be set up negative control, strong positive contrast, weak positive control and calibration object No. 1 ~ No. 5.Amplification detection method is as follows:
A, prepare reaction solution by reaction sample number (reaction sample number=sample number+reference substance to be checked 3+calibration object 5+1) n: get RT-PCR reaction solution n × 12.5 μ l, primer probe n × 2 μ l, RT-PCR enzyme mixture n × 0.5 μ l mixes in a centrifuge tube; The low-speed centrifugal several seconds, install in reaction tubes by 15 μ l/ pipes point;
B, sample this extract, reference substance extract, the each 10 μ l of calibration object extract and add respectively in reaction tubes, the low-speed centrifugal several seconds, take out and put on full-automatic fluorescent PCR instrument;
C, response procedures are: 37 DEG C of reaction 10 min, and 50 DEG C of reaction 15 min, then 95 DEG C of insulation 2 min, then by 94 DEG C of 10 s → 60 DEG C 45 s, circulate 45 times, and in 60 DEG C of signals that gather respectively FAM, JOE fluorescence channel;
D, instrument PCR program are carried out result preservation and data analysis by instrument and software requirement after having moved.To get fluorescent value higher than sample noise line and negative control as detection threshold; Determine that in each sample reaction tubes, reference gene detects Ct value within 30 ~ 32 scopes, avoids the false negative of detected result simultaneously; The HIV-1 rna content C (IU/ml) of the each sample extract of the automatic combined standard curve calculation of analysis software.
The inventive method principle is based on TaqMan hydrolysis probes fluorescent PCR principle, taking viral HIV-1 RNA as template, adopts virogene group-specific primers probe, be aided with reversed transcriptive enzyme,
taqarchaeal dna polymerase and UNG enzyme, through single stage method fluorescence RT-PCR experiment, can be fast, accurately human immunodeficiency virus type 1 (HIV-1) RNA template is analyzed; From reverse transcription to fluorescent PCR, a step can complete, and can effectively prevent that multiple operation from polluting.So detection method of the present invention and test kit high specificity, susceptibility is high, easy and simple to handle, result is distinct, reliability is high, can be used for the detection by quantitative of human immunodeficiency virus type 1 in serum (HIV-1) RNA.
Embodiment
embodiment 1a kind of human immunodeficiency virus type 1 single stage method fluorescent quantificationally PCR detecting kit.
1. the extraction of human immunodeficiency virus type 1 (HIV-1) RNA
Use Shanghai Xingyao Medical Technology Development Co., Ltd.'s post method purified reagent (pellosil absorption method) to carry out nucleic acid extraction to HIV-1 positive serum sample.
2. reverse transcription and fluorescent quantitative PCR (every person-portion 25ul system)
The preparation of a, single stage method fluorescence quantitative RT-RCR reaction solution:
B, single stage method fluorescence quantitative RT-RCR response procedures:
[1] 37℃ 10 min
[2] 50℃ 15 min
[3] 95℃ 2 min
[4] 94℃ 10 s
[5] 60℃ 45 s
[6] Go to [4] ，45 cycles
Gather FAM and JOE passage fluorescent signal in the 5th step,
[7] End。
3. detect:
The present invention is applicable to ABI StepOne, ABI 7500 types and ABI 7300 type quantitative real time PCR Instruments and carries out fluorescent quantitation detection.
4. result judgement:
Instrument PCR program is carried out result preservation and data analysis by instrument and software requirement after having moved.To get fluorescent value higher than sample noise line and negative control as detection threshold; Determine that in each sample reaction tubes, reference gene detects Ct value within 30 ~ 32 scopes, avoids the false negative of detected result simultaneously; The HIV-1 rna content C (IU/ml) of the each sample extract of the automatic combined standard curve calculation of analysis software.
embodiment 2clinical detection.
130 routine clinical samples are detected with aforesaid method, wherein human immunodeficiency virus type 1 patient 32 examples, detect positive rate 24.6%, accuracy rate 100%, and can carry out accurate quantitative analysis analysis to human immunodeficiency virus type 1 (HIV-1) RNA, be far superior to the traditional methods such as enzyme linked immunological.Detection method of the present invention and test kit high specificity, susceptibility is high, easy and simple to handle, degree of repeatability is high, can carry out fast qualitative detection by quantitative to human immunodeficiency virus type 1 (HIV-1) RNA, and the alternative traditional E LISA diagnostic method of always continuing to use.Use Shanghai Xingyao Medical Technology Development Co., Ltd.'s post method purified reagent (pellosil absorption method) to carry out nucleic acid extraction to HIV-1 positive serum sample, ensured the purity of template.Meanwhile, utilize single stage method fluorescence quantitative RT-RCR technology.After nucleic acid extraction finishes, directly nucleic acid is joined in reaction system, the synthetic and PCR of cDNA reacts at a pipe, without increasing additional step; And use reference gene to avoid false negative detected result, use UNG enzyme to avoid amplified production to pollute.The accuracy of amplification had both been guaranteed in this operation, sensitivity, shorten again the time, reduced pollution, improve the simplicity of fluorescent quantitative PCR detection method, not only can be used for HIV-1 detection by quantitative, also can be used as clinical labororatory to HIV-1 infect aided diagnosis method and the monitoring means of clinical therapeutic efficacy.
Nucleotides sequence list
SEQUENCE LISTING
<110> Shanghai Xingyao Medical Technology Development Co., Ltd.
Shanghai Fosun Pharmaceutical (Group) Co., Ltd.
Late, sharp greatly
Wu, great order
Summer, virtuous
<120> human immunodeficiency virus type 1 single stage method fluorescent quantificationally PCR detecting kit
<130> HIV-1 is quantitative
<140> cn
<141> 2012-12-21
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 137
<212> DNA
<213> Human immunodeficiency virus type 1
<400> 1
ggctgttgga aatgtggcaa ggaaggacac caaatgaaag aktgtactga gagacaggct 60
aattttttag ggaaaatctg gccttcccac aaggggaggc cagggaattt tcttcagaac 120
agaccagagc caacagc 137
<210> 2
<211> 137
<212> DNA
<213> Artificial
<220>
<223> Artificial Sequence
<400> 2
ggctgttgga aatgtggcaa ggaaggacac caaatgaaag aktgtactga gagacaggct 60
aattttttag ggaaaatctg gccttccacc aattggttcc tctctggtgt tcttcagaac 120
agaccagagc caacagc 137
<210> 3
<211> 17
<212> DNA
<213> Artificial
<220>
<223> Artificial Sequence
<400> 3
ggctgttgga aatgtgg 17
<210> 4
<211> 18
<212> DNA
<213> Artificial
<220>
<223> Artificial Sequence
<400> 4
gctgttggct ctggtctg 18
<210> 5
<211> 25
<212> DNA
<213> Artificial
<220>
<223> Artificial Sequence
<220>
<221> HIV-1 quantitation SEQ ID No.5
<222> (2)..(24)
<223> b=FAM; d=TAMRA
<400> 5
baattccccg gccttccttt gttgd 25
<210> 6
<211> 25
<212> DNA
<213> Artificial
<220>
<223> Artificial Sequence
<220>
<221> HIV-1 quantitation SEQ ID No.6
<222> (2)..(24)
<223> b=JOE; d=TAMRA
<400> 6
bccaccaatt ggttcctctc tgtgd 25
Claims (5)
1. a human immunodeficiency virus type 1 single stage method fluorescent quantificationally PCR detecting kit, it is characterized in that: the present invention is by investigating human immunodeficiency virus type 1 genom sequence, and to each genotype consistence conserved sequence of retrieval gained HIV-1, design a pair of HIV-1 Auele Specific Primer, a HIV-1 specificity fluorescent probe and an internal reference probe according to this conserved sequence, adopt real time fluorescence quantifying PCR method amplifying target genes.
2. human immunodeficiency virus type 1 single stage method fluorescent quantificationally PCR detecting kit claimed in claim 1, is characterized in that: gene order and the reference gene sequence of the amplification of human immunodeficiency virus type 1 (HIV-1) RNA specific PCR are respectively:
〈SEQ ID No.1〉
5’-GGCTGTTGGAAATGTGGCAAGGAAGGACACCAAATGAAAGAKTGTACTGAGAGACAGGCTAATTTTTTAGGGAAAATCTGGCCTTCCCACAAGGGGAGGCCAGGGAATTTTCTTCAGAACAGACCAGAGCCAACAGC-3’，
〈SEQ ID No.2〉
5’-GGCTGTTGGAAATGTGGCAAGGAAGGACACCAAATGAAAGAKTGTACTGAGAGACAGGCTAATTTTTTAGGGAAAATCTGGCCTTCCACCAATTGGTTCCTCTCTGGTGTTCTTCAGAACAGACCAGAGCCAACAGC-3’，
Human immunodeficiency virus type 1 fluorescence quantitative detection kit amplification gene sequence total length 137 bp, belong to HIV-1 gag structure gene district, are single-copy sequence; Reference gene sequence total length 137 bp, only with amplification gene in probe sequence different.
3. human immunodeficiency virus type 1 single stage method fluorescent quantificationally PCR detecting kit as claimed in claim 1, a sequence in a pair of HIV-1 specific primer sequence is identical with the sequence shown in SEQ ID No.1, the sequence complementation shown in another sequence and SEQ ID No.1; Sequence complementation shown in HIV-1 specific probe and SEQ ID No.1: wherein, described a pair of HIV-1 Auele Specific Primer, its sequence can be selected from SEQ ID No. 3 and SEQ ID No.4,
< SEQ ID No.3 > is: 5 '-GGCTGTTGGAAATGTGG-3 ',
< SEQ ID No.4 > is: 5 '-GCTGTTGGCTCTGGTCTG-3 ';
A pair of HIV-1 specific primer sequence can be also the sequence that above-mentioned sequence is extended 10～20 Nucleotide forward or backward, or is greater than more than 85% with above-mentioned sequence homology; Described HIV-1 specific probe, can be selected from the sequence of SEQ ID No.5, and < SEQ ID No.5 > is: 5 '-FAM-AATTCCCCGGCCTTCCTTTGTTG-TAMRA-3 ',
Its middle probe 5 '-end flag F AM fluorophor, the equal mark quenching group of probe 3 '-hold; HIV-1 gene fragment specific probe sequence can be also the sequence that above-mentioned sequence is extended 10～20 Nucleotide forward or backward, or is greater than more than 85% with above-mentioned sequence homology; Described internal reference probe, can be selected from the sequence of SEQ ID No.6, < SEQ ID No.6 > is: 5 '-JOE-CCACCAATTGGTTCCTCTCTGTG-TAMRA-3 ', its middle probe 5 '-end mark JOE fluorophor, the equal mark quenching group of probe 3 '-hold; Reference gene sequence can be also the sequence that above-mentioned sequence is extended 10 ~ 20 Nucleotide forward or backward, or is greater than more than 85% with above-mentioned sequence homology.
4. human immunodeficiency virus type 1 single stage method fluorescent quantificationally PCR detecting kit as claimed in claim 1, it is characterized in that: test kit comprises following composition: RT-PCR reaction solution, RT-PCR enzyme mixture, primer probe, negative control, strong positive contrast, weak positive control, calibration object No. 1 ~ No. 5: wherein, described RT-PCR reaction solution is the mixed solution of Tris-HCl (pH8.3) 20 mM, KCl 100 mM, gelatin 0.2 mg/ml, dATP, dGTP, each 0.4 mM of dCTP, dUTP, MgCl2 6 mM; Described enzyme mixture is the mixture (reversed transcriptive enzyme 3 U/ul, Taq archaeal dna polymerase 2 U/ul, UNG enzyme 1 U/ul) of reversed transcriptive enzyme, Taq archaeal dna polymerase and UNG enzyme; Described primer probe is the mixed solution (each 6.25 uM of primer, HIV-1 specific probe 2.5 uM, internal reference probe 2.5 uM) of a pair of HIV-1 Auele Specific Primer, a HIV-1 specific probe and an internal reference probe; Described negative control is the normal human serum without HIV-1 RNA; The human serum that described strong positive contrast is the slow virus that contains HIV-1 gene fragment of high density; The human serum of the slow virus that contains HIV-1 gene fragment that described weak positive control is lower concentration; The human serum of the slow virus that contains HIV-1 gene fragment that described calibration object is concentration known gradient No. 1 ~ No. 5.
5. human immunodeficiency virus type 1 single stage method fluorescent quantificationally PCR detecting kit as described in claim 1, is characterized in that: preparation and the amplification program of described RT-PCR reaction solution are as follows:
The preparation of a, single stage method fluorescence quantitative RT-RCR reaction solution:
RT-PCR reaction solution, RT-PCR enzyme mixture, primer probe are mixed according to the ratio of 12.5:0.5:2, and reaction solution volume is 25 ul;
B, single stage method quantitative fluorescent PCR response procedures:
[1] 37℃ 10 min
[2] 50℃ 15 min
[3] 95℃ 2 min
[4] 94℃ 10 s
[5] 60℃ 45 s
[6] Go to [4] ，45 cycles
Gather FAM and JOE passage fluorescent signal in the 5th step,
[7] End。
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| CN106119413A (en) * | 2016-07-01 | 2016-11-16 | 浙江省疾病预防控制中心 | A kind of HIV (human immunodeficiency virus) multiple fluorescence PCR detection reagent box and detection method |
| CN106367494A (en) * | 2016-08-30 | 2017-02-01 | 广州维伯鑫生物科技有限公司 | Method for resisting product pollution through one-step RT-PCR (reverse transcription-polymerase chain reaction) amplification of kit |
| CN106367494B (en) * | 2016-08-30 | 2019-07-09 | 广州维伯鑫生物科技有限公司 | A kind of method of the anti-product pollution of RT-PCR one step amplification kit |
| CN107099620A (en) * | 2017-05-09 | 2017-08-29 | 广州和实生物技术有限公司 | A kind of human immunodeficiency virus type 1 Constant Temperature Detection kit |
| CN107385116A (en) * | 2017-09-13 | 2017-11-24 | 北京福安华生物科技有限公司 | A kind of method and its special complete reagent for detecting 1 type human immunodeficiency virus |
| CN109295261A (en) * | 2018-11-21 | 2019-02-01 | 温州医科大学附属第医院 | A kind of primer, probe and detection method for detecting lentivirus RCL |
| CN111074004A (en) * | 2020-01-17 | 2020-04-28 | 复旦大学 | Human immunodeficiency virus type 1 drug resistance genotype detection method and kit |
| CN111074004B (en) * | 2020-01-17 | 2023-02-10 | 复旦大学 | Human immunodeficiency virus type 1 drug-resistant genotype detection method and kit |
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Application publication date: 20140806 |