CN106636458A - RT-LAMP (reverse transcription and loop-mediated isothermal amplification) detection primer group, RT-LAMP detection kit and RT-LAMP detection method for simian immunodeficiency virus - Google Patents
RT-LAMP (reverse transcription and loop-mediated isothermal amplification) detection primer group, RT-LAMP detection kit and RT-LAMP detection method for simian immunodeficiency virus Download PDFInfo
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Abstract
The invention discloses an RT-LAMP (reverse transcription and loop-mediated isothermal amplification) detection primer group, an RT-LAMP detection kit and an RT-LAMP detection method for a simian immunodeficiency virus. The detection primer group comprises a pair of outer primers, a pair of loop primers and a pair of inner primers. The detection kit comprises the primer group, RT-LAMP reaction liquid, Bst DNA polymerase, reverse transcriptase as well as negative control and positive control. The detection method comprises the following steps: extracting to-be-detected virus RNA; conducting reverse transcription on the RNA under the action of revertase; then, amplifying a sample template by virtue of six specific primers and one Bst DNA polymerase having strand displacement activity at 63-65 DEG C; and judging whether a to-be-detected sample contains the simian immunodeficiency virus (SIV) RNA by observing whether a reaction tube solution becomes muddy or not with naked eyes or performing electrophoresis to analyze whether a ladder exists or not. The detection primer group, the detection kit and the detection method provided by the invention have the advantages of being highly specific, high in sensitivity, rapid and efficient, simple and convenient to operate, easy in result reading and the like, and are suitable for popularization and application in grassroots animal epidemic disease monitoring units and experimental monkey breeding enterprises in remote areas.
Description
Technical field
The invention belongs to technical field of molecular biology, is related to the detection method of animal used as test correlation cause of disease, and in particular to
A kind of RT-LAMP of SIV (SIV) (Reverse Transcription and Loop-mediated
Isothermal Amplification, reverse transcription loop-mediated isothermal amplification) detection primer group, detection kit and its inspection
Survey method.
Background technology
First SIV is found that within 1985, simian acquired immunodeficiency syndrome virus is also called, subsequently several Strain are from African monkey and Asia
Separate in the rhesus monkeys of continent.SIVcpz, SIVsm, SIVmnd, SIVsyk and SIVagm five is broadly divided on SIV science of heredity
Big offspring, SIV has Host Dependence evolution feature, so that it is not pathogenic in natural host, such as SIVsm infection is pitch-black white
Face monkey, SIVagm infection cercopithecus aethiopses, SIVcpz infection chimpanzees etc. all without making the latter fall ill, but when across the species propagation of SIV
To non-natural host, such as Asia rhesus macaque, it will usually make it fall ill.The course advancement of SIVmac infection rhesus macaquies and clinical manifestation
It is closely similar with people's AIDS-like disease.In incubation period, PBC CD4+T is persistently reduced, under CD4/CD8 lymphocyte ratios
Drop；To AIDS disease stage, may occur in which chronic diarrhea and become thin, Body weight loss 60% when dead, simultaneously and concurrently general pouring
Bar exhaustion；The whole end stage can also occur various opportunistic infections.
SIV has been identified as being the pathogen for causing SAIDS, therefore, in the previous of simian acquired immunodeficiency syndrome Animal Model
Surely the interference of this pathogen is excluded.The diagnosis of SIV infection also wants Binding experiment room diagnosis side except checking clinical symptoms
Method, conventional laboratory diagnostic method have PCR methods, EUSA (ELISA) method, indirect immunofluorescence (IFA) method,
Western blotting (WB) method, isolation of virus etc..PCR methods are quick, sensitive, but its amplified production needs to be verified again.Disease
Poison is separated needs to carry out cell culture, cycle length, testing equipment is had high demands, therefore, the method is not suitable for general detection.Mesh
Before, the detection to SIV commonly uses ELISA method and IFA methods as primary dcreening operation, it is necessary to could be finally true with WB detection specific antibodies
Examine.ELISA method and the false positive rate height and WB methods operation sequence of the detection of IFA methods are complicated, and its application is limited to a certain extent
Scope.Therefore, whether for ensureing quality or keeper, the personal safety of scientific research personnel of whole monkey group, having must
Set up a kind of convenient, fast, sensitive, high specificity detection method.
The content of the invention
It is an object of the present invention to provide a kind of RT-LAMP detection primer groups of SIV.
Further object is that providing a kind of RT-LAMP detection reagent box of SIV.
Further object is that providing the RT-LAMP detection methods of SIV.
The technical solution used in the present invention is：
A kind of RT-LAMP detection primer groups of SIV, including a pair of outer primers, a pair of inner primers and a pair
Ring primer.Its nucleotide sequence difference is as follows：
Outer primer：
SIV-F3：5’-GCATTCACGCAGAAGAGA-3’(SEQ ID NO:1)；
SIV-B3：5’-CTGGCACTACTTCTGCTC-3’(SEQ ID NO:2)；
Inner primer：
SIV-FIP：5’-TCTGCTGTTCCTGTTTCCACCGAAACACACTGAGGAAGCA-3’(SEQ ID NO:3)；
SIV-BIP：5’-ACCAACAGCACCATCTAGCGATGGCAGGTGGACATAGT-3’(SEQ ID NO:4)；
Ring primer：
SIV-LF：5’-ACTAGGTGTCTCTGCACTATCT-3’(SEQ ID NO:5)
SIV-LB：5’-ACCCAGTACAACAAATAGGTGG-3’(SEQ ID NO:6).
A kind of RT-LAMP detection reagent box of SIV, the detection kit includes detection described above
Primer sets.
Preferably, detection kit includes primer sets described above, archaeal dna polymerase, reverse transcriptase, RT-LAMP reactions
Liquid, positive control and negative control.
Preferably, outer primer, ring primer, the mol ratio of inner primer are 1：4：8.
Preferably, archaeal dna polymerase is Bst archaeal dna polymerases.
Preferably, reverse transcriptase is AMV reverse transcriptases.
Preferably, RT-LAMP reactant liquors contain 10mM dNTP, 10 × ThermoPol reaction buffers, 150mM MgSO4
The aqueous solution.
Preferably, positive control is the DNA containing genes of interest fragment, and negative control is DEPC water.
A kind of RT-LAMP detection methods of SIV, comprise the steps：
1) measuring samples RNA is extracted；
2) reverse transcription loop-mediated isothermal amplification：25 μ l reaction systems are prepared, containing 0.2 μM of SIV-F3, SIV-B3
0.2 μM, 1.6 μM of SIV-FIP, 1.6 μM of SIV-BIP, 0.8 μM of SIV-LF, 0.8 μM of SIV-LB, RT-LAMP reactant liquors 12.5
μ l, archaeal dna polymerase 8U, reverse transcriptase 2.5U, 1～100ng of DNA to be checked, with DEPC water polishing to 25 μ l；Then in 63～65
DEG C reaction 45～60min；
3) interpretation of result：Observe by the naked eye whether solution in reaction tube is become cloudy or analyzed by agarose gel electrophoresis
Whether scalariform band is occurred.It is negative otherwise as the scalariform band as positive occur in solution turned cloudy or electrophoresis in reaction tube.
The invention has the beneficial effects as follows：
The present invention have high specific, high sensitivity, rapidly and efficiently, the beneficial effect such as easy to operate, result is readable：
1) high specific：
Essential core egg of the present invention according to SIV (SIV) (GenBank accession number is KC522234.1)
The partial sequence of white gene (gag) devises 6 specific primers, using above-mentioned 6 primers, expands 8 regions of target sequence,
Any region mismatches with primer and can not carry out nucleic acid amplification in 8 regions, therefore its specificity is high, and highly stable, shape
It is into primer dimer probability low, it is ensured that reaction is smoothed out；
2) high sensitivity：The lowest detection limit can reach DNAs of the 1fg/ μ l containing SIV target fragments；
3) rapidly and efficiently：Whole amplification only can be completed with 45～60min, and amplification yield is up to 109～1010Individual copy；
4) it is easy to operate：Complicated instrument is not needed, it is not necessary to special reagent, it is not necessary to carry out reverse transcription etc. in advance loaded down with trivial details
Step, it is only necessary to which thermostat water bath can react, and condition is gentleer；
5) result readability：Observe by the naked eye whether solution in reaction tube is become cloudy or analyzed by agarose gel electrophoresis
Scalariform band whether occur carries out interpretation to testing result, is adapted to the Site Detection of layman.
Description of the drawings
Fig. 1 is the testing result figure (1 of the specificity of embodiment 3：DEPC water is compareed；2：Negative sample is compareed；3：Monkey D types are inverse
Retroviral SRV1RNA；4：Monkey D type retrovirus SRV2RNA；5：Monkey D type retrovirus SRV3RNA；6：Monkey D types are reversed
Record virus SRV4RNA；7：The thermophilic T lymphotropic virus type Is STLV-I RNA of monkey；8：B virus of monkey BV DNA；9：Simian immunodeficiency defect disease
Malicious SIV RNA；10：SIV SIV DNAs；M-Marker DL2000)；
Fig. 2 is the testing result figure (1 of the sensitivity of embodiment 4：100pg/ μ l DNAs (SIV)；2：10pg/ μ l plasmids
DNA(SIV)；3：1pg/ μ l DNAs (SIV)；4：100fg/ μ l DNAs (SIV)；5：10fg/ μ l DNAs (SIV)；6：
1fg/ μ l DNAs (SIV)；7：100ag/ μ l DNAs (SIV)；8：100ag/ μ l DNAs (SIV)；9：DEPC water pair
According to).
Specific embodiment
With reference to embodiments the present invention is further illustrated, but is not limited thereto.
The foundation of the SIV RT-LAMP detection reagent box of embodiment 1
RT-LAMP (the Reverse Transcription and Loop-mediated of SIV
Isothermal Amplification, reverse transcription loop-mediated isothermal amplification) detection kit, including RT-LAMP primers
Group, RT-LAMP reactant liquors, BstDNA polymerases, reverse transcriptase, positive control and negative control.
(1) RT-LAMP design of primers：Essential core protein gene sequence (gag) with SIV (SIV) is
Target gene, using Software for Design SIV specificity RT-LAMP primers.Primer sequence is shown in Table 1.
The RT-LAMP primer sequence tables of the SIV of table 1. specificity
(2) RT-LAMP reactant liquors：10mM dNTP, 10 × ThermoPol reaction buffers, 150mM MgSO4The aqueous solution.
(3) Bst archaeal dna polymerases and AMV reverse transcriptases.
(4) positive control is the DNA that core protein gene (gag) fragment is wanted containing SIV (SIV),
Its preparation method is：To separate the SIV RNA for identifying as template, using outer primer (the SEQ ID NO in table 1:
1 and SEQ ID NO:2) reverse transcription PCR is carried out to SIVRNA, gained genetic fragment length is 258bp, sequence such as SEQ ID NO:
Shown in 7, the amplified fragments are reclaimed, be connected in carrier T using conventional method and build plasmid, as positive control.
GCATTCACGCAGAAGAGAAAGTGAAACACACTGAGGAAGCAAAACAGATAGTGCAGAGACACCTAGTGG
TGGAAACAGGAACAGCAGAAACTATGCCAAAAACAAGTAGACCAACAGCACCATCTAGCGGCAGAGGAGGAAATTAC
CCAGTACAACAAATAGGTGGTAACTATGTCCACCTGCCATTAAGCCCGAGAACATTAAATGCCTGGGTAAAATTGAT
AGAGGAAAAGAAATTTGGAGCAGAAGTAGTGCCAG(SEQ ID NO:7)
(5) negative control is DEPC water.
The RT-LAMP detection methods of the SIV of embodiment 2
With above-mentioned kit SIV (SIV) is detected by the following method：
(1) extraction of measuring samples RNA：Measuring samples are extracted using the viral RNA extracts kit of Mei Ji biotech firms
RNA；
(2) reverse transcription loop-mediated isothermal amplification：
25 μ l reaction systems contain：1.6 μM of 0.2 μM of 0.2 μM of SIV-F3, SIV-B3, SIV-FIP, SIV-BIP 1.6
0.8 μM of 0.8 μM of Μ m, SIV-LF, SIV-LB, the μ l of RT-LAMP reactant liquors 12.5, BstDNA polymerase 8U, AMV reverse transcriptases
2.5U, RNA1～100ng to be checked, with DEPC water polishing to 25 μ l；Positive control and negative control are set；By the PCR for preparing
Pipe is centrifuged after mixing, and in 65 DEG C 60min is reacted.
(3) result judges：Observe by the naked eye whether solution is become cloudy or analysed whether out by agarose gel electrophoresis
Existing scalariform band result of determination；It is cloudy otherwise as the scalariform band as positive occur in solution turned cloudy or electrophoresis in reaction tube
Property.
The specificity experiments of embodiment 3
With the method for embodiment 2 respectively to SIV (SIV) RNA, monkey D type retrovirus (SRV) RNA,
Monkey lymphocyte taxis virus (STLV) RNA, b virus of monkey (BV) DNA are detected.
Qualification result shows：With SIV (SIV) RT-LAMP primer sets as primer, SIVRNA and DNA
Solution substantially becomes cloudy in reaction tube, occurs obvious scalariform band after electrophoresis, and the reaction tube such as negative control and SRV, STLV, BV is molten
There is not muddiness in liquid, while scalariform band does not occur yet in electrophoresis, shows that detection primer of the present invention and method have good spy
The opposite sex.(see Fig. 1).
The sensitivity experiment of embodiment 4
The SIV for having built (SIV) DNA is made into 10 times of gradient dilutions, with the operation side of embodiment 2
Method is detected respectively to SIV (SIV) DNA after dilution.
Qualification result shows：With SIVRT-LAMP primer sets as primer, SIV DNAs are diluted by 10 times of gradients, knot
Fruit shows that 2 reaction tube solution of SIV DNAs of the concentration of 100pg/ μ l to 1fg/ μ l become cloudy, and occurs after electrophoresis obvious
There is not muddiness in the reaction tube solution such as scalariform band, 100ag/ μ l, the concentration of 10ag/ μ l and negative water control, while electrophoresis
There is not scalariform band.(see Fig. 2).
The comparison of the different detection primers of embodiment 5 and method Detection results
Detected using difference LAMP with the method for embodiment 2 and detected according to the step in embodiment 4, at the same using and
Fluorescence quantification PCR primer is detected that the Detection results of the different detection primer groups of comparison and different detection methods, other detections are drawn
The sequence and Detection results of thing group such as table 2 and table 3.
Other detection primer groups of table 2 and fluorescence quantification PCR primer
The different detection primers of table 3 and method Detection results
| Detection primer A of the present invention | Detection primer group B | Detection primer C | Fluorescence quantification PCR primer | |
| Detection sensitivity | 1fg/ul(+/+) | 10fg/ul(+/-) | 1fg/ul(+/-) | 10fg/ul(+/+) |
| Negative control testing result | Negative (-/-) | Negative (-/-) | Positive (+/-) | Negative (-/-) |
Remarks：(+/+) represent 2 repetitions and be the positive, it is once feminine gender that (+/-) represents 2 repetitions, is once sun
Property, (-/-) represent and repeat to be feminine gender twice
Testing result shows：The primer sets of the present invention have compared with other primer sets and fluorescent quantitative PCR detection method
The high feature of sensitivity, while also non-false positive phenomenon occurs.
Above example shows, the method for the present invention have high specific, high sensitivity, rapidly and efficiently, easy to operate, knot
The advantages of fruit readability, it is suitable to carry out popularization and application in each basic animal Disease monitor unit and experimental monkey groups breeding enterprise.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any Spirit Essences without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
<110>Experimental Animals Supervising Station, Guangdong Prov.
<120>The RT-LAMP detection primer groups of SIV, kit and its detection method
<130>
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<170> PatentIn version 3.5
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Claims (8)
1. RT-LAMP detection primer groups of a kind of SIV, including a pair of outer primers, a pair of inner primers and a pair of rings
Primer, its nucleotide sequence difference is as follows：
Outer primer：
SIV-F3：5’- GCATTCACGCAGAAGAGA- 3’（SEQ ID NO:1）；
SIV-B3：5’- CTGGCACTACTTCTGCTC- 3’（SEQ ID NO:2）；
Inner primer：
SIV-FIP：5’- TCTGCTGTTCCTGTTTCCACCGAAACACACTGAGGAAGCA- 3’（SEQ ID NO:3）；
SIV-BIP：5’- ACCAACAGCACCATCTAGCGATGGCAGGTGGACATAGT- 3’（SEQ ID NO:4）；
Ring primer：
SIV-LF：5’- ACTAGGTGTCTCTGCACTATCT- 3’（SEQ ID NO:5）
SIV-LB：5’- ACCCAGTACAACAAATAGGTGG- 3’（SEQ ID NO:6）.
2. the RT-LAMP detection reagent box of a kind of SIV, it is characterised in that：The detection kit will including right
Seek the detection primer group described in 1.
3. the RT-LAMP detection reagent box of SIV according to claim 2, it is characterised in that：The detection
Kit also includes archaeal dna polymerase, reverse transcriptase, RT-LAMP reactant liquors, positive control and negative control.
4. the RT-LAMP detection reagent box of SIV according to claim 3, it is characterised in that：Outer primer,
Ring primer, the mol ratio of inner primer are 1：4：8.
5. the RT-LAMP detection reagent box of SIV according to claim 3, it is characterised in that：The DNA
Polymerase is Bst archaeal dna polymerases, and the reverse transcriptase is AMV reverse transcriptases.
6. the RT-LAMP detection reagent box of SIV according to claim 3, it is characterised in that：The RT-
LAMP reactant liquors contain 10mM dNTP, 10 × ThermoPol reaction buffers, 150mM MgSO4The aqueous solution.
7. the RT-LAMP detection reagent box of SIV according to claim 3, it is characterised in that：The sun
Property control be the DNA containing genes of interest fragment, negative control be DEPC water.
8. a kind of RT-LAMP detection methods of SIV, comprise the steps：
1）Extract measuring samples RNA；
2）Reverse transcription loop-mediated isothermal amplification：25 μ l reaction systems are prepared, containing 0.2 μM of SIV-F3, the μ of SIV-B3 0.2
0.8 μM of 0.8 μM of 1.6 μM of 1.6 μM of M, SIV-FIP, SIV-BIP, SIV-LF, SIV-LB, the μ l of RT-LAMP reactant liquors 12.5,
Archaeal dna polymerase 8U, reverse transcriptase 2.5U, 1 ~ 100ng of RNA to be checked, with DEPC water polishing to 25 μ l；Then it is anti-in 63～65 DEG C
Answer 45～60min；
3）Interpretation of result：Observe by the naked eye whether solution in reaction tube is become cloudy or analysed whether by agarose gel electrophoresis
There is scalariform band result of determination；It is cloudy otherwise as the scalariform band as positive occur in solution turned cloudy or electrophoresis in reaction tube
Property；
The detection kit used detection kit described in any one of claim 2 ~ 7 claim of above-mentioned detection method.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107475457A (en) * | 2017-09-29 | 2017-12-15 | 福建省农业科学院畜牧兽医研究所 | Duck batai virus ring mediated isothermal amplification detection primer group and kit |
| CN111304370A (en) * | 2020-04-22 | 2020-06-19 | 广东省实验动物监测所 | Primer and probe for rapidly detecting Simian Immunodeficiency Virus (SIV), kit and using method thereof |
| CN111850162A (en) * | 2019-04-25 | 2020-10-30 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | RPA kit, primer, probe and method for detecting simian immunodeficiency virus |
-
2016
- 2016-09-21 CN CN201610839424.6A patent/CN106636458B/en active Active
Non-Patent Citations (9)
| Title |
|---|
| 丛喆等: "PCR 技术在猴免疫缺陷病毒(SIV)感染模型中的应用", 《中国实验动物学报》 * |
| 丛喆等: "SYBR Green I实时荧光定量RT—PCR测定猴免疫", 《中国实验动物学报》 * |
| 何伏秋等: "PCR在猴艾滋病模型中检测SIV基因的应用", 《中国实验动物学杂志》 * |
| 何伏秋等: "巢式PCR检测猴免疫缺陷病毒基因", 《中国人兽共患病杂志》 * |
| 曾亚岚: "基于逆转录环介导等温扩增技术的高灵敏度HIV-1 RNA检测", 《中国优秀硕士学位论文全文数据库》 * |
| 朱丽妍等: "实时荧光定量PCR 技术快速评价恒河猴", 《中国微生物学和免疫学杂志》 * |
| 李晓燕等: "套式PCR 在检测SHIV 动物模型中的应用", 《动物医学进展》 * |
| 王静等: "猴免疫缺陷病毒( SIV) 实时荧光定量", 《中国比较医学杂志》 * |
| 谭贵良等: "《现代分子生物学及组学技术在食品安全监测中的应用》", 30 June 2014 * |
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| CN107475457A (en) * | 2017-09-29 | 2017-12-15 | 福建省农业科学院畜牧兽医研究所 | Duck batai virus ring mediated isothermal amplification detection primer group and kit |
| CN107475457B (en) * | 2017-09-29 | 2021-04-20 | 福建省农业科学院畜牧兽医研究所 | Duck Batai virus loop-mediated isothermal amplification detection primer group and kit |
| CN111850162A (en) * | 2019-04-25 | 2020-10-30 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | RPA kit, primer, probe and method for detecting simian immunodeficiency virus |
| CN111304370A (en) * | 2020-04-22 | 2020-06-19 | 广东省实验动物监测所 | Primer and probe for rapidly detecting Simian Immunodeficiency Virus (SIV), kit and using method thereof |
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