CN103173577A - Method for detecting porcine sapeloviruses based on reverse transcription loop-mediated isothermal amplification technology - Google Patents
Method for detecting porcine sapeloviruses based on reverse transcription loop-mediated isothermal amplification technology Download PDFInfo
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Abstract
The invention relates to the application field of biotechnology, and provides a method for detecting porcine sapeloviruses based on a reverse transcription loop-mediated isothermal amplification technology. The method comprises the following steps: screening a conserved sequence block 5' UTR of the porcine sapeloviruses and designing four primers according to the conserved sequence block; preparing a sample to be detected; preparing a reaction liquid and carrying out a reverse transcription loop-mediated isothermal amplification reaction (RT-LAMP); and identifying a reaction result according to the real-time detection of an amplification curve output by a turbidity meter, the color changing conditions of the calcein of a fluorescent dye or the direct observation of the turbidity change of the reaction liquid. For relevant detection departments or companies, the method which is low in price, rapid in speed, high in efficiency and high in specificity is provided.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method that detects pig Sa Peiluo virus.
Background technology
Pig Sa Peiluo virus (Porcine Sapelovirus) is the virus of a boar of microRNA Viraceae Sa Peiluo Tobamovirus.It can cause the syndromes such as nervous disorders, reproductive failure, respiratory insufficiency, pneumonia and diarrhoea of pig.This virus can with the virus generation polyinfection of other pigs, be brought very huge economic threat to pig industry.Therefore, to this kind Detecting, be the important measures of mutually infecting between early prevention, early control and releasing swinery.This experiment for be Chinese the first strain Sa Peiluo virus (PSV-Csh) (gene numbering: HQ875059), this viral genome total length is 7502 base pairs, only has an open reading frame, the polyprotein precursor of encoding, holding at genomic 5' end and 3' is genomic non-translational region 5'UTR and 3'UTR.
At present, have much for these Detecting means, as separation, enzyme-linked immunosorbent assay, the various PCR means of virus.Virus is separated and enzyme-linked immunosorbent assay is all most basic detection method, but is detection means the most consuming time also, for the diagnosis in advance of paroxysmal epidemic disease, can not accomplish detection as early as possible, may delay like this control of epidemic disease.Molecular biological detection method more and more comes into one's own, and has become a kind of detection means of routine as RT-PCR, but these methods need high-accuracy instrument and masterful technique.For those feeler mechanisies from far-off regions and animal clinic, these methods can not be brought into play their effect.The loop-mediated isothermal amplification technique for detection of pig Sa Peiluo virus of the present invention's report, it is a kind of simple, quick, highly sensitive, high special detection method, it is worth noting most that this detection method does not need expensive instrument, only need a water-bath, can complete whole detection.
Loop-mediated isothermal amplification technique (LAMP) is to have a high special and high efficiency nucleic acid amplification technologies by what the people such as Japanese scholars Notoni research and development a kind of completed under constant temperature.The method is for 4 primers of 6 special zone design of goal gene, and under the effect of strand displacement archaeal dna polymerase-Bst archaeal dna polymerase, constant temperature keeps dozens of minutes, can complete amplification.Its principle is the F2 sequence initial action of inner primer FIP, outer primer F3 carries out strand replacement reaction, replace the chain that makes new advances, then inner primer BIP opens the stem ring with new chain combination, B3 finally forms the DNA structure of a similar dumbbell shape at BIP outside formation complementary strand, and this structure is as the initial structure of amplified reaction, begun circulation and extended, having guaranteed constantly carrying out of reaction.
Reverse transcription loop-mediated isothermal amplification technique (RT-LAMP) is to add archaeal dna polymerase and AMV reversed transcriptive enzyme in reaction tubes, and single stage method is completed the amplification of RNA.This has not only saved the RNA reverse transcription is the time and experiment consumptive material, reagent of DNA, has also improved the amplification efficiency of RNA.RT-LAMP has the incomparable advantage of traditional detection method in viral context of detection: 1. quick: completing of whole experiment only needs 2h, only need 1.5h for staff expertly, it does not want special instrument simultaneously, as long as can complete this experiment in the equipment of a constant temperature, need not complicated temperature control device; 2. amplification efficiency is high: after several copy reactions finished, product can reach 10
9Copy; 3. easy to detect quick: because reaction product is a lot, can form the white opacity thing in reaction tubes, can judge accordingly whether reaction occurs, also can utilize the fluorescence dye fluorexon to carry out stopped pipe and detect, so not only avoided the environmental pollution that nucleic acid causes but also saved the electrophoresis required time; 4. to have saved the RNA reverse transcription be the process of DNA to RT-LAMP, avoided the secondary pollution of nucleic acid and the pollution of RNA enzyme; 5. specificity is high: 4 primers need the template combination, just dumbbell shape can occur, circulation and a large amount of amplifications could occur.
Summary of the invention
The purpose of this invention is to provide a kind of method that detects efficiently, fast, simply pig Sa Peiluo virus, can be applicable to zooprophylazis at different levels control center, animals at different levels clinic and enterprise.
For achieving the above object, the invention provides a kind of method that detects pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique.
The method that detects pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique of the present invention comprises following steps:
(1) 4 primers of design
Described primer comprises outer primer F3, outer primer B3, inner primer FIP and inner primer BIP, and described primer sequence is as shown in SEQ ID NO:1-4;
(2) preparation testing sample
Adopt commercial RNA the extraction test kit of DNA virus nucleic acid extract the RNA of sample;
(3) preparation reaction solution
Adopt commercial RT-LAMP test kit, add reaction buffer, the mixed liquid of enzyme, 4 primers, fluorescence dye fluorexon, described RNA and water according to specification sheets in reaction tubes, cumulative volume is 25 μ L;
(4) react
The described reaction tubes for preparing is placed in conversion unit, 60～65 ℃ of isothermal reaction 1h, 83 ℃ of 5min finish reaction;
(5) result detects.
Concrete operations are as follows:
(1) 4 primers of design
Filter out the relative conserved sequence district 5'UTR of Sa Peiluo virus by on-line search engine BLAST and sequence analysis software DNAstar.According to this section sequence, utilize Primer Explorer VE4.0 to design 4 primers, comprising outer primer F3, outer primer B3, inner primer FIP and inner primer BIP, primer sequence is as shown in SEQ ID NO:1-4.
(2) preparation testing sample
Adopt commercial RNA the extraction test kit of DNA virus nucleic acid extract the RNA of sample.
(3) preparation reaction solution
Adopt commercial RT-LAMP test kit, add 12.5 μ L2 * reaction buffers (RM) according to specification sheets in reaction tubes; 1.0 μ L enzyme mixation (EM); Final concentration is outer primer F3 and the B3 of 0.20 μ M; Final concentration is: inner primer FIP and the BIP of 1.6 μ M; 1.0 μ L fluorescence dye fluorexon; The 5 described RNA of μ L; Supply deionized water to cumulative volume 25 μ L.
(4) react
The reaction tubes that configures is placed in Real-Time Monitoring turbidimeter or water-bath, 60～65 ℃ of isothermal reaction 1h, 83 ℃ of 5min finish reaction.
(5) result judgement.
Reaction tubes is placed in the Real-Time Monitoring turbidimeter, according to the response situation in the amplification curve judgement reaction solution of Real-Time Monitoring turbidimeter output.
Reaction tubes is placed in water-bath, and owing to having added the fluorescence dye fluorexon in reaction solution, whether so can occur according to the variation judgement reaction of reaction solution color before and after reaction: a) example reaction pipe liquid is yellow-green colour, and this pattern detection result is positive; B) example reaction pipe liquid is orange red, and this pattern detection result is negative.
In addition, also can adopt the opacity observation, if react, the color of reaction solution can be by the transparent oyster white that becomes, but the tolerance range of the method is not high.
According to the method that detects pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique of the present invention, preferably, temperature of reaction is 63 ℃.
The method that detects the detection of pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique of the present invention, easy to detect quick, only just can reach testing goal through single step reaction; Detection sensitivity is high, and amplification template only needs 2.97 * 10
2Copy/microlitre; Identify easyly, can judge according to the variable color situation of Real-Time Monitoring turbidimeter or fluorescence dye fluorexon whether reaction occurs.
Be described further below with reference to the technique effect of accompanying drawing to design of the present invention, concrete structure and generation, to understand fully purpose of the present invention, feature and effect.
Description of drawings
Fig. 1 is the monitoring result of the Real-Time Monitoring turbidimeter in specific embodiment 1.1：63℃；2：65℃；3:61℃。
Fig. 2 is the monitoring result of the Real-Time Monitoring turbidimeter in specific embodiment 2.1: pig Sa Peiluo virus.
Fig. 3 is the monitoring result of the Real-Time Monitoring turbidimeter in specific embodiment 3.Be followed successively by from left to right: 2.97 * 10
8Copy, 2.97 * 10
9Copy, 2.97 * 10
7Copy, 2.97 * 10
6Copy, 2.97 * 10
5Copy, 2.97 * 10
4Copy, 2.97 * 10
3Copy, 2.97 * 10
2Copy, 2.97 * 10
1Copy (sea line), negative control (sea line).
In the present invention, agents useful for same, instrument all can be from commercially available acquisitions.Virus strain used is preserved by this laboratory.
Embodiment
The present invention will be further described by embodiment below in conjunction with accompanying drawing and subordinate list.
Embodiment 1:RT-LAMP detects pig Sa Peiluo virus optimal reaction temperature
Obtain the full genome (accession number is HQ875059) of Chinese the first strain pig Sa Peiluo virus on GeneBank, according to LAMP design of primers principle, filter out this genomic conserved sequence district 5'UTR, for six distinguished sequence districts of this sequence designing 4 primers and synthetic.The acquisition of the RNA template of Sa Peiluo virus is the RNA DNA extraction test kit that utilizes day root, extracts to specifications viral pure liquid.The process specifications configuration reaction system of the RT-LAMP reaction kit of buying according to Beijing genealogical records exchanged by those who have sworn brotherhood bio tech ltd, cumulative volume is 25 μ L.The final concentration of outer primer F3 and B3 is 0.2 μ M, and the final concentration of inner primer FIP and BIP is 1.6 μ M.
According to as above reaction system configuration reaction solution, design three temperature of reaction: 61 ℃, 63 ℃ and 65 ℃.Parallel two group reaction pipes are positioned over respectively in Real-Time Monitoring turbidimeter and water-bath, and the reaction times is 60min.The detected result of Real-Time Monitoring turbidimeter as shown in Figure 1, amplification curve is obviously indicated 63 ℃ for optimal reaction temperature.The the second group reaction pipe that is placed in water-bath shows that other reaction tubes of fluorescence intensity ratio of the reaction solution that is placed in 63 ℃ is strong, although the opacity visual inspection of reaction difference is not obvious, can illustrate that also 63 ℃ are optimal reaction temperature.
Embodiment 2:RT-LAMP detects the specificity of pig Sa Peiluo virus
choose the pig that does not contain Sa Peiluo virus prompt Shen virus (PTV Harbin veterinary institute be so kind as to give), pig enterovirus 9(PEV9 the laboratory preserve), swine foot-and-mouth disease virus (FMDV buy in Chinese Zhongmu Industry Co.,Ltd), pig enterovirus 9(PEV9 the laboratory preserve), encephalomyocarditis virus (EMCV Lanzhou veterinary institute be so kind as to give), transmissible gastroenteritis of swine (TGEV laboratory preserve), porcine epizootic diarrhea (PEDV preserves in the laboratory), pig blue-ear disease poison (PRRSV laboratory preserve) nucleic acid, according to reaction system configuration reaction solution as above, two groups of parallel reaction tubess, be placed on respectively in Real-Time Monitoring turbidimeter and water-bath, temperature arranges 63 ℃, preserve 70min, 80 ℃ of 5min finish reaction.The detected result of Real-Time Monitoring turbidimeter as shown in Figure 2, amplification curve obviously indication only has the sample of pig Sa Peiluo virus that amplification has occured.The the second group reaction pipe that is placed in water-bath shows that the color of the reaction solution that only contains pig Sa Peiluo virus is by the orange red yellow-green colour that becomes.The specificity that the primer that the present invention designs is described is very high.
Embodiment 3:RT-LAMP detects the sensitivity of pig Sa Peiluo virus
Use the water without RNase to dilute with 10 times of gradients the RNA of the pig Sa Peiluo virus extracted, the concentration of RNA mother liquor is: 2.97 * 10
9Copy/microlitre.Dilution is 2.97 * 10 successively
8Copy/microlitre, 2.97 * 10
7Copy/microlitre, 2.97 * 10
6Copy/microlitre, 2.97 * 10
5Copy/microlitre, 2.97 * 10
4Copy/microlitre, 2.97 * 10
3Copy, 2.97 * 10
2Copy/microlitre and 2.97 * 10
1Copy/microlitre.According to reaction system configuration reaction solution as above, two groups of parallel reaction tubess are positioned over respectively in Real-Time Monitoring turbidimeter and water-bath, and temperature setting is set to 63 ℃, preserves 60min, and 80 ℃ of 5min finish reaction.The interpretation of result method is the same, and experimental result shows that finally the minimum detectability degree of the RT-LAMP of pig Sa Peiluo virus is 2.97 * 10
2Copy/microlitre, but the best reaction density of this reaction is 2.97 * 10
8Copy/microlitre is not that template concentrations is more high better.
Embodiment 4:RT-LAMP detects the clinical verification of pig Sa Peiluo virus
Choose the excrement sample of 28 parts of healthy boars (Hunan), extract RNA.According to reaction system configuration reaction solution as above, two groups are carried out parallel reactor.Be positioned over respectively in Real-Time Monitoring turbidimeter and water-bath, temperature is made as 63 ℃, preserves 60min, and 80 ℃ of 5min finish reaction.The interpretation of result method is the same, and experimental result shows to only have 3 duplicate samples amplification to occur, and positive rate is 10.7%.Identical sample is verified with conventional RT-PCR, and the RT-PCR result shows 2 duplicate samples and amplification occurs, and positive rate is 7.1%.Illustrate that RT-LAMP is sharper than the sensitivity of RT-PCR method, the virus of low levels in sample can be detected.
More than describe preferred embodiment of the present invention in detail.The ordinary skill that should be appreciated that this area need not creative work and just can design according to the present invention make many modifications and variations.Therefore, all technician in the art all should be in the determined protection domain by claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.
Claims (10)
1. method that detects pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique comprises following steps:
(1) 4 primers of design
Described primer comprises outer primer F3, outer primer B3, inner primer FIP and inner primer BIP, and described primer sequence is as shown in SEQ ID NO:1-4;
(2) preparation testing sample
Adopt commercial RNA the extraction test kit of DNA virus nucleic acid extract the RNA of sample;
(3) preparation reaction solution
Adopt commercial RT-LAMP test kit, add mixing 1 liquid of reaction buffer, enzyme, described 4 primers, fluorescence dye fluorexon, described RNA and water according to specification sheets in reaction tubes, cumulative volume is 25 μ L;
(4) react
The described reaction tubes for preparing is placed in conversion unit, 60～65 ℃ of isothermal reaction 1h, 83 ℃ of 5min finish reaction;
(5) result detects.
2. detect as described in claim 1 the method for pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique, it is characterized in that: in described step (1), described 4 primers are filter out the relative conserved sequence district 5'UTR of Sa Peiluo virus and utilize Primer Explorer VE4.0 to design according to this section sequence by on-line search engine BLAST and sequence analysis software DNAstar.
3. detect as described in claim 1 the method for pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique, it is characterized in that: described outer primer is 0.20 μ M at the final concentration of described reaction solution.
4. detect as described in claim 3 the method for pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique, it is characterized in that: described inner primer is 1.6 μ M at the final concentration of described reaction solution.
5. detect as described in claim 1 the method for pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique, it is characterized in that: in described step (4), described conversion unit is the Real-Time Monitoring turbidimeter.
6. detect as described in claim 5 the method for pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique, it is characterized in that: in described step (5), the method that described result detects is according to the response situation in the described reaction solution of amplification curve judgement of described Real-Time Monitoring turbidimeter output.
7. detect as described in claim 1 the method for pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique, it is characterized in that: in described step (4), described conversion unit is water-bath.
8. detect as described in claim 7 the method for pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique, it is characterized in that: in described step (5), whether the method that described result detects occurs for the variation judgement reaction according to the described reaction solution color before and after reaction: a) described example reaction pipe liquid is yellow-green colour, and described sample detection result is positive; B) described example reaction pipe liquid is orange red, and described sample detection result is negative.
9. detect as described in claim 1 the method for pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique, it is characterized in that: temperature of reaction is 63 ℃.
10. detect as described in claim 1 the method for pig Sa Peiluo virus based on the reverse transcription loop-mediated isothermal amplification technique, it is characterized in that: the final concentration of described RNA in described reaction solution is 2.97 * 10
8Copy/microlitre.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525948A (en) * | 2013-10-08 | 2014-01-22 | 上海交通大学 | Porcine Sapelovirus real-time fluorescent quantitative RT-PCR detection method |
| CN103614493A (en) * | 2013-11-12 | 2014-03-05 | 西南民族大学 | Fluorescent quantitative PCR (Polymerase Chain Reaction) kit for rapidly detecting porcine sapelovirus (PSV) and application thereof |
| CN106771237A (en) * | 2016-12-13 | 2017-05-31 | 湖南农业大学 | A kind of ELISA kit for detecting porcine sapelo virus antibody |
-
2013
- 2013-04-12 CN CN2013101277286A patent/CN103173577A/en active Pending
Non-Patent Citations (3)
| Title |
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| DAOLIANG LAN等: ""Isolation and characterization of the first Chinese porcine sapelovirus strain"", 《ARCH VIROL》 * |
| 张兴娟等: "猪瘟病毒野毒株RT-LAMP可视化检测方法的建立", 《中国预防兽医学报》 * |
| 鑫婷等: ""猪呼吸与繁殖综合症病毒RT-LAMP检测方法的建立"", 《中国农业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525948A (en) * | 2013-10-08 | 2014-01-22 | 上海交通大学 | Porcine Sapelovirus real-time fluorescent quantitative RT-PCR detection method |
| CN103614493A (en) * | 2013-11-12 | 2014-03-05 | 西南民族大学 | Fluorescent quantitative PCR (Polymerase Chain Reaction) kit for rapidly detecting porcine sapelovirus (PSV) and application thereof |
| CN106771237A (en) * | 2016-12-13 | 2017-05-31 | 湖南农业大学 | A kind of ELISA kit for detecting porcine sapelo virus antibody |
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Application publication date: 20130626 |