CN101358246A - LAMP kit for detecting hogcholera virus and preparation method thereof - Google Patents
LAMP kit for detecting hogcholera virus and preparation method thereof Download PDFInfo
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- CN101358246A CN101358246A CNA2008100422173A CN200810042217A CN101358246A CN 101358246 A CN101358246 A CN 101358246A CN A2008100422173 A CNA2008100422173 A CN A2008100422173A CN 200810042217 A CN200810042217 A CN 200810042217A CN 101358246 A CN101358246 A CN 101358246A
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Abstract
The invention belongs to the field of sanitary examination, and relates to a LAMP kit for testing classical swine fever virus and an establishing method and an application thereof. The kit contains a test system which is composed of the LAMP reaction liquid of six LAMP primers. The tests prove that the kit of the invention has good specificity and sensitivity, fast amplification speed, high efficiency and simple and convenient identification. The test system of the invention can rapidly and conveniently test the classical swine fever virus in high-efficiency, high-specificity and high-sensitivity under the isothermal condition of 65 DEG C without complicate instruments, can better satisfy the spot tests for the classical swine fever virus, provides a novel technical platform for food safety testing, can better meet the urgent requirements for the spot testing of foot and mouth diseases at present, is used for the spot testing of import and export quarantine, food sanitary departments, animal breeding farms, etc, and is easy to be popularized in a wide range.
Description
Technical field
The invention belongs to the sanitary inspection field, relate to a kind of test kit that is used to detect Pestivirus suis, particularly a kind of LAMP test kit and establishment method and application that is used to detect Pestivirus suis.
Technical background
(Swine Fever SF), claims hog cholera virus again to swine fever, and Europe is called classic swine fever.Found swine fever in the U.S. first, and now propagated into many countries and regions in the world in 1833.According to the data that food and agricultural organization-International Office of Epizootics-World Health Organization (FAO-OIE-WHO) animal health yearbook (1992) is announced, there is swine fever in existing 44 countries and regions.Mainly be distributed in the countries and regions in South America, Europe and Asia.The financial loss that swine fever causes is huge.In Holland, 1983 and 1984 degree are in order to control swine fever, are used to transport and disappear ruin the evaluation of infecting swinery, sterilization room, reparations peasant loss, epidemic prevention pig and registration etc. and spend 1.27 hundred million Francs altogether.In addition, stop the live pig allocation and transportation as production loss, the infected zone of infecting the pig farm and the indirect loss of aspect such as restrict export then is difficult to estimate.In Britain, carried out in 1963 mandatory butcher policy after, eliminated swine fever in 1966.For this reason, cost 1,200 ten thousand pounds altogether.After this, except that once taking place 3 times in 1971 on a small scale the outburst, still kept no swine fever country up to 1986.But in April, 1986 typical acute swine fever takes place, slaughtered 26 groups of totally 7781 first-born pigs, only recovering damage of this just reaches more than 450,000 dollar.
The isothermal amplification (LAMP) of ring mediation is a kind of novel nucleic acids amplification technique by T.Notomi (2000) invention, this technology relies on primer and a kind of archaeal dna polymerase with strand displacement characteristic of 4 special designs, can be efficiently under isothermal condition, quick, high amplified target sequence specifically.In recent years, this technology is widely used in pathogen detection abroad.System is made a definite diagnosis in the laboratory that people (2007) such as Masaki Imai utilize LAMP to set up avian influenza virus; People (2007) such as Nobuyuki Hayashi at four kinds of cordiale zymic ITS sequences Design the LAMP Auele Specific Primer, set up LAMP detection architecture efficiently.LAMP can also detect other and human relevant virus, as viral hemorrhagic septicemia (VHS), cytomegalovirus (CMV), Ebola virus (EBOV), chronic burkitt's lymphoma virus (EBV), irido virus, human herpes virus type 8, hematopoietic tissue necrosis virus (IHHNV), tomato spotted wilf virus, tomato yellow leaf curl virus etc.There is no both at home and abroad at present and be useful on LAMP test kit and the application in Pestivirus suis detects that detects Pestivirus suis.
Summary of the invention
The object of the invention is to provide a kind of LAMP test kit that is used to detect Pestivirus suis.
Test kit of the present invention comprises following component:
(1) LAMP reaction solution:
Contain in per 23 μ L LAMP reaction solutions: DEPC water 1.9 μ L; 10 * ThermoPol buffer, 2.5 μ L; 25m,MMg,SO4 6 μ L; 5M Betaine 5 μ L; 10mM dNTP 3 μ L; 50 μ M Primer F3,0.1 μ L; 50 μ M PrimerB3,0.1 μ L; 50 μ M Primer FIP, 0.8 μ L; 50 μ M Primer BIP, 0.8 μ L; 50 μ M Primer LF, 0.4 μ L; 50 μ M Primer LB, 0.4 μ L; 8U/ μ L Bst enzyme 1 μ L; 5U/ μ L AMV reversed transcriptive enzyme 1 μ L;
Article (2) six, LAMP primer (collating sequence 1-6):
Forward?Outer F3 AGCTCCCTGGGTGGTCTA
Reverse?Outer B3 CCTAATAGTGGGCCTCTGCA
Forward?Inner FIP GCCCTCGTCCACATAGCATCTCAGTACAGGACAGTCGTCAGT
Reverse?Inner BIP GCCCAAGACACACCTTAACCCTTCAGGTCGTACTCCCATCAC
Forward?Loop LF GGGCTTCTGCTCACGTCG
Reverse?Loop LB GGTCGCTAGGGTGAAATC
Another object of the present invention is to provide the establishment method of above-mentioned LAMP test kit.
Described LAMP test kit prepares by following method and step:
1) obtain all Pestivirus suis genome sequences from the gene data library searching, carry out homology analysis, obtain the specific conservative target sequence of the Pestivirus suis gene of sequence 7 by BLAST software,
2) according to the dna sequence dna of the conservative target of step 1) gained, six LAMP primers of implementation sequence 1-6;
3) according to six LAMP primer configuration LAMP reaction solutions of gained, constitute detection architecture.
Table 1 is the specific configuration of 23 μ L LAMP reaction solutions.
Table 1
Annotate: listed each constituent concentration is a mother liquid concentration in the table.
Further purpose of the present invention provides the application method of described test kit.
The method that the above-mentioned LAMP test kit that is used to detect Pestivirus suis detects Pestivirus suis is: at first dispose the LAMP reaction solution, extract the nucleic acid in the testing sample then, get this nucleic acid extraction liquid add above-mentioned prepared the LAMP reaction solution in, put 65 ℃ of constant temperature 45min and carry out the LAMP amplification; Picogreen or SYBRgreen are added in the amplification back in reaction solution, according to the colour-change of reaction solution, judged result: green illustrates to have Pestivirus suis in the testing sample; Orange illustrates not have Pestivirus suis in the testing sample.
The above-mentioned application method that is used to detect the LAMP test kit of Pestivirus suis specifically may further comprise the steps:
(1) the above-mentioned LAMP reaction solution of preparation 23 μ L;
(2) extract nucleic acid in the testing sample according to a conventional method;
(3) extracting solution of getting the described nucleic acid of 2 μ L adds in the LAMP reaction solution that step (1) makes, and making the end reaction volume is 25 μ L, and the instantaneous centrifugal 30S of 10000rpm is with the mixing reaction solution;
(4) put 65 ℃ of constant temperature 45min and carry out the LAMP amplification;
(5) take out the amplified reaction pipe, in reaction solution, add 5 μ L Picogreen or SYBRgreen,, then illustrate to have Pestivirus suis in the testing sample, if then there is not Pestivirus suis in orange in the testing sample if reaction solution becomes green.
The present invention is the specificity of template detection system with six kinds with the similar viral nucleic acid of Pestivirus suis, the result shows, the inventive method can only increase Pestivirus suis nucleic acid and can not detect other non-target viral nucleic acid, confirms that the LAMP that is set up has good specificity; With 10
-3, 10
-410
-9Dilution Pestivirus suis nucleic acid is that template is carried out LAMP and RT-PCR respectively, the relatively sensitivity of two kinds of detection methods, and the result shows that this LAMP can detect 10
-5Concentration nucleic acid, the RT-PCR method can only detect 10
-4Concentration nucleic acid confirms that the LAMP that is set up has good sensitivity; Determine the specificity of product with Sph I restriction endonuclease, the result shows that the LAMP amplified production is stepped on gel electrophoresis spectrum, through restriction endonuclease digestion, the 100bp electrophoretic band occurs, is consistent with theoretical expected value, and explanation is a specific amplification.
The present invention has following outstanding advantage:
1) high specific: 6 primers have guaranteed the high degree of specificity of LAMP amplification to the identification in 8 special zones of target sequence, and promptly LAMP can find out corresponding target sequence and increase, (Fig. 1) from the gene sample that differs a Nucleotide only.
2) highly sensitive: LAMP sensitivity can compare favourably with PCR or is higher, its required amplification template can reach 10 the copy or still less, (Fig. 2).
3) identify easy: when in reaction solution, adding picogree fluorescence dyes such as (or SYBRgreen), if amplification is arranged, picogreen will with the DNA combination, get final product by visual inspection, green if reaction solution turns, then positive; As reaction solution is orange, the reaction that is negative then is described, (Fig. 3).
4) simple to operate: in case the success of LAMP design of primers, as long as test sample (target nucleic acid) and reagent are put into 65 ℃ of thermostat water baths together, whether about 1h just can judge amplification.
5) efficient amplification fast: whole amplified reaction can be finished in one hour, and productive rate can reach 0.5mg/ml.
Detection architecture of the present invention can be under isothermal condition, fast, conveniently, efficiently, high special, detect Pestivirus suis with sensitivity, do not need complex instrument, for food safety detection provides new technology platform, can better satisfy at present to on-the-spot the pressing for of detecting of foot and mouth disease, be used to import and export the scene detection of quarantine, food sanitation department, livestock rearing field etc., be easy to apply on a large scale, have vast market prospect and bigger economical, societal benefits.
Description of drawings
Fig. 1 is the specific detection electrophorogram as a result of Pestivirus suis LAMP detection architecture,
Wherein, M.DNA Marker; 1～7. is respectively Pestivirus suis, pig breeding and breathing syndrome virus, PRV (Pseudorabies virus), transmissible gastro-enteritis virus, pig parvoviral, Schweineseuche virus; Negative control.
Fig. 2 is the sensitivity checking electrophorogram of Pestivirus suis LAMP detection architecture,
Wherein, M.DNA Marker; 1～7. is respectively 10
-3, 10
-410
-9Dilution transmissible gastroenteritis of swine viral nucleic acid; 8. negative control.
Fig. 3 confirms electrophorogram for the LAMP specific amplification, wherein,
Swimming lane 1:LAMP amplified production is stepped on gel electrophoresis spectrum,
Swimming lane 2: through restriction endonuclease digestion, the 100bp electrophoretic band occurs, be consistent with theoretical expected value.
Embodiment
1, specific DNA sequences is searched
Obtain many strains Pestivirus suis genome sequence, carry out homology analysis by BLAST software and promptly find specific conservative target sequence to carry out the LAMP design of primers from U.S.'s gene database-GenBank retrieval.
2, the LAMP design of primers
By special LAMP primer-design software design primer
Primer?design?V3(http://primerexplorer.jp/elamp3.0.0/index.html)，
The concrete LAMP primer of design is (collating sequence 1-6):
3, set up detection architecture
The Mg of different final concentrations is set
2+(2,4,6,8,10, mM), dNTP (0,0.2,0.4,0.6,0.8,1.0.1.2,1.4mM), inside and outside primer concentration ratio (1: 1,1: 2,1: 4,1: 61: 8,1: 10,1: 12), respectively in the differential responses time (10,20,30,45,60min) and temperature (57 ℃, 60 ℃, 63 ℃, 65 ℃, 68 ℃) reaction down, finally obtain the optimum response parameter, thereby set up Pestivirus suis LAMP detection architecture.
The detection architecture of optimizing (23 μ l) is as follows:
1 * ThermoPol buffer, 8mM Mg
2+, 1M betaine, 1.2mM dNTP, 0.2 μ M outer primer (F3 and B3), 1.6 μ M inner primers (FIP and BIP), 0.8 μ M encircles primer (LF and LB), 2 μ l template ribonucleic acids, 5U AMV reversed transcriptive enzyme, 8U Bst polysaccharase.Detection reaction condition: 65 ℃ of constant temperature 45min.
Described Mg
2+ final concentration=(containing 20mM * 2.5 μ l among mother liquid concentration 25mM * 4 μ l+ThermoPol buffer)/25 μ l.
4, the susceptibility of analyzing and testing system, specificity
With pig breeding and breathing syndrome virus, PRV (Pseudorabies virus), transmissible gastro-enteritis virus, pig parvoviral and five kinds of specificitys that viral nucleic acid is the template detection system of Schweineseuche virus, the result shows, the test of the specificity of LAMP detection architecture is good, can be in numerous viruses the specific Pestivirus suis that detects.
With 10
-1, 10
-210
-7Dilution transmissible gastroenteritis of swine viral nucleic acid is that template is carried out the relatively sensitivity of two kinds of detection methods of LAMP and RT-PCR respectively, and the result shows that the common RT-PCR method of LAMP remolding sensitivity is high 10 times.
Its trace routine is:
(1) by following proportioning preparation reaction system (23 μ L):
(2) LAMP amplification
With the nucleic acid in Trizon or the RNeasy Mini Kit commercialization RNA extraction test kit extraction testing sample.Get 2 μ L nucleic acid extraction liquid and be added in the above-mentioned LAMP reaction solution that has prepared, making the end reaction volume is 25 μ L, 10000rpm, and instantaneous centrifugal 30S is with the mixing reaction solution.Be put in 65 ℃ of constant temperature 45min then and carry out the LAMP amplification.
(3) result judges
Take out reaction tubes, add 5 μ L Picogreen (or SYBRgreen) in reaction solution, if reaction solution becomes green, then the explanation reaction is positive, if orange is then reacted negative.
Be the reliability of checking effect of the present invention, carry out following evaluation:
1, the LAMP specific amplification is confirmed
Judge product structure with Sph I restriction endonuclease, determine that it is specific amplification products.Through restriction endonuclease digestion, 100bp electrophoretic band (swimming lane 2) appears, is consistent with theoretical expected value, explanation is specific amplification (Fig. 3).
2, the specificity of Pestivirus suis LAMP detection architecture,
With pig breeding and breathing syndrome virus, PRV (Pseudorabies virus), transmissible gastro-enteritis virus, the tiny disease of pig pig and five kinds of specificitys (Fig. 1) that viral nucleic acid is the template detection system of Schweineseuche virus.The result shows that swine fever LAMP detection method can only increase Pestivirus suis nucleic acid and can not detect other non-target viral nucleic acid, confirms that the LAMP that is set up has the good detection specificity.
3, the sensitivity of Pestivirus suis LAMP detection architecture reaches with common RT-PCR and compares
With 10
-1, 10
-210
-7Dilution Pestivirus suis nucleic acid is that template is carried out LAMP and RT-PCR respectively, relatively the sensitivity (Fig. 2) of two kinds of detection methods.The result shows, highly sensitive 10 times than RT-PCR of the Pestivirus suis LAMP detection methods of being set up.
Sequence table:
＜110〉Shanghai Bureau of Emigration ﹠. Engression Examination ﹠. Quarantine People's R
＜120〉be used to detect the LAMP test kit and the using method thereof of Pestivirus suis
<160>7
<210>1
<211>18
<212>DNA
<213>Forward?Outer?F3
＜223〉artificial sequence
<400>1
AGCTCCCTGG?GTGGTCTA
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<223>Reverse?Outer?B3
<400>2
CCTAATAGTG?GGCCTCTGCA
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<223>Reverse?Outer?B3
<400>2
CCTAATAGTG?GGCCTCTGCA
<210>3
<211>42
<212>DNA
＜213〉artificial sequence
<223>Forward?Inner?FIP
<400>3
GCCCTCGTCC?ACATAGCATC?TCAGTACAGG?ACAGTCGTCA?GT
<210>4
<211>42
<212>DNA
＜213〉artificial sequence
<223>Reverse Inner?BIP
<400>4
GCCCAAGACA?CACCTTAACC?CTTCAGGTCG?TACTCCCATC?AC
<210>5
<211>18
<212>DNA
＜213〉artificial sequence
<223>Forward?Loop?LF
<400>5
GGGCTTCTGC?TCACGTCG
<210>6
<211>18
<212>DNA
＜213〉artificial sequence
<223>Reverse?Loop?LB
<400>6
GGTCGCTAGG?GTGAAATC
<210>7
<211>
<212>DNA
＜213〉artificial sequence
＜223〉the conservative target DNA sequence of Pestivirus suis gene specific
<400>7
AGCTCCCTGG?GTGGTCTAAG?TCCTGAGTAC?AGGACAGTCG?TCAGTAGTTC?GACGTGAGCA?60
GAAGCCCACC?TCGAGATGCT?ATGTGGACGA?GGGCATGCCC?AAGACACACC?TTAACCCTAG?120
CGGGGGTCGC?TAGGGTGAAA?TCACACCACG?TGATGGGAGT?ACGACCTGAT?AGGGCGCTGC?180
AGAGGCCCAC?TATTAGG 197
Claims (5)
1, a kind of LAMP test kit that is used to detect Pestivirus suis, its feature comprises following component:
(1) LAMP reaction solution:
Contain in per 23 μ L LAMP reaction solutions: DEPC water 1.9 μ L; 10 * ThermoPol buffer, 2.5 μ L; 25m,MMg,SO4 6 μ L; 5M Betaine 5 μ L; 10mM dNTP 3 μ L; 50 μ M Primer F3,0.1 μ L; 50 μ M PrimerB3,0.1 μ L; 50 μ M Primer FIP, 0.8 μ L; 50 μ M Primer BIP, 0.8 μ L; 50 μ M Primer LF, 0.4 μ L; 50 μ M Primer LB, 0.4 μ L; 8U/ μ L Bst enzyme 1 μ L; 5U/ μ L AMV reversed transcriptive enzyme 1 μ L;
(2) the LAMP primer of collating sequence 1-6:
Forward?Outer F3 AGCTCCCTGGGTGGTCTA
Reverse?Outer B3 CCTAATAGTGGGCCTCTGCA
Forward?Inner FIP GCCCTCGTCCACATAGCATCTCAGTACAGGACAGTCGTCAGT
Reverse?Inner BIP GCCCAAGACACACCTTAACCCT?TCAGGTCGTACTCCCATCAC
Forward?Loop LF GGGCTTCTGCTCACGTCG
Reverse?Loop LB GGTCGCTAGGGTGAAATC 。
2, be used to detect the preparation method of the LAMP test kit of Pestivirus suis, it is characterized in that comprising the steps:
1) obtains the Pestivirus suis genome sequence from the gene data library searching, carry out homology analysis, obtain the specific conservative target sequence of the Pestivirus suis gene of sequence 7 by BLAST software;
2) according to the dna sequence dna of the conservative target of step 1) gained, the LAMP primer of implementation sequence 1-6;
3) according to the LAMP primer configuration LAMP reaction solution of gained, constitute detection architecture.
3, by the described preparation method of claim 2, it is characterized in that described LAMP reaction solution is the described 23 μ L LAMP reaction solutions of claim 1.
4, be used to detect the using method of the LAMP test kit of Pestivirus suis according to claim 1, it is characterized in that may further comprise the steps:
(1) the above-mentioned 23 μ L LAMP reaction solutions of preparation;
(2) nucleic acid in the extraction testing sample;
(3) extracting solution of getting 2 μ L described (2) nucleic acid adds in the LAMP reaction solution of step (1) preparation, and making the end reaction volume is 25 μ L, and the instantaneous centrifugal 30S of 10000rpm is with the mixing reaction solution;
(4) put 65 ℃ of constant temperature 45min and carry out the LAMP amplification;
(5) take out the amplified reaction pipe, in reaction solution, add 5 μ L Picogreen or SYBRgreen, according to the colour-change of reaction solution, judged result.
5, using method as claimed in claim 4, wherein said step (5) is described result be judged as: green, for there being Pestivirus suis in the testing sample; Orange is not for existing Pestivirus suis in the testing sample.
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| CN111455113B (en) * | 2020-05-21 | 2021-04-02 | 云南省畜牧兽医科学院 | Primer group and kit for detecting foot-and-mouth disease virus by RT-LAMP method and application of primer group and kit |
| CN111676319A (en) * | 2020-06-22 | 2020-09-18 | 河北三狮生物科技有限公司 | LAMP primer group and kit for detecting classical swine fever virus and using method thereof |
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