CN102912033A - Loop-mediated isothermal amplification (LAMP) kit for detecting porcine bocavirus (PBoV) and preparation method of kit - Google Patents
Loop-mediated isothermal amplification (LAMP) kit for detecting porcine bocavirus (PBoV) and preparation method of kit Download PDFInfo
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Abstract
The invention relates to a loop-mediated isothermal amplification (LAMP) kit for detecting porcine bocavirus (PBoV) and a preparation method of the kit. The kit comprises an LAMP reagent prepared from aseptic double distilled water, wherein 24 MuL of the LAMP reagent is added into each reaction; 24 MuL of the LAMP reaction solution comprises the following components with corresponding concentrations: 10 U of BstDNA polymerase, 2.5 MuL of polymerase buffer solution (200 mM Tris-HCl, pH 8.8), 100 mM of KCl, 100 mM of (NH4)2SO4, 25 mmol of betaine, 30 mmol of dNTP, 5 pmol of primer P1, 5 pmol of primer P2, 40 pmol of primer P3 and 40 pmol of primer P4. The invention has the benefits that the kit can detect suspicious PBoV infection efficiently and quickly, has a simple detection method, does not need expensive test instruments and is suitable for being popularized in clinical disease diagnosis.
Description
Technical field
The invention belongs to the veterinary biologics field, be specifically related to LAMP test kit of a kind of detection pig bocavirus (PBoV) and preparation method thereof.
Background technology
Pig bocavirus (Porcine bocavirus, PBoV) is a kind of nonencapsulated single-stranded DNA viruses, belongs to the Parvoviridae bocavirus and belongs to.2005, Allander separated the bocavirus that obtains the people first from the infant who suffers from respiratory tract infection.Henceforth, all over the world one after another at respiratory tract, blood, ight soil, urines etc. locate to find bocavirus.There is the probability of patient infection's bocavirus of respiratory passage diseases obviously high than the normal people.2009, in the Sweden swinery, chance on and have PBoV in the swinery, also detect PBoV in China swinerys in 2010.Bocavirus mainly causes lower respiratory infection and the diarrhoea of the animals such as people, ox, pig clinically.From in April, 2010 so far, Korea S, Thailand; Severe diarrhea occurs the piglet of the Guangdong of China, Fujian, Guangxi, Jiangxi, Hubei, Hebei and Shandong large-scale pig farm successively: sucking piglets is many to be vomitted after birth in 2-3 days; violent diarrhoea occurs subsequently; mortality ratio is higher, and mortality ratio reached 100% after diarrhoea appearred in some areas.And sow and growing and fattening pigs non-evident sympton.Treatment measures during according to epidemic diarrhea, transmissible gastroenteritis and rotavirus infection are substantially without unusual effect.The Clinical anatomic cardinal symptom is GI severe haemorrhage.The sample analysis that the severe diarrhea piglet gathers is found, epidemic diarrhea, transmissible gastroenteritis and rotavirus Positive rate are lower, positive rate less than 5%, and pig bocavirus (porcine bocavirus, PBoV) and Kobu virus Positive rate reach 86.6%.This shows, PBoV and Kobu virus are the arch-criminals of current grice diarrhoea.But up to the present, because deep not enough to the understanding of this two-strain, there is no effective vaccine and prophylactico-therapeutic measures and business-like detection kit and come out, therefore developing the PBoV detection kit can carry out quick diagnosis to this disease, and clinical treatment and disease control are had huge meaning.
Isothermal amplification technique (the loop-mediated isothermal amplification of ring mediation, LAMP) be by a kind of novel nucleic acids amplification technique of T.Notomi in invention in 2000, this technology relies on four specific primers and increases for four zones of aim sequence, the archaeal dna polymerase that uses has the strand displacement function, therefore can realize constant-temperature amplification.Because its amplification depends on four specific regions of aim sequence, therefore its specificity and susceptibility are all high than regular-PCR and quantitative fluorescent PCR, and owing to this reaction is carried out under constant temperature, therefore do not need expensive instrument, and use common water-bath can carry out amplified reaction, and can within one hour, finish at amplified reaction in addition, required time is short, and its reaction product is easy to observe, and can judge whether amplified sample is positive by naked eyes.Based on above advantage, LAMP to invention day its, be widely used in the detection of cause of disease, such as the detection of the virus diseases such as viral hemorrhagic septicemia, VHS, cytomegalovirus, Ebola virus, hepatitis C virus, simplexvirus, tubercule bacillus, Salmonellas, intestinal bacteria, bird flu, porcine influenza and bacteriosis.Up to the present, do not find to use the LAMP method to detect the report of PBoV.
Summary of the invention
The objective of the invention is LAMP test kit of a kind of detection pig bocavirus (PBoV) and preparation method thereof, can efficiently infect suspicious PBoV fast and detect, and detection means is simple, does not need expensive laboratory apparatus, is adapted at promoting in the clinical disease diagnosis.
The objective of the invention is to be achieved through the following technical solutions:
The LAMP test kit of a kind of detection pig bocavirus (PBoV), comprise the LAMP reaction reagent with the aseptic double-distilled water preparation, each reaction adds 24 μ L LAMP reaction reagents, wherein the component and the concentration that comprise of LAMP reaction solution 24 μ L is respectively: the BstDNA polysaccharase of 10U, 2.5 the polymerase buffer of μ L (200mM Tris-HCl (pH 8.8), 100mM KCl, 100mM (NH
4)
2SO
4, 1%Triton X-100), the MgSO of 150mmol
4, the trimethyl-glycine of 25mmol, the dNTP of 30mmol, the primer P1 of 5pmol, the primer P2 of 5pmol, the primer P3 of 40pmol, the primer P4 of 40pmol, wherein each primer sequence is respectively:
P1：CCAATCTGGCCAAGAGCAT，
P2：CGGCAACAGCATAGAGTCC，
P3：CCACACGATCAGCTTGGCCGGCTATACGGCTGCGTGAAC，
P4：TGGGAAGAAGCGCTGATGCAC-TCGGTCGATCCTGAACTCG。
The preparation method of the LAMP test kit of a kind of detection pig bocavirus (PBoV) may further comprise the steps:
1) from ncbi database Genbank, searches the PBoV sequence, use sequence alignment software DNAstar to carry out homology analysis, obtain the conservative target sequence of PBoV;
2) according to step 1) sequence that obtains uses the online primer-design software of PrimerExplore V4 to carry out design of primers, chooses the conserved sequence district as making up primer that test kit uses, and primer sequence is respectively:
P1：CCAATCTGGCCAAGAGCAT，
P2：CGGCAACAGCATAGAGTCC，
P3：CCACACGATCAGCTTGGCCGGCTATACGGCTGCGTGAAC，
P4：TGGGAAGAAGCGCTGATGCAC-TCGGTCGATCCTGAACTCG；
3) with step 2) designed primer synthesizes, use aseptic double-distilled water preparation LAMP reaction reagent, wherein the component and the concentration that comprise of LAMP reaction solution 24 μ L is respectively: the BstDNA polysaccharase of 10U, 2.5 the polymerase buffer of μ L (200mM Tris-HCl (pH 8.8), 100mM KCl, 100mM (NH
4)
2SO
4, 1%Triton X-100), the MgSO of 150mmol
4, the trimethyl-glycine of 25mmol, the dNTP of 30mmol, the primer P1 of 5pmol, the primer P2 of 5pmol, the primer P3 of 40pmol, the primer P4 of 40pmol.
After in the LAMP reaction reagent, adding 1 μ L template mixing to be detected, put 65 ℃ of reactions 1 hour, after finishing, reaction whether becomes turbid to judge amplification in the observing response pipe, observe for convenient, can be after reaction reaction tubes be put in the whizzer 12000 rev/mins centrifugal 1 minute, have or not white precipitate at the bottom of the observation tube, if observe white precipitate then be judged to the positive.
Beneficial effect of the present invention is: high specific and hypersensitivity, use four primers, and increase for four conservative regions of PBoV sequence, its specificity and susceptibility are all than high with the PCR under the condition and quantitative PCR; Simple to operate, be easy to judge, amplified reaction only needs a thermostat water bath to get final product, and can finish amplified reaction in one hour; The result judges simply, can use naked eyes to determine whether and become turbid to have judged whether specific amplification, compare with regular-PCR and quantitative PCR, do not need expensive plant and instrument, and amplification can be in the situation that covered result of determination have been avoided the pollution of amplified production to subsequent detection after finishing.
Embodiment
PBoV LAMP design of primers and screening:
Login NCBI website http://www.ncbi.nlm.nih.gov/, search the sequence of the PBoV of Genbank announcement, after using DNAstar to carry out sequence alignment, take pig bocavirus genome sequence HM053694 as template, use the online primer-design software of PrimerExplore V4 to carry out LAMP reaction design of primers, from the primer sequence of design, choose following 4 primers and carry out the experiment of specificity, susceptibility and the primer component that final Confirmation reagent box uses
P1：CCAATCTGGCCAAGAGCAT，
P2：CGGCAACAGCATAGAGTCC，
P3：CCACACGATCAGCTTGGCCGGCTATACGGCTGCGTGAAC，
P4:TGGGAAGAAGCGCTGATGCAC-TCGGTCGATCCTGAACTCG, use orthogonal experiment to carry out the optimization of each component, establish ratio and reaction density thereof that the above primer uses in the LAMP test kit: the reaction density of each primer in each reaction system, the primer P1 of 5pmol, the primer P2 of 5pmol, the primer P3 of 40pmol, the primer P4 of 40pmol.
The assembling of test kit (take 50 reactions as example):
Test kit of the present invention only is made of the LAMP reaction solution, and this reaction solution is by sterilization distilled water preparation, and wherein specification is to contain 1.2ml LAMP reaction solution in the test kit of 50 reactions, and each reaction consumption is 24 μ L.Each component and concentration are as follows in this 24 μ L reaction solution: the BstDNA polysaccharase of 10U, the polymerase buffer of 2.5 μ L (200mM Tris-HCl (pH 8.8), 100mM KCl, 100mM (NH
4)
2SO
4, 1%Triton X-100), the MgSO of 150mmol
4, the trimethyl-glycine of 25mmol, the dNTP of 30mmol, the primer P1 of 5pmol, the primer P2 of 5pmol, the primer P3 of 40pmol, the primer P4 of 40pmol.The LAMP reaction solution for preparing is put-20 ℃ or-80 ℃ of freezing preservation transportations, avoid multigelation as far as possible.
The use of PBoV LAMP test kit:
Extract DNA according to ordinary method, then get the DNA sample that 1 μ L extracts, join in the 0.2ml thin-walled PCR reaction tubes, add subsequently 24 μ L LAMP reaction solutions, slight mixing.Put in the water-bath, 65 ℃ were reacted 45-60 minute.React complete rear taking-up PCR pipe, 12000 rev/mins centrifugal 1 minute, then observe PCR reaction tubes bottom and have or not white precipitate, produce if any precipitation and then be judged to be the positive.
The sensitivity experiments of test kit:
According to conventional DNA extraction method, extract the positive pathological material of disease DNA of PBoV, carry out 10 times of serial dilutions, choose 10
1～7Deng 7 extent of dilution, the test kit for preparing with this invention carries out sensitivity experiments, reaction result is used the methods such as gel electrophoresis and visual inspection precipitation verify.The electrophoresis result demonstration, along with the reduction of template concentrations, its electrophoresis result difference is not remarkable, and centrifugal observation precipitation capacity is also without significant difference.10
1Doubly～10
6Doubly dilution all can detect PBoV nucleic acid.
The specificity experimental verification of test kit:
Use respectively DNA or the RNA of the viruses such as the extraction pig breeding of conventional DNA and RNA extracting method and breathing syndrome virus, Pestivirus suis, Transmissible gastroenteritis virus, Porcine epidemic diarrhea virus, rotavirus, PCV-II, pig parvoviral, with the sterilization distilled water as negative control, after using the ordinary method reverse transcription to be cDNA the RNA that extracts, together use this test kit to detect with the DNA that extracts.Detected result finds that PBoV is positive, and other all becomes negative.This result shows, the test kit of the detection PBoV of the present invention's preparation has specificity.
Claims (4)
1. LAMP test kit that detects pig bocavirus (PBoV), it is characterized in that, it comprises the LAMP reaction reagent with the aseptic double-distilled water preparation, each reaction adds 24 μ L LAMP reaction reagents, wherein the component and the concentration that comprise of LAMP reaction solution 24 μ L is respectively: the BstDNA polysaccharase of 10U, 2.5 the polymerase buffer of μ L, the MgSO of 150mmol
4, the trimethyl-glycine of 25mmol, the dNTP of 30mmol, the primer P1 of 5pmol, the primer P2 of 5pmol, the primer P3 of 40pmol, the primer P4 of 40pmol, wherein each primer sequence is respectively:
P1：CCAATCTGGCCAAGAGCAT，
P2：CGGCAACAGCATAGAGTCC，
P3：CCACACGATCAGCTTGGCCGGCTATACGGCTGCGTGAAC，
P4：TGGGAAGAAGCGCTGATGCAC-TCGGTCGATCCTGAACTCG。
2. the LAMP test kit of detection pig bocavirus according to claim 1 (PBoV) is characterized in that, described polymerase buffer is made by following component: the 200mM Tris-HCl of pH 8.8,100mM KCl, 100mM (NH
4)
2SO
4, and 1%Triton X-100.
3. a preparation method who detects the LAMP test kit of pig bocavirus (PBoV) is characterized in that, may further comprise the steps:
1) from ncbi database Genbank, searches the PBoV sequence, use sequence alignment software DNAstar to carry out homology analysis, obtain the conservative target sequence of PBoV;
2) according to step 1) sequence that obtains uses the online primer-design software of PrimerExplore V4 to carry out design of primers, chooses the conserved sequence district as making up primer that test kit uses, and primer sequence is respectively:
P1：CCAATCTGGCCAAGAGCAT，
P2：CGGCAACAGCATAGAGTCC，
P3：CCACACGATCAGCTTGGCCGGCTATACGGCTGCGTGAAC，
P4：TGGGAAGAAGCGCTGATGCAC-TCGGTCGATCCTGAACTCG；
3) with step 2) designed primer synthesizes, use aseptic double-distilled water preparation LAMP reaction reagent, wherein the component and the concentration that comprise of LAMP reaction solution 24 μ L is respectively: the BstDNA polysaccharase of 10U, the polymerase buffer of 2.5 μ L, the MgSO of 150mmol
4, the trimethyl-glycine of 25mmol, the dNTP of 30mmol, the primer P1 of 5pmol, the primer P2 of 5pmol, the primer P3 of 40pmol, and the primer P4 of 40pmol.
4. a preparation method who detects the LAMP test kit of pig bocavirus (PBoV) is characterized in that, described polymerase buffer is made by following component: the 200mM Tris-HCl of pH 8.8,100mMKCl, 100mM (NH
4)
2SO
4, and 1%Triton X-100.
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Cited By (2)
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| CN105219887A (en) * | 2015-11-09 | 2016-01-06 | 北京市农林科学院 | For the primer sets of typing detection of porcine bocavirus, test kit and detection method |
| CN105713995A (en) * | 2016-04-25 | 2016-06-29 | 广西壮族自治区兽医研究所 | Porcine bocavirus loop-mediated isothermal amplification (LAMP) kit and application thereof |
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Patent Citations (5)
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| WO2001071009A2 (en) * | 2000-03-17 | 2001-09-27 | Anticancer, Inc. | Whole-body optical imaging of gene expression and uses thereof |
| CN101096704A (en) * | 2006-06-01 | 2008-01-02 | 杭州致远医学检验所有限公司 | A set of fluorescent quantitative PCR primer and probe for detecting human boca virus |
| CN101550452A (en) * | 2008-03-31 | 2009-10-07 | 中山大学达安基因股份有限公司 | Human bocavirus real-time fluorescence PCR detection reagent kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219887A (en) * | 2015-11-09 | 2016-01-06 | 北京市农林科学院 | For the primer sets of typing detection of porcine bocavirus, test kit and detection method |
| CN105219887B (en) * | 2015-11-09 | 2019-02-05 | 北京市农林科学院 | For the primer sets of typing detection of porcine bocavirus, kit and detection method |
| CN105713995A (en) * | 2016-04-25 | 2016-06-29 | 广西壮族自治区兽医研究所 | Porcine bocavirus loop-mediated isothermal amplification (LAMP) kit and application thereof |
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Application publication date: 20130206 |