CN105821161A - Primer, kit and method for detecting porcine reproductive and respiratory syndrome virus - Google Patents

Primer, kit and method for detecting porcine reproductive and respiratory syndrome virus Download PDF

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CN105821161A
CN105821161A CN201610266369.6A CN201610266369A CN105821161A CN 105821161 A CN105821161 A CN 105821161A CN 201610266369 A CN201610266369 A CN 201610266369A CN 105821161 A CN105821161 A CN 105821161A
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primer
prrs virus
test kit
detecting
dna
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胡军瑜
王健斌
龚贻洲
周军
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Wuhan Jinghong Wanfangtang Medical Technology Co Ltd
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Abstract

The invention discloses a primer for detecting porcine reproductive and respiratory syndrome virus. The primer aims at conserved domains of the porcine reproductive and respiratory syndrome virus and has nucleotide sequences as follows: an outer primer F3: TCTTCTGGGAGGAGGATTC; an outer primerB3: GAGGATGCATAAGCTCGA; an inner primer FIP: TCTAAACAAAACTCAACAGGCCACCAATTGAACCTTTTTAGTTTCT; and an inner primer BIP: CCTGATCGTCGTCATTGCGGACTGTCTGTCGTATTATGTGTC. The invention further discloses a kit for detecting the porcine reproductive and respiratory syndrome virus. The kit comprises a DNA/RNA extracting reagent, the primer, a reaction buffer solution, Bst DNA polymerase and a nucleic acid dye. The kit provided by the invention has strong anti-fouling performance and good specificity.

Description

A kind of for detecting the primer of PRRS virus, test kit and method
Technical field
The invention belongs to animal virus Molecular Detection biological technical field, relate to a kind of for detecting the primer of PRRS virus, test kit and method.
Background technology
Porcine reproductive and respiratory syndrome (PorcineReproductiveandRespiratorySyndrome, PRRS) it is a kind of acute infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), clinic is mainly characterized by the breeding difficultys such as farrowing sow miscarriage, premature labor, stillborn fetus and mummy tire and piglet dyspnea, high mortality and growing and fattening pigs adnormal respiration are characterized, also referred to as " swine infertility and miscarriage syndrome ".This disease has been dispersed throughout the main pig-raising countries in the whole world and area with PRRSV, has become as one of main epidemic disease of threat pig industry sound development.PRRSV is the single strand plus RNA virus of Arteriviridae, Arterivirus.Its full-length genome is about 15kb, containing 9 open reading frame (ORFs).Wherein, N protein is also referred to as nucleocapsid protein, about 15kD, 0RF7 encode, and in virion, content is higher, N gene high conservative.Forming homodimer between N protein, the assembling to virus is most important, and body can be induced to produce immunization of cell, is the preferable target antigen detecting this special viral antibody.
Europe class (with LelystadVirus strain as representative) and two kinds of genotype of american type (with ATCCVR-232 strain as representative) can be classified as according to the difference of PRRSV structural gene sequence, and according to the difference of its pathogenicity, american type porcine reproductive and respiratory syndrome virus therein can be divided into again classical PRRS virus and two kinds of hypotypes of high-pathogenicity porcine reproductive and respiratory syndrome virus, although PRRSV can cause same disease, but there is the biggest difference in the genome sequence between Europe, the United States' amphitypy strain and antigenicity, Serologic test shows there is certain cross reaction.At present, the strain great majority that isolated in China obtains belong to american type, in recent years, have scholar to identify Europe class PRRSV by molecular biology and serological method, show to have existed in China swinery Europe class PRRSV.Owing to clinical symptoms is complicated, and it mostly is mixed infection, it is thus desirable to set up a kind of fast and convenient, differential diagnostic method of high specificity, PRRSV in domestic swinery is carried out epidemiological surveillance, follows the tracks of cause of disease, understanding Europe class, american type strain, in the popular of China different regions and the regularity of distribution, preferably prevent, control the popular of PRRSV and distribute.
Ring mediated constant temperature nucleic acid amplification technology (loop-mediatedisothermalamplificationofDNA, LAMP) it is that a kind of novel nucleic acid amplification method grown up in recent years is (see document NotomiT, OkayamaH, MasubuchiH, YonekawaT, WatanabeK, AminoN, HaseT.Loop-mediatedisothermalamplificationofDNA.NucleicA cidsRes.2000Jun15；28(12):E63.).Ring mediated isothermal amplification (LAMP) technology is for 4 special primers of 6 region designs of target gene, a kind of strand displacement archaeal dna polymerase (BstDNApolymerase) is utilized to be incubated about 60min at constant temperature (about 65 DEG C), nucleic acid amplification reaction can be completed, quickly realize the nucleic acid amplification of a large amount of copy number.Compared with temperature varied cyclical amplification technique, the demand of instrument and equipment is more simplified by isothermal amplification technique, simultaneously as reaction need not denaturation renaturation process, thus can effectively shorten the response time.
Loop-mediated isothermal amplification technique has the advantage that compared with conventional nucleic acid amplification method
1) rapidly and efficiently, because need not double-stranded DNA thermal denaturation in advance. avoiding temperature cycles and loss of time of causing, nucleic acid amplification all can complete in 1h.
2) simple to operate, LAMP nucleic acid amplification is to carry out under isothermal conditions, water-bath is had only to for middle and small hospital, it is not required to high-end instrument and equipment and professional technique, amplification by-product magnesium pyrophosphate precipitation directly can be judged through naked eyes or detect its turbidity by amplification, it is also possible to combine the fluorescent dye SYBRGreenI dyeing of double-stranded DNA.Being judged by naked eyes under uviol lamp or daylight, if containing amplified production, reactant mixture turns green；Otherwise, then keep that SYBRGreenI's is orange constant.The amplification of RNA is had only to add reverse transcriptase in reaction system just can synchronize to carry out (RT-LAMP), it is not necessary to special reagent and instrument.
3) high sensitivity and specificity, owing to being 4 species-specific primers for the design of 6 regions of target sequence, in 6 regions, any region is not mated with primer and all can not be carried out nucleic acid amplification, therefore its specificity is high, for virus amplification template up to several copies, exceed the difference of the order of magnitude than PCR.
Summary of the invention
It is an object of the invention to provide a kind of for detecting the primer of PRRS virus, test kit and method；Described test kit detection process is simple, resistance tocrocking strong, need not special instrument, and result is the most highly sensitive, specificity is good, is to detect the extraordinary a kind of alternative method of high-pathogenicity porcine reproductive and respiratory syndrome virus at present.
To achieve these goals, the technical solution used in the present invention is as follows:
A kind of primer for detecting PRRS virus, described primer is the primer for PRRS virus conservative region, and its nucleotide sequence is as follows:
Outer primer F3:TCTTCTGGGAGGAGGATTC (SEQIDNO:1)；
Outer primer B3:GAGGATGCATAAGCTCGA (SEQIDNO:2)；
Inner primer FIP:TCTAAACAAAACTCAACAGGCCACCAATTGAACCTTTTTAGTTTCT (SEQIDNO:3)；
Inner primer BIP:CCTGATCGTCGTCATTGCGGACTGTCTGTCGTATTATGTGTC (SEQIDNO:4).
A kind of test kit for detecting PRRS virus, described test kit includes that DNA/RNA extracts reagent, described primer, reaction buffer, BstDNA polymerase and nucleic acid dye for PRRS virus conservative region.By PrimerExplorerV4 (http://primerexplorer.jp/e/v4_manual/index.html) specific fragment can expand by two pairs of specific primers of Photographing On-line the most fast and accurately, in turn ensure that the specificity of experiment simultaneously.
Described DNA/RNA extracts reagent and includes: BloodViralDNA/RNAkit (promegaWizard), extracts pig blue-ear disease poison DNA or RNA in the cell of cultivation to specifications.
Described reaction buffer includes 10mMdNTP, 10 × ThermoPol reaction buffer, 200mMTris-HCl (pH8.825 DEG C), 100mMKCl, 100mM (NH4)2SO4、20mMMgSO4, 1%TritonX-100.
The concentration of described BstDNA polymerase is 8U/ μ l.
Described nucleic acid dye is 1000 × SYBRGreenI.
A kind of use described test kit for the method detecting PRRS virus, comprise the following steps:
Use DNA/RNA to extract reagent from the cell cultivated and extract viral DNA template, it is subsequently adding the described primer for PRRS virus conservative region, reaction buffer, BstDNA polymerase configuration LAMP reactant, carry out LAMP reaction, reaction adds nucleic acid dye after terminating, color according to reactant liquor differentiates, completes the detection to PRRS virus.
Owing to have employed technique scheme, the present invention has the following advantages and beneficial effect:
Test kit provided by the present invention detection process is simple, resistance tocrocking strong, need not special instrument, and result is the most highly sensitive, specificity is good, is to detect the extraordinary a kind of alternative method of high-pathogenicity porcine reproductive and respiratory syndrome virus at present.
The primer that the present invention provides has good specificity；The lowest detection limit of detection method reaches 102 copies/microlitre DNA, and sensitivity is the highest.
Detailed description of the invention
In order to be illustrated more clearly that the present invention, below in conjunction with preferred embodiment, the present invention is described further.It will be appreciated by those skilled in the art that following specifically described content is illustrative and be not restrictive, should not limit the scope of the invention with this.
Design of primers: by high-pathogenicity porcine reproductive and respiratory syndrome virus genome sequence compares analysis, selects without secondary structure and the section of high conservative, designs multipair primer, interior with primer without complementary series between primer.Optimum primer sequence combination is following (seeing appendix shown in 1 with reference to ref sequence): owing to genome sequence and antigenicity exist the biggest difference, carry out multiple ratio pair according to PRRSV gene order (NC_001961.1) glycosylation envelope protein, select high conservative section.
Outer primer F3:TCTTCTGGGAGGAGGATTC (SEQIDNO:1)；
Outer primer B3:GAGGATGCATAAGCTCGA (SEQIDNO:2)；
Inner primer FIP:TCTAAACAAAACTCAACAGGCCA-CCAATTGAACCTTTTTAGTTTCT (SEQIDNO:3)；
Inner primer BIP:CCTGATCGTCGTCATTGCGG-ACTGTCTGTCGTATTATGTGTC (SEQIDNO:4).
The foundation of reaction system and optimization: utilize the high-pathogenicity porcine reproductive and respiratory syndrome virus of inactivation as measuring samples, extract pig blue-ear disease poison DNA with the extracting method of BloodViralDNA/RNAkit (promegaWizard) nucleic acid extraction reagent, store for future use under conditions of temperature is for-20 DEG C after extracting subpackage.
The optimization of primer concentration is in the case of in reaction system, other condition is identical, the primer concentration of high-pathogenicity porcine reproductive and respiratory syndrome virus is made multiple proportions serial dilution from 0.2 μm ol/L to 2.5 μm ol/L respectively, by the com-parison and analysis of result of the test, determine final concentration of 0.5 μm ol/L of optimal primer.
The optimization of BstDNA polymerase consumption is by comparing the optimization experiment result of enzyme dosage (in terms of unit Unit), and selected 8U is as the consumption of BstDNA enzyme in test kit reaction system.
Utilize above-mentioned primer to carry out the foundation of reaction system, finally determine that the high-pathogenicity porcine reproductive and respiratory syndrome virus LAMP reaction system of employing is 25 μ l systems.25 μ l reaction systems are prepared in PCR pipe, wherein four kinds of each 1 μ l of primer, reaction buffer 2.5 μ l, the BstDNA polymerase 1 μ l of 8U/ μ l, template DNA 2 μ l are (according to detection samples sources difference, can suitably adjust template dosage), add water and be supplemented to 25 μ l.Using genomic DNA as positive control, with ddH2O is as negative control.
LAMP reacts: above-mentioned reaction system PCR pipe is put in 62 DEG C of isothermal reaction 60min.
Analytical reactions result: treat that LAMP reaction terminates, containing a large amount of purpose fragments in 0.25mleppendorf pipe.In eppendorf pipe, add 1 μ l nucleic acid dye, if reactant liquor color is orange, represent that result is negative, if reactant liquor color is green, represent that result is positive.
Embodiment 1
Pig blue-ear disease PRRS sample is provided by Hua Zhong Agriculture University, and BstDNA polymerase and 10 × ThermoPol reaction buffer are purchased from NEB, the preparation of template to be measured: extract with the extracting method of BloodViralDNA/RNAkit nucleic acid extraction reagent.Primer is synthesized by Shanghai Sheng Gong biological engineering company limited.
1) specific primer is designed and synthesized
With two couple of online LAMP primer design software PrimerExploerV4 design PRRS to primer (FIP, BIP, F3, B3), it is noted that the setting of Tm value, the stability of primer termination, G/C content, secondary structure and the length of primer during design primer.The primer designed is synthesized by Shanghai biological engineering company limited.
2) purpose fragment obtains:
1. preparation LAMP reaction system: prepare 25 μ l reaction systems in PCR pipe, 10 × ThermopolReactionBuffer reaction buffer 2.5 μ L, 4 each 1 μ L of specific primer (FIP, BIP, F3, B3), the BstDNA polymerase 1 μ l of 8U/ μ l, template DNA 2 μ l, deionized water is added to cumulative volume 25 μ L.Using pig genomic DNA as positive control, with ddH2O is as negative control.Described reaction buffer includes 10mMdNTP, 10 × ThermoPol reaction buffer, 200mMTris-HCl (pH8.825 DEG C), 100mMKCl, 100mM (NH4)2SO4、20mMMgSO4, 1%TritonX-100.
2. LAMP reaction: above-mentioned PCR pipe is put in 62 DEG C of isothermal reaction 60min.
3. analytical reactions result: treat that LAMP reaction terminates, containing a large amount of purpose fragments in 0.25mleppendorf pipe.In eppendorf pipe, add 1 μ l nucleic acid dye 1000 × SYBRGreenI, if reactant liquor color is orange, represent that result is negative, if reactant liquor color is green, represent that result is positive.Specific detection:
Specific detection is done and using pig genomic DNA as positive control for template, with ddH with the DNA of PCV2, PCV1, PRV, PRRS of 105 copy/microlitres2O shows as negative control, result, presents green, and other template DNA reaction tubes are orange, show that the specific primer that the present invention designs has good specificity in experimentation in the reaction tube of pig PRRSDNA template and genomic DNA.
Sensitivity technique:
Extract PRRSDNA according to the extracting method of BloodViralDNA/RNAkit nucleic acid extraction reagent, and be diluted to do LAMP reaction with the PRRSDNA of 1010,109,108,107,106,105,104,103,102,101 copy/microlitres for template with 10 times of concentration series dilution methods.
LAMP reaction system and reaction condition and interpretation of result are with embodiment 1.
Testing result: except PCR pipe that DNA concentration is 101 copies/microlitre show orange in addition to, the PCR pipe of remaining concentration all presents green, shows that the lowest detection limit of the detection method of the present invention reaches 102 copies/microlitre DNA, sensitivity is the highest.
Adnexa 1 (ref sequence)
ATGGTGTCGAGCTTATGCATCCTCGATATTAGTATCAAGTGTCTTTTTTTGTCGTTCATTTTGTTGTGTTTTTTTTTTGTTTTTATGATTACATATTATATTAGGTTCTTTCGCTTCGGACTGGCATGGTTTTTTGAGCTCTTAGACATCAATTGGGTTTGTTTTCCTTTCCTGACCTCGGATGCTGCCCTGCATATCTTTCAAATCAGTAAGGCGCTATGTTGTGTGGTGTTTGTTGCCCTCTTCTGGGAGGAGGATTCTGCCAATTGAACCTTTTTAGTTTCTGTGTTCATCTGCTGCTTTGGCCTGTTGAGTTTTGTTTAGATTAGGGTGTCGGGCCTGATCGTCGTCATTGCGGCAGCGTCAGGTCTCGTTGC。

Claims (7)

1. the primer being used for detecting PRRS virus, it is characterised in that:
Described primer is the primer for PRRS virus conservative region, and its nucleotide sequence is as follows:
Outer primer F3:TCTTCTGGGAGGAGGATTC；
Outer primer B3:GAGGATGCATAAGCTCGA；
Inner primer FIP:TCTAAACAAAACTCAACAGGCCACCAATTGAACCTTTTTAGTTTCT；
Inner primer BIP:CCTGATCGTCGTCATTGCGGACTGTCTGTCGTATTATGTGTC.
2. the test kit being used for detecting PRRS virus, it is characterised in that: described test kit includes that DNA/RNA extracts primer, reaction buffer, BstDNA polymerase and the nucleic acid dye described in reagent, claim 1.
Test kit for detecting PRRS virus the most according to claim 2, it is characterised in that: described DNA/RNA extracts reagent and includes: BloodViralDNA/RNAkit, extracts pig blue-ear disease poison DNA or RNA in the cell cultivated.
Test kit for detecting PRRS virus the most according to claim 2, it is characterised in that: described reaction buffer includes 10mMdNTP, 10 × ThermoPol reaction buffer, 200mMTris-HCl, 100mMKCl, 100mM (NH4)2SO4、20mMMgSO4, 1%TritonX-100.
Test kit for detecting PRRS virus the most according to claim 2, it is characterised in that: the concentration of described BstDNA polymerase is 8U/ μ l.
Test kit for detecting PRRS virus the most according to claim 2, it is characterised in that: described nucleic acid dye is 1000 × SYBRGreenI.
7. one kind uses test kit described in any one of claim 2～6 for the method detecting PRRS virus, it is characterised in that: comprise the following steps:
Use DNA/RNA to extract reagent from the cell cultivated and extract viral DNA template, it is subsequently adding the described primer for PRRS virus conservative region, reaction buffer, BstDNA polymerase configuration LAMP reactant, carry out LAMP reaction, reaction adds nucleic acid dye after terminating, color according to reactant liquor differentiates, completes the detection to PRRS virus.
CN201610266369.6A 2016-04-26 2016-04-26 Primer, kit and method for detecting porcine reproductive and respiratory syndrome virus Pending CN105821161A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109355436A (en) * 2018-12-10 2019-02-19 绵阳师范学院 A kind of RT-LAMP Primer composition and its application detecting PRRSV

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101319253A (en) * 2008-07-18 2008-12-10 中国农业科学院兰州兽医研究所 Method for detecting high-pathogenicity blue ear disease
CN102277445A (en) * 2010-12-10 2011-12-14 中华人民共和国珠海出入境检验检疫局 PRRS RT-LAMP detection method and detection kit
CN103509880A (en) * 2013-10-15 2014-01-15 江苏省农业科学院 LAMP detection kit of highly-pathogenic porcine reproductive and respiratory syndrome viruses
CN104862418A (en) * 2015-05-29 2015-08-26 中国农业科学院兰州兽医研究所 Specific primers for detecting European-type porcine reproductive and respiratory syndrome viruses and corresponding detection kit

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101319253A (en) * 2008-07-18 2008-12-10 中国农业科学院兰州兽医研究所 Method for detecting high-pathogenicity blue ear disease
CN102277445A (en) * 2010-12-10 2011-12-14 中华人民共和国珠海出入境检验检疫局 PRRS RT-LAMP detection method and detection kit
CN103509880A (en) * 2013-10-15 2014-01-15 江苏省农业科学院 LAMP detection kit of highly-pathogenic porcine reproductive and respiratory syndrome viruses
CN104862418A (en) * 2015-05-29 2015-08-26 中国农业科学院兰州兽医研究所 Specific primers for detecting European-type porcine reproductive and respiratory syndrome viruses and corresponding detection kit

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109355436A (en) * 2018-12-10 2019-02-19 绵阳师范学院 A kind of RT-LAMP Primer composition and its application detecting PRRSV

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Application publication date: 20160803