CN109593893A - African hog cholera virus fluorescent PCR quick detection kit - Google Patents

African hog cholera virus fluorescent PCR quick detection kit Download PDF

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CN109593893A
CN109593893A CN201910108734.4A CN201910108734A CN109593893A CN 109593893 A CN109593893 A CN 109593893A CN 201910108734 A CN201910108734 A CN 201910108734A CN 109593893 A CN109593893 A CN 109593893A
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asfv
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赵林萍
娄亚坤
任宝红
杨楠
侯亚博
付燕峰
崔兴涛
张�杰
曾小宇
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Zhongbiao Testing Henan Service Ltd
ZHENGZHOU ZHONGDAO BIOLOGICAL TECHNOLOGY Co Ltd
Zhengzhou University
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Zhongbiao Testing Henan Service Ltd
ZHENGZHOU ZHONGDAO BIOLOGICAL TECHNOLOGY Co Ltd
Zhengzhou University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q1/686—Polymerase chain reaction [PCR]

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Abstract

The invention discloses a kind of African hog cholera virus fluorescent PCR quick detection kits, including specific primer ASFV-F and ASFV-R, and TaqMan probe ASFV-P, 5 ' end label reporter fluorescence dyestuffs of probe are FAM, and 3 ' end mark fluorescent quenching groups are BHQ-1.Kit provided by the invention can be used for the Nasal swabs of pig, blood, serum, blood plasma, the African swine fever virus detection in tissue, realize the quick detection of African swine fever virus.The present invention extracts from DNA during the entire process of detecting ASFV and testing result occurs, only need 40min, greatly shortens detection time, improves detection efficiency.

Description

African hog cholera virus fluorescent PCR quick detection kit
Technical field
The present invention relates to a kind of African hog cholera virus fluorescent PCR detection methods, specifically, being related to a kind of African swine fever virus The foundation of fluorescent PCR quick detection kit detection method and application method, belong to technical field of bioengineering.
Background technique
African swine fever (African swine fever, ASF) is domestic pig and the open country as caused by African swine fever virus (ASFV) A kind of strong, bleeding sexually transmitted disease of pig.Acute is died of illness characterized by high fever, reticuloendothelial system bleeding and high lethality rate Rate is close to 100%.World Organization for Animal Health (OIE) be classified as must notifiable a kind of epidemic disease, pig breeding industry is endangered larger. 2018 Liaoning Province, the China Nian8Yue report African swine fever epidemic situation for the first time, indicate that African swine fever is passed to China, cause to my pig breeding industry Massive losses, due to there is no effective vaccine control, so establishing one kind, fast and accurately detection method just seems especially heavy It wants.
African swine fever virus is the unique member of ASF sample Viraceae, and it is in 20 that the diameter of virion, which is 175~215 nanometers, Face body is symmetrical, there is cyst membrane, and genome is bifilar linear DNA, 170~190kb of size.The virus is mainly infection with macrophage Target cell, and with its virus protein that peculiar gene dosage is numerous, mechanism is complicated participate in ASFV absorption, invasion host cell, The functions such as host immune defenses system are escaped in virus replication and assembly, so that virus is more effectively infected host cell, are caused Acute, hot, the hemorrhagic clinical symptoms and pathological lesion of pig.In addition, there is complicated infection endless form in African swine fever, Subclinical infection pig and the swine products polluted by swine fever virus and its product are the weights that African swine fever introduces the regional swinery of health Communication media is wanted, which can also infect wild boar and soft ticks, can be between domestic pig and wild boar, between wild boar and soft ticks and domestic pig The circulating propagation between soft ticks.
Currently, ASF there is no effective vaccine prevention and treatment method, in a short time, by early diagnosis and the anti-keyholed back plate of compartmentalization Reason is to carry out anti-system, and using stringent monitoring plan, one it is found that take a wide range of mode of slaughtering to cut off the infection sources.OIE recommends The Serology tests such as HAD, ELISA, IFA, time-consuming, complicated for operation, is not suitable for the quick inspection with African swine fever virus It surveys.Real-time fluorescence quantitative PCR (Real-time Quantitative PCR qPCR) has easy to operate, quick and convenient, sensitive The advantage high, reproducible, pollution rate is low is spent, genetic test field is widely used in.
Summary of the invention
The purpose of the invention is to provide a kind of African swine fever fluorescent PCR quick detection kit, African swine fever is realized The quick detection of virus.The present invention extracts from DNA during the entire process of detecting ASFV and testing result occurs, only needs 40min greatly shortens detection time, improves detection efficiency.
To achieve the above object, the present invention provides a kind of African hog cholera virus fluorescent PCR quick detection kit, specific to walk It is rapid as follows:
(1) the p72 genome sequence for referring to 24 genotype Reference strains of African swine fever virus, screens conservative region, if Meter ASFV-F and ASFV-R is upstream and downstream primer, PCR amplification p72 gene；The reaction system of PCR amplification is 20 μ l, wherein 2 × Taq PCR Mix 10 μ l, primer ASFV-F (20 μM) and ASFV-R (20 μM) each 1 μ l, template 1 μ l, ddH27 μ l, PCR expansion of O The response procedures of increasing are 94 DEG C of initial denaturations 5min, 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 60s, 25 circulations, 72 DEG C of 10min；Wherein Primer ASFV-F and ASFV-R are as follows:
ASFV-F:5 '-ATCCGATCACATTACCTA-3 '；SEQ ID NO:1；
ASFV-R:5 '-GTGGTCTTCAAAGCAAAGG-3 '；SEQ ID NO:2；
(2) pEASY-T1-p72 carrier is constructed, sequencing is carried out to p72 gene；The correct bacterial strain of sequencing result expands Culture extracts plasmid, as positive control.
(3) using p72 gene as target gene, specific primer ASFV-F and ASFV-R and TaqMan probe ASFV- is designed 5 ' end label reporter fluorescence the dyestuffs of P, probe are FAM, and 3 ' end mark fluorescent quenching groups are BHQ-1.Wherein specific primer It is as follows with TaqMan probe sequence:
ASFV-F:5′-ATCCGATCACATTACCTA-3′；SEQ ID NO:3
ASFV-R:5′-GTGGTCTTCAAAGCAAAGG-3′；SEQ ID NO:4
ASFV-P:5′-FAM-TCCGTAACTGCTCATGGTATCAAT-3′-BHQ-1；SEQ ID NO:5
(4) pass through many experiments, optimize reaction condition, the best PCR reaction system of reagent are as follows: 2 × TransStart Probe qPCR SuperMix 12.5 μ l, 20 μm of ol/L ASFV-F/ASFV-R each 0.5 μ l, 20 μm of 0.4 μ of ol/L ASFV-P Then 1 μ l positive control/negative control is added in l, sterilizing distilled water is added to make volume to 25 μ l.The PCR reaction interval of the kit Sequence are as follows: UNG processing: 50 DEG C, 2min；Initial denaturation: 95 DEG C of 3min, 1 circulation；Pre- amplification: 95 DEG C of 8s, 55 DEG C of 8s, 1 circulation； PCR amplification: 95 DEG C of 8s；55 DEG C of 8s, 40 circulations, acquire fluorescence signal herein.
(5) another aspect of the present invention further relates to a kind of kit, and it includes the groups of primer pair as described above and probe Product is closed, it is also preferable to include one of sample Fluorescence PCR liquid, enzyme mixation, negative control and positive controls or a variety of. Preferably, the archaeal dna polymerase can be added in the fluorescence quantitative PCR reaction solution, and composition Fluorescence PCR liquid carries out integration packet Dress.It is highly preferred that the archaeal dna polymerase is Taq archaeal dna polymerase.Kit of the present invention also includes positive control And/or negative control；Positive control is pEASY-T1-p72 recombinant plasmid；The negative control is seedless sour water.The present invention mentions The kit of confession can be used in detecting African swine fever virus, can be used for the Nasal swabs of pig, blood, serum, blood plasma, in tissue African swine fever virus detection.
Positive control plasmid doubling dilution is 10 by 5.1 sensitivity verifying-1~10-6, detected, tested by method in (4) Demonstrate,prove the sensitivity of this kit.
5.2 specificity verifications hinder swine fever virus nucleic acid, foot and mouth disease virus nucleic acid, pig breeding with breathing by method in (4) Hinder syndrome virus nucleic acid, Pseudorabies virus nucleic acid to be detected, verifies the specificity of this kit.
The present invention chooses the highly conserved p72 gene of African swine fever virus (coding B646L structural proteins) and is used as target gene, Design specific primer and probe establish the special fluorescence PCR detecting method of ASFV.African swine fever fluorescent PCR of the invention is quick Detection kit is extracted from DNA during the entire process of detecting African swine fever and testing result occurs, then only needs 40min, significantly It reduces manual operations and shortens the time.And the present invention once can detect multiple samples, the detection particularly suitable for gross sample.
Specific embodiment
The present invention is described in further detail below, the given examples are served only to explain the present invention, is not intended to limit The scope of the present invention.
The building of 1 design of primers of embodiment and African swine fever virus pEASY-T1-p72 gene recombination plasmid
According to 24 genotype Reference strains p72 gene orders (being shown in Table 1) of the African swine fever virus included on GenBank, Screen conservative region, design primer and probe, primer ASFV-F ASFV-R and probe ASFV-P it is as follows:
ASFV-F:5 '-ATCCGATCACATTACCTA-3 ' SEQ ID NO:3
ASFV-R:5 '-GTGGTCTTCAAAGCAAAGG-3 ' SEQ ID NO:4
ASFV-P:5 '-FAM-TCCGTAACTGCTCATGGTATCAAT-3 '-BHQ-1SEQ ID NO:5.
1 African swine fever Reference strains of table
Strain name Genotype Genebank accession number Separately point
Kongo73 I KJ671545 The Congo
Georgia2007 II AM999764 Georgia
BOT/1/99 III AF504886 Mozambique
RSA/1/99/W IV AF449477 South Africa
Tengani V AF301541 Malawi
SPEC265 VI AF270710 Mozambique
RSA/1/98 VII AF302818 South Africa
Malawi/1978 VIII AF270707 Malawi
Ken07.Eld1 IX FJ154441 Spain
MWHOG/1 X AY351548 East Africa
KAB/62 XI AY351522 East Africa
MFUE6/1 XII AY351561 East Africa
SUM/1411 XIII AY351542 East Africa
NYA/12 XIV AY351555 East Africa
TAN/1/01 XV AY494552 East Africa
TAN/2003/1 XVI AY494550 East Africa
ZIM/92/1 XVII DQ250119 South Africa
NAM/1/95 XVIII DQ250122 Namibia
SPEC/125 XIX DQ250112.1 South Africa
RSA/1/95 XX DQ250123 South Africa
SPEC/53 XXI DQ250111 South Africa
SPEC/245 XXII DQ250117 South Africa
ETH/1 XXIII KT795354 Ethiopia
MOZ-10/2006 XXIV KY353989 Mozambique
Gene II type Georgia2007 strain (accession number: AM999764) is referred to according to the ASFV logged on GenBank P72 gene design primer, and using it as template PCR amplifications p72 gene；Wherein the reaction system of PCR amplification is 20 μ l, wherein 2 × Taq PCR Mix 10 μ l, primer ASFV-F (20 μM) and ASFV-R (20 μM) each 1 μ l, template 1 μ l, ddH2O 7μl；PCR Amplified reaction program: 94 DEG C of initial denaturations 5min, 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 60s, 25 circulations, 72 DEG C of 10min；Recycle mesh Gene, connect pEASY-T1 carrier, 10 μ l systems: target gene 4.5 μ l, pEASY-T1Vector 0.5 μ l, 2 × 5 μ l of Connect buffer, 16 DEG C of connections are overnight；Connection product is transformed into DH5 α competent cell；Picking single colonie carries out PCR detection carries out sequencing by Hua Da gene biological company, the Georgia2007 poison included on sequencing result and GenBank Strain (accession number: AM999764) sequence identity is up to 99% or more.
The foundation of 2 African hog cholera virus fluorescent PCR quick detection reagent reaction system of embodiment
The correct pEASY-T1-p72 bacterial strain of 1 sequencing result of embodiment is expanded into culture, extracts plasmid, and using it as template Carry out fluorescent PCR amplification.Reaction system: 2 × TransStart Probe qPCR SuperMix 12.5 μ l, 20 μm of ol/L Then 1 μ l pEASY-T1-p72 Plasmid DNA work is added in ASFV-F/ASFV-R each 0.5 μ l, 20 μm of 0.4 μ l of ol/L ASFV-P For template, sterilizing distilled water is added to make volume to 25 μ l.The PCR response procedures of the reagent are as follows: UNG processing: 50 DEG C, 2min；In advance Denaturation: 95 DEG C of 3min, 1 circulation；Pre- amplification: 95 DEG C of 8s, 55 DEG C of 8s, 1 circulation；PCR amplification: 95 DEG C of 8s；55 DEG C of 8s, 40 Circulation acquires fluorescence signal.
The foundation of 3 African hog cholera virus fluorescent PCR detection kit detection method of embodiment
(1) this kit reaction system: it is right that positive control/feminine gender is added in 17 μ l of PCR reaction solution, 3 μ l of enzyme mixation According to/5 μ l of measuring samples, total volume is 25 μ l.PCR response procedures are as follows: the PCR response procedures of the reagent are as follows: UNG processing: 50 DEG C, 2min；Initial denaturation: 95 DEG C of 3min, 1 circulation；Pre- amplification: 95 DEG C of 8s, 55 DEG C of 8s, 1 circulation；PCR amplification: 95 DEG C of 8s； 55 DEG C of 8s, 40 circulations, acquire fluorescence signal herein.
(2) judgement of this kit test result: a. is positive: there are obvious exponential increase in value≤35 detection result of specimen Ct Phase determines detection African swine fever virus nucleic acid.B. suspicious: detection result of specimen Ct value is in 35~38 ranges.Reply mark at this time This carries out repeating detection, if repeating experimental result Ct value still in 35~38 ranges, has obvious Exponential growth stage, is then determined as Otherwise the positive is feminine gender.C. negative: detection result of specimen Ct value > 38 or without Ct value determines that African swine fever virus core is not detected Acid.
The verifying of 4 kit sensitivity of embodiment
The concentration of recombinant plasmid pEASY-T1-p72, adjustment concentration to 10 are surveyed with ultraviolet absorption method6Copies/ μ l is used ddH2O carries out 10 times of doubling dilutions, dilution range 10 to positive plasmid-1~10-6, PCR detection is carried out respectively as template.It presses The reaction system reagent preparation and setting response procedures of kit.The results show that the minimum detectability of this kit is 10copies/μl。
The verifying of 5 kit specificity of embodiment
Extract swine fever virus nucleic acid, foot and mouth disease virus nucleic acid, pig breeding and respiratory disorder syndrome virus nucleic acid, pseudo- mad dog Viral nucleic acid, respectively template carry out PCR detection, by kit system reagent preparation and setting response procedures.The results show that inspection Surveying result is feminine gender, this kit specificity is good.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Claims (3)

1. a kind of African hog cholera virus fluorescent PCR quick detection kit, including specific primer ASFV-F and ASFV-R, and 5 ' end label reporter fluorescence the dyestuffs of TaqMan probe ASFV-P, probe are FAM, and 3 ' end mark fluorescent quenching groups are BHQ-1； Wherein specific primer and TaqMan probe sequence are as follows:
ASFV-F:5′-ATCCGATCACATTACCTA-3′；
ASFV-R:5′-GTGGTCTTCAAAGCAAAGG-3′；
ASFV-P:5′-FAM-TCCGTAACTGCTCATGGTATCAAT-3′-BHQ-1。
2. kit according to claim 1 further includes sample Fluorescence PCR liquid, enzyme mixation, negative control and sun Property control one of or it is a variety of.
3. a kind of preparation method of African hog cholera virus fluorescent PCR quick detection kit, the specific steps are as follows:
(1) the p72 genome sequence for referring to the multiple genotype Reference strains of African swine fever virus screens conservative region, design ASFV-F and ASFV-R is upstream and downstream primer, PCR amplification p72 gene；The reaction system of PCR amplification is 20 μ l, wherein 2 × Taq PCR Mix 10 μ l, primer ASFV-F (20 μM) and ASFV-R (20 μM) each 1 μ l, template 1 μ l, ddH27 μ l of O, PCR amplification Response procedures are 94 DEG C of initial denaturations 5min, 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 60s, 25 circulations, 72 DEG C of 10min；Wherein primer ASFV-F and ASFV-R are as follows:
ASFV-F:5 '-ATCCGATCACATTACCTA-3 '；SEQ ID NO:1；
ASFV-R:5 '-GTGGTCTTCAAAGCAAAGG-3 '；SEQ ID NO:2；
(2) pEASY-T1-p72 carrier is constructed, sequencing is carried out to p72 gene；The correct bacterial strain of sequencing result expands culture, Plasmid is extracted, as positive control；
(3) using p72 gene as target gene, specific primer ASFV-F and ASFV-R and TaqMan probe ASFV-P is designed, is visited 5 ' end label reporter fluorescence dyestuffs of needle are FAM, and 3 ' end mark fluorescent quenching groups are BHQ-1, wherein specific primer sequence Under entering:
ASFV-F:5′-ATCCGATCACATTACCTA-3′；SEQ ID NO:3
ASFV-R:5′-GTGGTCTTCAAAGCAAAGG-3′；SEQ ID NO:4
ASFV-P:5′-FAM-TCCGTAACTGCTCATGGTATCAAT-3′-BHQ-1；SEQ IDNO:5
(4) pass through many experiments, optimize reaction condition, the best PCR reaction system of reagent are as follows: 2 × TransStart Probe QPCR SuperMix 12.5 μ l, 20 μm of ol/L ASFV-F/ASFV-R each 0.5 μ l, 20 μm of 0.4 μ l of ol/L ASFV-P, then 1 μ l positive control/negative control is added, sterilizing distilled water is added to make volume to 25 μ l；
PCR response procedures are as follows: UNG processing: 50 DEG C, 2min；Initial denaturation: 95 DEG C of 3min, 1 circulation；Pre- amplification: 95 DEG C of 8s, 55 DEG C 8s, 1 circulation；PCR amplification: 95 DEG C of 8s；55 DEG C of 8s, 40 circulations, acquire fluorescence signal herein.
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