CN105671211A - Peste des petits ruminants virus (RRPV), Schmallenberg virus (SBV) and Kobuvirus (KBV) molecule differential diagnosis method and application thereof - Google Patents

Peste des petits ruminants virus (RRPV), Schmallenberg virus (SBV) and Kobuvirus (KBV) molecule differential diagnosis method and application thereof Download PDF

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CN105671211A
CN105671211A CN201610212457.8A CN201610212457A CN105671211A CN 105671211 A CN105671211 A CN 105671211A CN 201610212457 A CN201610212457 A CN 201610212457A CN 105671211 A CN105671211 A CN 105671211A
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virus
kbv
sbv
pprv
primer pair
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翟少伦
魏文康
吕殿红
温肖会
贾春玲
周秀蓉
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Institute of Animal Health of Guangdong Academy of Agricultural Sciences
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Institute of Animal Health of Guangdong Academy of Agricultural Sciences
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

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Abstract

The invention discloses a multiplex PCR (polymerase chain reaction) method for simultaneously differentiating and detecting Peste des petits ruminants virus, Schmallenberg virus and Kobuvirus. The multiplex PCR method primers comprise an RRPV primer pair, an SBV primer pair and a KBV primer pair. According to the method, different primer pairs are utilized to amplify viral nucleic acid segments with different sizes, thereby achieving the goal of differential diagnosis. The method can simultaneously detect and differentiate RRPV, SBV and KBV, has the advantages of high specificity, high sensitivity, time saving, labor saving and the like, and can be used for detecting clinical samples of flocks and herds.

Description

PPR virus, execute Maron shellfish lattice virus and storehouse cloth virus molecular diagnosis method and application thereof
Technical field
Patent of the present invention belongs to animal pathogenic technical field of microbial detection, specifically a kind of differentiate simultaneously PPR virus, execute Maron shellfish lattice virus and storehouse cloth virus multiple RT-PCR detection method.
Background technology
In recent years, the Ministry of Agriculture and each province and city agricultural sector have put into effect file and the measure of a series of promotion herbivorous stock raising development, and cattle and sheep industry obtains unprecedented development opportunity. But, some cattle and sheep exotic diseases, emerging infectious disease (such as PPR virus, executing Maron shellfish lattice virus and storehouse cloth virus) often cause cattle and sheep to fall ill in a large number, death, and plant's loss is serious.
PPR (Pestedespetitsruminants, PPR), have another name called and little ruminate the false Cattle plague of beast (pseudorinderpest), pneumoenteritis (pneumoenteritis), stomatitis pneumoenteritis compound disease (stomatitis-pneumoenteritiscomplex), it is by PPR virus (Pestedespetitsruminantsvirus, PPRV) a kind of acute viral infectious disease caused, main infection small ruminant, including sheep, goat, Babalus bubalis L., blue sheep, argali etc., with heating, stomatitis, diarrhoea, pneumonia is feature, case fatality rate is high. the country such as Africa, Asia, Europe is popular extensively, and impact is serious.
Execute Maron shellfish lattice sick (Schmallenbergdisease, SBD), by executing Maron shellfish lattice virus (Schmallenbergvirus, SBV) infection causes, clinical symptoms is heating, diarrhoea, weak etc., and milk production of cow reduces, and defect etc. occurs in newborn young stock. At present, this disease sweeps across whole Europe, portions of Africa and Asian countries and is also involved.
Storehouse cloth virus (Kobuvirus, KBV) it is Picornaviridae storehouse cloth Tobamovirus member, for without cyst membrane RNA viruses, storehouse cloth Tobamovirus is at least divided into 4 kinds: people Ku Bu virus (also referred to as Aichi virus), Niu Kubu virus, sheep storehouse cloth virus and pig storehouse cloth virus. At present, the clinical symptoms that cloth virus in storehouse causes is mainly diarrhoea. In world pop.
Currently, in the plant such as cattle and sheep, cause of disease mixed infection is serious. And existing detection method detection cause of disease is single, also existing wastes time and energy, can not differentiate PPR virus simultaneously, executes the shortcoming three kinds viral such as Maron shellfish lattice virus and storehouse cloth virus.
Summary of the invention
The purpose of this patent is in that to use the method such as molecular biology, bioinformatics to carry out design of primers, sequence alignment and reaction system optimization, build PPR virus, execute Maron shellfish lattice virus and storehouse cloth virus molecular diagnosis method, it is achieved PPR virus, execute the Maron shellfish lattice virus quick Detection results with storehouse cloth virus " the same three examine ". The method is PPR virus, executes Maron shellfish lattice virus and a kind of method for quick of storehouse cloth viral diagnosis offer, fill up the blank that three kinds of viruses can not diagnose simultaneously, provide certain technical support and help for the diagnosis of cattle and sheep epidemic disease, investigation, prevention and control and purification.
Patent of the present invention to solve the technical problem that and to be achieved through the following technical solutions:
The detection primer that the present invention relates to has 3 pairs, it is PPRV primer pair, SBV primer pair and KBV primer pair respectively, wherein the PPRV primer pair for PPRV detection includes two primers of PPRV-F and PPRV-R, and the SBV primer pair for SBV detection includes two primers of SBV-F and SBV-R and the KBV primer pair for KBV detection includes two primers of KBV-F and KBV-R.
Inventive feature also relates to following steps.
Configure 50 μ LPCR reaction systems: 2 × Buffer25 μ L, Mix1 μ L, PPRV-F, PPRV-R, SBV-F, each 0.5 μ L, the PPRV of SBV-R, KBV-F and KBV-R, SBV and KBV template 3 μ L, RNAse-freeH respectively2O12 μ L;
PCR response procedures: 50 DEG C of reverse transcription 30min; 95 DEG C of denaturation 5min; 95 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, amount to 35 circulations; 72 DEG C re-extend 10min;
Result is observed: takes 10 μ LPCR products and carries out 1.5% agarose gel electrophoresis analysis, observed result under gel imaging system.
Further, the method that the present invention sets up, it is characterised in that include primer of the present invention.
Further, primer of the present invention is used for detecting PPR virus, executes Maron shellfish lattice virus and storehouse cloth virus.
Described step is for detecting PPR virus, executing Simple infection or the mixed infection of Maron shellfish lattice virus and storehouse cloth virus.
Application of the present invention or detection method are used not only for the clinical sample detection of cattle and sheep plant, it is also possible to be applied to the ruminant sample detection in Urban Zoo, Safari Park.
The invention discloses and differentiate detection PPR virus simultaneously, execute Maron shellfish lattice virus and the molecular method of storehouse cloth virus, there is the advantages such as high specificity, sensitivity are high, time saving and energy saving. The method is PPR virus, executes a kind of method for quick of diagnosis offer of Maron shellfish lattice virus and storehouse cloth virus, fill up the blank that three kinds of viruses can not diagnose simultaneously, provide certain technical support and help for the diagnosis of cattle and sheep epidemic disease, investigation, prevention and control and purification.
Accompanying drawing explanation
Fig. 1 is multiplexed PCR amplification and specific test result result; M:DNAMarker2000; 1:PPRV positive plasmid; 2:SBV positive plasmid; 3:KBV positive plasmid; 4:PPRV+SBV positive plasmid; 5:PPRV+KBV positive plasmid; 6:KBV+SBV positive plasmid; 7:PPRV+KBV+SBV positive plasmid; 8: canine distemper virus; 9: Avian pneumo-encephalitis virus; 10: capripox virus; 11: sheep of virus; 12: foot and mouth disease virus; 13: bovine parainfluenza virus; 14: negative control
Fig. 2 multiplex PCR susceptibility results; M:DNAMarker2000; 1-12: 10-1-10-12Plasmid doubling dilution.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention will be further described, and its operating procedure can be clear and definite further.
Optimal enforcement example:
1 material and method
1.1PPRV, SBV and KBV positive recombiant plasmid, canine distemper virus, Avian pneumo-encephalitis virus, capripox virus, sheep of virus, foot and mouth disease virus, bovine parainfluenza virus strain are preserved by Institute of Animal Health,Guangdong Academy Of Agricultural Sciences's preparation.
1.2 main agents
DNA/RNA extracts test kit purchased from Axygen, One step RT-PCR reagent and DNAMarker2000 purchased from Takara company.
1.3 design of primers
Respectively according to the sequence of PPRV, SBV, KBV in current paper and GenBank, design three is to primer. Primer is synthesized by Jin Weizhi bio tech ltd, Suzhou. Primer sequence is in Table 1.
Table 1 multiple PCR primer sequence
Remarks: S=G/C; R=A/G
The extraction of 1.4RNA
Extract test kit description according to DNA/RNA and carry out viral DNA/RNA extraction.
1.5 multiplexed PCR amplification and specific test
With PPR virus, execute Maron shellfish lattice virus and (or) storehouse cloth virus-positive recombiant plasmid mixture be multiplex PCR template, set up multiple PCR method.Adopt 50 μ LPCR reaction systems: 2 × Buffer25 μ L, PCRMix1 μ L, PPRV-F, PPRV-R, SBV-F, each 0.5 μ L, the PPRV of SBV-R, KBV-F and KBV-R, SBV and KBV positive template 3 μ L, RNAse-freeH respectively2O12 μ L;
PCR response procedures: 50 DEG C of reverse transcription 30min; 95 DEG C of denaturation 5min; 95 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, amount to 35 circulations; 72 DEG C re-extend 10min;
Result is observed: takes 10 μ LPCR products and carries out 1.5% agarose gel electrophoresis analysis, observed result under gel imaging system.
The sensitivity tests of 1.6 multiplex PCRs
Measure PPR virus respectively, execute the template concentrations of Maron shellfish lattice virus and (or) storehouse cloth virus-positive recombiant plasmid, then carry out doubling dilution, measure the sensitivity of the method.
2 results
2.1 multiplexed PCR amplification and specific test
Adopt the multiplex PCR that optimized to PPR virus, execute that Maron shellfish lattice are viral and (or) storehouse cloth virus-positive recombiant plasmid and mixed liquor expand, result obtains size and is about 750bp, the fragment of 500bp and 200bp, matching with the fragment of sequencing result 766bp, 474bp and 206bp, result is as shown in Figure 1. And canine distemper virus, Avian pneumo-encephalitis virus, capripox virus, sheep of virus, foot and mouth disease virus, bovine parainfluenza virus strain etc. do not present band, it was shown that the method has good specificity (as shown in Figure 1).
The sensitivity tests of 2.3 multiplex PCRs
After testing, the sensitivity of the multiplex PCR after optimization respectively 9.268pg/ml (PPRV), 12.45pg/ml (SBV) and 16.21pg/ml (KBV) (as shown in Figure 2).
The detection of 2.4 clinical samples
The 52 parts of cattle and sheep diarrheic stools gathered are detected, wherein positive 2 parts of PPRV nucleic acid, positive 10 parts of KBV nucleic acid, do not detect SBV. Additionally, there is 1 part of PPRV and KBV mixed infection.

Claims (5)

1. a PPR virus, execute Maron shellfish lattice virus and storehouse cloth virus molecular diagnosis method, described method is PPR virus, executes Maron shellfish lattice virus and storehouse cloth virus One step RT-PCR, it is characterized in that: the Primer composition used includes PPRV primer pair, SBV primer pair and KBV primer pair, wherein PPRV primer pair includes two primers of PPRV-F and PPRV-R, and SBV primer pair includes two primers of SBV-F and SBV-R and KBV primer pair includes two primers of KBV-F and KBV-R.
2. PPR virus as claimed in claim 1 a kind of, execute Maron shellfish lattice virus and storehouse cloth virus molecular diagnosis method, described Primer composition particularly as follows:
PPRV-F:GGTACCATAGCATCACCGACTCSEQIDNO.1
PPRV-R:CCCTTGCTCCTAAGTTTTTTGTAATSEQIDNO.2
SBV-F:GGCTTACACTCCCATTGATTTCTSEQIDNO.3
SBV-R:ATTGACCGTCCATAACCTTTGATSEQIDNO.4
KBV-F:GGATTACAAGTGTTTTGATGCCASEQIDNO.5
KBV-R:TGATGGTGTTGATGATSGARGTRSEQIDNO.6.
3. PPR virus as claimed in claim 1 or 2 a kind of, execute Maron shellfish lattice virus and storehouse cloth virus molecular diagnosis method, described RT-PCR comprises the following steps: configure 50 μ LPCR reaction systems: 2 × Buffer25 μ L, Mix1 μ L, PPRV-F, PPRV-R, SBV-F, each 0.5 μ L of SBV-R, KBV-F and KBV-R, PPRV, SBV and KBV positive template is 3 μ L, RNAse-freeH respectively2O12 μ L;
Wherein, PPRV-F and PPRV-R is for the detection of PPR, and SBV-F and SBV-R is for executing the detection of Maron shellfish lattice virus, and KBV-F and KBV-R is for the detection of storehouse cloth virus;
PCR response procedures: 50 DEG C of reverse transcription 30min; 95 DEG C of denaturation 5min; 95 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, amount to 35 circulations; 72 DEG C re-extend 10min;
Result is observed: takes 10 μ LPCR products and carries out 1.5% agarose gel electrophoresis analysis, observed result under gel imaging system.
4. a PPR virus, execute the Primer composition used in Maron shellfish lattice virus and storehouse cloth virus molecular diagnosis method, it is characterized in that described Primer composition includes: PPRV primer pair, SBV primer pair and KBV primer pair, wherein PPRV primer pair includes two primers of PPRV-F and PPRV-R, and SBV primer pair includes two primers of SBV-F and SBV-R and KBV primer pair includes two primers of KBV-F and KBV-R.
5. Primer composition as claimed in claim 5, described Primer composition particularly as follows:
PPRV-F:GGTACCATAGCATCACCGACTCSEQIDNO.1
PPRV-R:CCCTTGCTCCTAAGTTTTTTGTAATSEQIDNO.2
SBV-F:GGCTTACACTCCCATTGATTTCTSEQIDNO.3
SBV-R:ATTGACCGTCCATAACCTTTGATSEQIDNO.4
KBV-F:GGATTACAAGTGTTTTGATGCCASEQIDNO.5
KBV-R:TGATGGTGTTGATGATSGARGTRSEQIDNO.6.
CN201610212457.8A 2016-04-07 2016-04-07 Peste des petits ruminants virus (RRPV), Schmallenberg virus (SBV) and Kobuvirus (KBV) molecule differential diagnosis method and application thereof Pending CN105671211A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013009084A2 (en) * 2011-07-12 2013-01-17 주식회사 파나진 Composition for simultaneously detecting mycobacterium tuberculosis and nontuberculous mycobacteria by means of real-time multiplex polymerase chain reaction comprising nested hybridization pna probe system having parallel binding structure, and method for detection using same

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013009084A2 (en) * 2011-07-12 2013-01-17 주식회사 파나진 Composition for simultaneously detecting mycobacterium tuberculosis and nontuberculous mycobacteria by means of real-time multiplex polymerase chain reaction comprising nested hybridization pna probe system having parallel binding structure, and method for detection using same

Non-Patent Citations (3)

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李小鹏等: "小反刍兽疫RT-PCR的鉴定及N基因序列分析", 《亚洲兽医病例研究》 *
王建昌等: "施马伦贝格病毒实时荧光RT-PCR检测方法的建立及应用", 《中国兽医学报》 *
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Application publication date: 20160615