CN101220397A - Reagent kit special for testing high pathogenicity pig replication and syndrome virus variation strain - Google Patents
Reagent kit special for testing high pathogenicity pig replication and syndrome virus variation strain Download PDFInfo
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- CN101220397A CN101220397A CNA2008100140622A CN200810014062A CN101220397A CN 101220397 A CN101220397 A CN 101220397A CN A2008100140622 A CNA2008100140622 A CN A2008100140622A CN 200810014062 A CN200810014062 A CN 200810014062A CN 101220397 A CN101220397 A CN 101220397A
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Abstract
The invention provides a special kit for detecting highly pathogenic porcine reproductive and syndrome virus mutation strain. The highly pathogenic porcine reproductive and syndrome virus mutation strain is detected by self-designing specific primers PSX1/PSX2, extracting virus RNA and carrying out reverse transcription into cDNA and utilizing a PCR method; the size of the estimated amplification segment of an amplification mutation strain is 404bp and an amplification traditional PRRSV amplification segment is 494bp. Compared with the prior art, the special kit has strong specificity and sensitivity, and the coincidence rate can achieve more than 90 percent compared with a virus separation and IPMA method. The amplification results of the primers to hogcholera virus, pseudorabies virus and porcine parvovirus are negative, which can distinguish a NSP2 mutation strain and a traditional strain; the method can detect the PRRSV with 12.8ng/L, thus the invention can be widely used for the clinical detection of the highly pathogenic porcine reproductive and syndrome virus mutation strain.
Description
1, technical field
Patent of the present invention relates to the diagnostic techniques in Vet Biotechnology field, specifically is a kind of detection high-pathogenicity porcine reproductive and syndrome virus variation strain diagnostic method and dedicated kit.
2, background technology
China's outburst summer in 2006 pig " hyperpyrexia disease ", the Ministry of Agriculture has determined that the sick for this reason protopathy of high-pathogenicity porcine reproductive and syndrome virus variation strain is former at present.Done a large amount of work at high-pathogenicity porcine reproductive and the domestic a lot of scholars of syndrome virus variation strain.The method that detects high-pathogenicity porcine reproductive and syndrome virus variation strain at present mainly contains viral isolation identification, peroxidase monolayer assay (IPMA) and RT-PCR diagnostic method.Isolation identification and peroxidase monolayer assay (IPMA) technical requirements are higher, can only carry out in the laboratory, and the experimenter is had relatively high expectations, and are difficult to large-scale application and check in terrain.At the present situation of the domestic generally popular hyperpyrexia disease of present China, be badly in need of a kind of diagnostic method that can be applied to extensive terrain sample.
3, summary of the invention
The objective of the invention is ubiquity, bring the present situation of serious financial loss, a kind of diagnostic method that can be applied to extensive terrain sample is provided to pig industry at present China " hyperpyrexia disease ".
Patent of the present invention is achieved through the following technical solutions:
1.1 design of primers: according to high-pathogenicity porcine reproductive that takes place after 2006 and respiratory syndrome all is the characteristics that lack to occur in the Nsp2 zone, designed a pair of Auele Specific Primer, this primer can be included this deletion fragment, the amplification variant estimates that the amplified fragments size is 404bp, and the PRRSV amplification segment that increases traditional is 494bp.Auele Specific Primer is as follows:
PSX1：5’-TGGTCCTAACGGTTCGGAAGAAAC-3’
PSX2：5’-GTGAGCTGAGTATTTTGGGCGTGT-3’
1.2 the processing of pathological material of disease: pathological material of disease shredded in the mill that is put in sterilization grind,, get viral suspension-20 ℃ multigelation 3 times with 1: 4 times of dilution of aseptic cell culture fluid, the centrifugal 10min of 8000rpm, it is standby to get supernatant liquor-80 ℃ preservation.
1.3 the extraction of viral RNA
1. get lapping liquid 300 μ L and add 600 μ L RNAout, vibration mixing room temperature leaves standstill 5min;
2. add 200 μ L chloroforms and firmly put upside down, leave standstill 5min behind the centrifuge tube mixing, the centrifugal 10min of 12000rpm.
3. get supernatant, add 500 μ L Virahols, behind the vibration mixing, room temperature is placed 10min, the centrifugal 10min of 12000rpm.
4. supernatant discarded gently adds 1mL 75% ethanol (preparation of DEPC water) piping and druming and dissolves the centrifugal 5min of 7500rpm.
5. abandon supernatant, room temperature leaves standstill 5-15min, makes the RNA precipitation dry.
6. add the DEPC water dissolution RNA of 10 μ L, be used for reverse transcription.
1.4 reverse transcription
Get the RNA7 μ L of extraction, 70 ℃ of effects of Oligo (dT) 1 μ L 10min, the back adds 5 * buffer4 μ L, dNTP Mixture 3 μ L, Rnase Inhibitor 1 μ L, ddH on ice
2O 3 μ L, M-MLV 1 μ L, 42 ℃ of water-bath 1h.Carry out reverse transcription with downstream primer PSX2.With this synthetic cDNA is that template is carried out the PCR reaction.
Polymerase chain reaction 1.5 (PCR)
10 * reaction buffer, 5.0 μ L
dNTP(2.5pMol/μL) 4.0μL
Primer(+)(25pMol/μL) 1.0μL
Primer(-)(25pMol/μL) 1.0μL
MgCl
2(25mMol) 4.0μL
cDNA 5.0μL
Taq archaeal dna polymerase (5u/ μ L) 0.5 μ L
Aseptic H
2O adds to 50.0 μ L
Reaction conditions is: (1) 95 ℃ of 5min; (2) 94 ℃ of 1min, 54.5 ℃ of 1min, 72 ℃ of 1min, totally 28 circulations; (3) 72 ℃ of 5min.Pcr amplification product is in-20 ℃ of preservations.
1.6 electrophoresis: voltage 100V electrophoresis 40min, get the gel 15min that in containing the ethidium bromide of 10mg/mL (EB) aqueous solution, dyes, under dna gel imaging system instrument, observe and take pictures.
The present invention designs Auele Specific Primer voluntarily, extracting viral RNA and reverse transcription is cDNA, utilize PCR method to detect high-pathogenicity porcine reproductive and syndrome virus variation strain on this basis, and succeeded in developing dedicated kit on this basis, compared with prior art, this diagnostic method and dedicated kit have very strong specificity, susceptibility, separate with virus and to compare coincidence rate with the IPMA method and can reach more than 90%, can be widely used in the detection of clinical high-pathogenicity porcine reproductive and syndrome virus variation strain.
4, description of drawings
Fig. 1 is the RT-PCR detection figure of clinical sample.
M:DL2000 among the figure (2000,1000,750,500,250,100bp);
1-8: clinical sample, all the other are empty swimming lane.
5, embodiment:
1) primer
According to high-pathogenicity porcine reproductive that takes place after 2006 and respiratory syndrome all is the characteristics that lack to occur in the Nsp2 zone, designed a pair of Auele Specific Primer, this primer can be included this deletion fragment, the amplification variant estimates that the amplified fragments size is 404bp, and the PRRSV amplification segment that increases traditional is 494bp.Synthetic by TaKaRa Bioisystech Co., Ltd.
PSX1：5’-TGGTCCTAACGGTTCGGAAGAAAC-3’
PSX2：5’-GTGAGCTGAGTATTTTGGGCGTGT-3’
2) processing of pathological material of disease
Pathological material of disease shredded in the mill that is put in sterilization grind,, get viral suspension-20 ℃ multigelation 3 times with 1: 4 times of dilution of aseptic cell culture fluid, the centrifugal 10min of 8000rpm, it is standby to get supernatant liquor-80 ℃ preservation.
3) extraction of viral RNA
1. get lapping liquid 300L and add 600 μ L RNAout, vibration mixing room temperature leaves standstill 5min;
2. add 200 μ L chloroforms and firmly put upside down, leave standstill 5min behind the centrifuge tube mixing, the centrifugal 10min of 12000rpm.
3. get supernatant, add the 500L Virahol, behind the vibration mixing, room temperature is placed 10min, the centrifugal 10min of 12000rpm.
4. supernatant discarded gently adds 1mL 75% ethanol (preparation of DEPC water) piping and druming and dissolves the centrifugal 5min of 7500rpm.
5. abandon supernatant, room temperature leaves standstill 5-15min, makes the RNA precipitation dry.
6. add the DEPC water dissolution RNA of 10 μ L, be used for reverse transcription.
4) reverse transcription
Get the RNA7 μ L of extraction, 70 ℃ of effects of Oligo (dT) 1 μ L 10min, the back adds 5 * buffer4 μ L, dNTP Mixture 3 μ L, Rnase Inhibitor 1 μ L, ddH on ice
2O 3 μ L, M-MLV 1 μ L, 42 ℃ of water-bath 1h.Carry out reverse transcription with downstream primer PSX2.With this synthetic cDNA is that template is carried out the PCR reaction.
5) polymerase chain reaction (PCR)
10 * reaction buffer, 5.0 μ L
dNTP(2.5pMol/μL) 4.0μL
Primer(+)(25pMol/μL) 1.0μL
Primer(-)(25pMol/μL) 1.0μL
MgCl
2(25mMol) 4.0μL
cDNA 5.0μL
Taq archaeal dna polymerase (5u/ μ L) 0.5 μ L
Aseptic H
2O adds to 50.0 μ L
6) different primers adopt following temperature program(me) respectively:
PSX1/PSX2：
(1) 95 ℃ of 5min; (2) 94 ℃ of 1min, 54.5 ℃ of 1min, 72 ℃ of 1min, totally 28 circulations; (3) 72 ℃ of 5min pcr amplification products are in-20 ℃ of preservations.
Pestivirus suis, porcine pseudorabies virus, pig parvoviral negative control are established in test.
7) electrophoresis
Voltage 100V electrophoresis 40min gets the gel 15min that dyes in containing the ethidium bromide of 10mg/mL (EB) aqueous solution, observe under dna gel imaging system instrument and take pictures.
The result
1) specificity of RT-PCR
Utilize the synthetic primer that Pestivirus suis, porcine pseudorabies virus, pig parvoviral are not all amplified and expect band of the same size.
2) detection of pathological material of disease
The doubtful pathological material of disease for high-pathogenicity porcine reproductive and respiratory syndrome that the Shandong Province was gathered later in 2006 carries out the RT-PCR detection with the PSX1/PSX2 primer, and the result amplifies the variant specific fragment in pathological material of disease, Figure 1 shows that the detected result of part sample.
Claims (1)
1. one kind is detected high-pathogenicity porcine reproductive and syndrome virus variation strain dedicated kit, it is characterized in that being used for the Auele Specific Primer PSX1/PSX2 of the detection of clinical high-pathogenicity porcine reproductive and syndrome virus variation strain, specific amplification PRRSV NSP2 variant, the amplification variant estimates that the amplified fragments size is 404bp, the PRRSV amplification segment that increases traditional is 494bp, and preparation process is as follows:
1) primer of test kit employing is
PSX1：5’-TGGTCCTAACGGTTCGGAAGAAAC-3’
PSX2：5’-GTGAGCTGAGTATTTTGGGCGTGT-3’，
Annealing temperature is 54.5 ℃, adopts 28 circulations, and the amplification variant estimates that the amplified fragments size is 404bp, and the PRRSV amplification segment that increases traditional is 494bp;
2) processing of pathological material of disease: pathological material of disease shredded in the mill that is put in sterilization grind,, get viral suspension-20 ℃ multigelation 3 times with 1: 4 times of dilution of aseptic cell culture fluid, the centrifugal 10min of 8000rpm, it is standby to get supernatant liquor-80 ℃ preservation.
3) extraction of viral RNA
1. get lapping liquid 300 μ L and add 600 μ L RNAout, vibration mixing room temperature leaves standstill 5min;
2. add 200 μ L chloroforms and firmly put upside down, leave standstill 5min behind the centrifuge tube mixing, the centrifugal 10min of 12000rpm.
3. get supernatant, add 500 μ L Virahols, behind the vibration mixing, room temperature is placed 10min, the centrifugal 10min of 12000rpm.
4. supernatant discarded gently adds 1mL 75% ethanol (preparation of DEPC water) piping and druming and dissolves the centrifugal 5min of 7500rpm.
5. abandon supernatant, room temperature leaves standstill 5-15min, makes the RNA precipitation dry.
6. add the DEPC water dissolution RNA of 10 μ L, be used for reverse transcription.
4) reverse transcription
Get the RNA7 μ L of extraction, 70 ℃ of effects of Oligo (dT) 1 μ L 10min, the back adds 5 * buffer4 μ L, dNTP Mixture 3 μ L, Rnase Inhibitor 1 μ L, ddH on ice
2O 3 μ L, M-MLV 1 μ L, 42 ℃ of water-bath 1h.Carry out reverse transcription with downstream primer PSX2.With this synthetic eDNA is that template is carried out the PCR reaction.
5) polymerase chain reaction (PCR)
10 * reaction buffer, 5.0 μ L
dNTP(2.5pMol/μL) 4.0μL
Primer(+)(25pMol/μL) 1.0μL
Primer(-)(25pMol/μL) 1.0μL
MgCl
2(25mMol) 4.0μL
cDNA 5.0μL
Taq archaeal dna polymerase (5u/ μ L) 0.5 μ L
Aseptic H
2O adds to 50.0 μ L
6) reaction conditions is: (1) 95 ℃ of 5min; (2) 94 ℃ of 1min, 54.5 ℃ of 1min, 72 ℃ of 1min, (3) 72 ℃ of 5min of totally 28 circulations.Pcr amplification product is in-20 ℃ of preservations.1.6 electrophoresis: voltage 100V electrophoresis 40min, get the gel 15min that in containing the ethidium bromide of 10mg/mL (EB) aqueous solution, dyes, under dna gel imaging system instrument, observe and take pictures.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101818212A (en) * | 2010-04-14 | 2010-09-01 | 中国农业科学院哈尔滨兽医研究所 | Loop-mediated isothermal amplification kit for rapidly detecting porcine parvovirus |
| CN102212623A (en) * | 2011-06-02 | 2011-10-12 | 广东省农业科学院兽医研究所 | Two-color fluorescence quantitative polymerase chain reaction (PCR) combined detection method of swine fever virus and blue ear disease virus and kit thereof |
| CN102297969A (en) * | 2011-05-25 | 2011-12-28 | 福建省农业科学院畜牧兽医研究所 | Preparation method of immunofluorescent reagent for differential diagnosis of traditional American type and variant porcine reproductive and respiratory syndrome |
| CN102483411A (en) * | 2008-11-26 | 2012-05-30 | 南达科他州立大学 | Identification of porcine reproductive and respiratory syndrome virus |
| CN102621319A (en) * | 2012-03-16 | 2012-08-01 | 吉林大学 | Colloidal gold quick diagnosis test paper for distinguishing classic porcine reproductive and respiratory syndrome (PRRS) from highly pathogenic PRRS (HPRRS) |
| CN103333970A (en) * | 2013-05-21 | 2013-10-02 | 广西壮族自治区动物疫病预防控制中心 | Reverse transcription-polymerase chain reaction (RT-PCR) primers for detecting highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) viruses and HP-PRRS virus TJ strain and kit with the RT-PCR primers |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100439516C (en) * | 2007-03-14 | 2008-12-03 | 中国动物疫病预防控制中心 | Pig breeding and respiration syndrome virus ultrastrong variation strain RT-PCR reagent case |
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- 2008-01-23 CN CN2008100140622A patent/CN101220397B/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102483411A (en) * | 2008-11-26 | 2012-05-30 | 南达科他州立大学 | Identification of porcine reproductive and respiratory syndrome virus |
| CN101818212A (en) * | 2010-04-14 | 2010-09-01 | 中国农业科学院哈尔滨兽医研究所 | Loop-mediated isothermal amplification kit for rapidly detecting porcine parvovirus |
| CN101818212B (en) * | 2010-04-14 | 2012-09-26 | 中国农业科学院哈尔滨兽医研究所 | Loop-mediated isothermal amplification kit for rapidly detecting porcine parvovirus |
| CN102297969A (en) * | 2011-05-25 | 2011-12-28 | 福建省农业科学院畜牧兽医研究所 | Preparation method of immunofluorescent reagent for differential diagnosis of traditional American type and variant porcine reproductive and respiratory syndrome |
| CN102212623A (en) * | 2011-06-02 | 2011-10-12 | 广东省农业科学院兽医研究所 | Two-color fluorescence quantitative polymerase chain reaction (PCR) combined detection method of swine fever virus and blue ear disease virus and kit thereof |
| CN102212623B (en) * | 2011-06-02 | 2013-01-16 | 广东省农业科学院兽医研究所 | Two-color fluorescence quantitative polymerase chain reaction (PCR) combined detection method of swine fever virus and blue ear disease virus and kit thereof |
| CN102621319A (en) * | 2012-03-16 | 2012-08-01 | 吉林大学 | Colloidal gold quick diagnosis test paper for distinguishing classic porcine reproductive and respiratory syndrome (PRRS) from highly pathogenic PRRS (HPRRS) |
| CN102621319B (en) * | 2012-03-16 | 2014-06-04 | 吉林大学 | Colloidal gold quick diagnosis test paper for distinguishing classic porcine reproductive and respiratory syndrome (PRRS) from highly pathogenic PRRS (HPRRS) |
| CN103333970A (en) * | 2013-05-21 | 2013-10-02 | 广西壮族自治区动物疫病预防控制中心 | Reverse transcription-polymerase chain reaction (RT-PCR) primers for detecting highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) viruses and HP-PRRS virus TJ strain and kit with the RT-PCR primers |
| CN103333970B (en) * | 2013-05-21 | 2015-02-04 | 广西壮族自治区动物疫病预防控制中心 | Reverse transcription-polymerase chain reaction (RT-PCR) primers for detecting highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) viruses and HP-PRRS virus TJ strain and kit with the RT-PCR primers |
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| Publication number | Publication date |
|---|---|
| CN101220397B (en) | 2012-07-04 |
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