WO2021164186A1 - 鉴别猪伪狂犬病毒变异毒株的pcr引物及方法 - Google Patents

鉴别猪伪狂犬病毒变异毒株的pcr引物及方法 Download PDF

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WO2021164186A1
WO2021164186A1 PCT/CN2020/100609 CN2020100609W WO2021164186A1 WO 2021164186 A1 WO2021164186 A1 WO 2021164186A1 CN 2020100609 W CN2020100609 W CN 2020100609W WO 2021164186 A1 WO2021164186 A1 WO 2021164186A1
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virus
pcr
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白娟
姜平
吕林
王先炜
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南京农业大学
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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  • the invention relates to the technical field of identification of biological strains, in particular to PCR primers and detection methods for identifying variant strains of porcine pseudorabies virus.
  • PRV Pseudorabies Virus
  • Herpesviridae family and alpha herpesvirus subfamily The genome is about 140kd and encodes 69 open reading frames.
  • the virus mainly causes abortion, stillbirth and respiratory symptoms in sows, and neurological symptoms and diarrhea in piglets, causing huge economic losses to the pig industry.
  • Many countries in Europe and America have eradicated the disease through immunization and purification technology.
  • the disease has also been effectively controlled in my country through the use of enhanced immunity and purification technologies in the past ten years.
  • the PRV vaccine immunized pig farms in my country broke out again.
  • the national standard method mainly targets the pseudorabies virus gD gene, which can specifically amplify a 220bp fragment. Because the gD gene is an important envelope protein in the process of pseudorabies virus infection, this gene exists in all types of pseudorabies viruses, including vaccine viruses and wild viruses (except for pseudorabies with gD deletion), so this method can It is used to identify whether the pseudorabies virus is infected or not, but it cannot distinguish between the vaccine virus and the wild strain.
  • the pseudorabies virus gE gene detection method is mainly used to distinguish vaccine viruses from wild virus strains.
  • Wild virus strains can amplify 632bp fragments, but because the vaccine virus lacks the gE gene, the corresponding fragments cannot be amplified. Because pig farms are immunized with Bartha-K61 and other vaccines at this stage, the national standard method is no longer suitable for identification, and the pseudorabies virus gE gene detection method for distinguishing vaccine viruses from wild viruses has been well applied, but due to pseudorabies The emergence of virulent mutant strains limits this method. This method cannot distinguish between variant strains and classic strains, and the distinction between variant strains and classic strains is very important in immunization and monitoring. Therefore, a method that can effectively distinguish between classic pseudorabies virus strains and variant viruses is needed. PCR diagnosis method for strains.
  • the technical scheme of the present invention includes the following contents:
  • a PCR primer set for identifying variant strains of porcine pseudorabies virus includes primers 1F-C, 1F-V, 1R, 2F, and 2R.
  • the sequence of each primer is as follows:
  • the application of the aforementioned PCR primer set in identifying variant strains of porcine pseudorabies virus includes the following steps: extracting virus DNA to be identified, and using primers 1F-C, 1F-V, and 1R in the PCR primer set to perform the first step Amplification, the two bands amplified are variant strains or HB98 vaccine strains, and the one band (178bp) amplified is the classic strain; two bands (178bp, 467bp) are amplified
  • the DNA is amplified in the second step using primers 2F and 2R in the PCR primer set.
  • the amplified product with a molecular weight of 211 bp is a variant strain
  • the amplified product with a molecular weight of 293 bp is a HB98 vaccine strain.
  • the variant strain has 21 more bases of sequence inserted than the classic strain.
  • a kit for identifying variant strains of porcine pseudorabies virus which contains the above PCR primer set.
  • a method to identify variant strains of porcine pseudorabies virus extract the virus DNA to be identified, use the primers 1F-C, 1F-V, 1R in the above PCR primer set for the first step to amplify, and amplify two bands
  • the one that is a mutant or HB98 vaccine strain, and the one that is amplified with 1 band is the classic strain; the two bands of viral DNA are amplified and then the primers 2F and 2R in the above PCR primer set are used for the second step.
  • the amplified product has a molecular weight of 211 bp as a variant strain, and the amplified product has a molecular weight of 293 bp as a HB98 vaccine strain.
  • reaction system for the first step amplification Taqmix 12.5 ⁇ l, distilled water 7.5 ⁇ l, DMSO1 ⁇ l, 10uM upstream primer 1F-C, downstream primer 1F-V, identification primer 1R each 1 ⁇ l, DNA template 1 ⁇ l.
  • the reaction conditions for the first amplification step are: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30s, 61°C annealing for 30s, 72°C extension for 30s, 30 cycles, and finally 72°C for final extension for 10 minutes.
  • the reaction system for the second step amplification Taqmix 12.5 ⁇ l, distilled water 8.5 ⁇ l, DMSO 1 ⁇ l, 10uM upstream primer F'and downstream primer R'are respectively 1 ⁇ l and 1 ⁇ l, DNA template 1 ⁇ l.
  • the reaction conditions for the second amplification step are: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30s, 62°C annealing for 30s, 72°C extension for 30s, a total of 30 cycles; finally 72°C for final extension for 10 minutes.
  • the sensitivity of the first amplification 10TCID 50 reaches the amount of virus or 0.01ng / ml plasmid amount; second amplification sensitivity 10TCID 50 reaches the amount of virus or 0.1ng / ml plasmid amount.
  • the PCR identification method of the present invention has an accuracy rate of 90% to 100%.
  • the principle to distinguish between classic strains and variant strains is: in the first PCR, if the amplified product has only one band of 178bp, it is a classic PRV strain, and if the amplified product contains two bands of 467bp and 178bp, it is a mutation. Strain or HB98 vaccine strain. If two bands appear, the second step of PCR is required. If the size of the amplified product of the second PCR is 211bp, it is a variant strain, and if the size of the amplified product is 293bp, it is the HB98 vaccine strain. The judgment method is simple and easy to implement.
  • the sensitivity of the first step PCR can reach 10TCID 50 virus amount or 0.01ng/ml plasmid amount, and the second step PCR sensitivity can reach 10TCID 50 virus amount or 0.1ng/ml plasmid amount. This sensitivity can effectively detect whether there is pseudorabies virus infection in the clinical material, and distinguish the classic strain or variant strain of pseudorabies virus;
  • Figure 1 shows the amplification of the UL44 segment of the classic strain and the variant strain.
  • 1F-C1 primer M, DL2000 DNA marker; 1, variant strain ZJ01; 2, classic vaccine strain HB98; 3, classic wild strain LA; 4, classic vaccine strain Bartha-K61; 5 classic vaccine strain Bucharest ; 6, negative control.
  • 1F-C2 primer M, DL2000 DNA marker; 1, classic wild strain LA variant strain ZJ01; 2, classic vaccine strain Bartha-K61; 3, variant strain ZJ01; 4, classic vaccine strain HB98; 5, Negative control
  • Figure 2 is the distinction between HB98 and pseudorabies mutant strain ZJ01, M, DL2000 DNA marker; 1, negative control; 2, ZJ01 strain; 3, HB98 strain;
  • Figure 3 is the first step of PCR specificity test, M, DL2000 DNA Marker; 1, porcine circovirus type 2; 2, porcine reproductive and respiratory syndrome virus; 3, swine fever virus; 4, porcine Japanese encephalitis Virus; 5, porcine encephalomyocarditis virus; 6, porcine epidemic diarrhea virus; 7, porcine Seneca virus; 8, negative control; 9, porcine pseudorabies virus ZJ01 strain; 10, porcine pseudorabies virus LA strain;
  • Figure 4 The second step of PCR specificity test, M, DL2000 DNA Marker; 1, porcine circovirus type 2; 2, porcine reproductive and respiratory syndrome virus; 3, swine fever virus; 4, porcine Japanese encephalitis virus 5, porcine encephalomyocarditis virus; 6, porcine epidemic diarrhea virus; 7, porcine Seneca virus; 8. negative control; 9, porcine pseudorabies virus ZJ01 strain; 10, porcine pseudorabies virus HB98 strain;
  • Figure 5 The first step of PCR to distinguish the susceptibility test of PRV classic strain (LA strain) and variant strain (ZJ01 strain).
  • M DL5000 DNA marker; 1,100000TCID 50 ; 2,10000TCID 50 ; 3,1000TCID 50 ; 4,100TCID 50 ; 5,10TCID 50 ; 6, negative control;
  • Figure 6 The second step PCR method to identify the sensitivity test of PRV variant strain and HB98, M, DL5000; 1,100000TCID 50 ; 2,10000TCID 50 ; 3,1000TCID 50 ; 4,100TCID 50 ; 5,10TCID 50 ; 6, negative Control
  • Figure 7 DNA sensitivity test of recombinant plasmid containing UL44 gene, M, DL5000 DNA marker; 1,100000ng/ml; 2,10000ng/ml; 3,1000ng/ml; 4,100ng/ml; 5,10ng/ml; 6 , 1ng/ml; 7, 0.1ng/ml; 8, 0.01ng/ml; 9, negative control;
  • Figure 8 DNA sensitivity test of recombinant plasmid containing UL36 gene, M, DL5000 DNA marker; 1,100000ng/ml; 2,10000ng/ml; 3,1000ng/ml; 4,100ng/ml; 5,10ng/ml; 6 , 1ng/ml; 7, 0.1ng/ml; 8, 0.01ng/ml; 9, negative control.
  • TaqMix was purchased from Novartis, and DMSO was purchased from Sigma.
  • the primers were synthesized from Nanjing GenScript Company.
  • the PCR instrument was purchased from eppendorf company.
  • the viral DNA extraction kit was purchased from Omega Company.
  • Virus PRV variant strain ZJ01 strain (isolated and identified in 2011), PRV traditional strain LA strain (provided by researcher Fan Weixing, China Animal Disease Health Center) (Dong et al., Virology 2018), HB98 vaccine strain ( Wuhan Keqian Company PRV commercial vaccine), Bartha-K61 strain (Meria Company PRV commercial vaccine), Bucharest strain (Suo Teng Company PRV commercial vaccine).
  • Clinical material 30 samples of brain or lung tissue suspension from diseased pigs in East China, stored at -20°C for later use.
  • the mutant and HB98 strains are expected to amplify two bands (467bp, 178bp), and the classical strain is expected to amplify 1 band (178bp).
  • the second step of PCR uses primers 2F and 2R.
  • the molecular weights of the amplified products of the mutant strain and the HB98 vaccine strain were 211bp and 293bp, respectively.
  • the primers were synthesized by Nanjing GenScript Company.
  • the 2R primer is a perfect match with the HB98 strain (EA strain), but there are some base mismatches with the 5'end of the ZJ01 strain, which does not affect the amplification efficiency.
  • the first PCR PCR reaction system: Taqmix 12.5 ⁇ l, distilled water 7.5 ⁇ l, DMSO ⁇ l, 10uM upstream primer 1F-C, downstream primer 1F-V, identification primer 1R each 1 ⁇ l, DNA template 1 ⁇ l.
  • PCR reaction conditions pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 61°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles, and final extension at 72°C for 10 minutes. Take 10ul of the PCR product in a 1.5% agarose gel for electrophoresis identification.
  • 1F-C1 set of primers ZJ01 mutant strain amplified two bands with molecular weights of approximately 835bp and 467bp.
  • the classic strain LA, classic vaccine strains Bucharest and Bartha-K61 all amplified only one band, the size of which It is about 813bp, which is consistent with the prediction.
  • two bands were also amplified in the classic vaccine strain HB98, and the molecular weight was consistent with the amplification result of the ZJ01 mutant strain.
  • the PCR products were cloned into the pMD19-T vector, and the gene sequencing results were correct.
  • the amplified band of the classic strain LA was 783 bp
  • the amplified band of the classic strain Bucharest and Bartha-K61 vaccine strains was 813 bp, which was consistent with the design.
  • 1F-C2 sets of primers ZJ01 mutant strain amplified two bands with molecular weights of about 178bp and 467bp.
  • LA classic strain and Bartha-K61 vaccine strain both amplified only one band with a size of about 178bp. Consistent with the forecast.
  • two bands were also amplified in the classic vaccine strain HB98, and the molecular weight was consistent with the amplification result of the ZJ01 mutant strain.
  • the PCR products were cloned into the pMD19-T vector, and the gene sequencing results were correct.
  • the 1F-C2 set of primers have clearer bands, and the distinction between mutant strains and classic strains is better. Therefore, the 1F-C2 set of primers was selected as the first step PCR of this method for all the following experimental studies.
  • Second step PCR PCR reaction system: Taqmix 12.5 ⁇ l, distilled water 8.5 ⁇ l, DMSO 1 ⁇ l, 10uM upstream primer F', downstream primer R'each 1 ⁇ l, DNA template 1 ⁇ l.
  • PCR reaction conditions 95°C pre-denaturation 5min; 95°C denaturation 30s, 62°C annealing 30s, 72°C extension 30s, a total of 30 cycles; finally 72°C final extension 10min. Take 10ul of the PCR product in a 1.5% agarose gel for electrophoresis identification.
  • PCV-2 porcine circovirus type 2
  • PRRSV porcine reproductive and respiratory syndrome virus
  • CSFV swine fever virus
  • EMCV encephalomyocarditis virus
  • PEDV porcine epidemic diarrhea virus
  • JEV Japanese encephalitis virus
  • SVV swine Seneca virus
  • the first step of PCR the PRV ZJ01 strain and LA strain were respectively inoculated on BHK21 cells, and the virus was collected after the lesions completely appeared, and the infection of half of the tissue cells (TCID 50 ) was determined. Dilute the virus solution of the two virus strains into 40 TCID 50 , 400 TCID 50 , 4 ⁇ 10 3 TCID 50, 4 ⁇ 10 4 TCID 50 and 4 ⁇ 10 5 TCID 50 viruses per ml, and take 250 ⁇ l through Omega’s virus DNA extraction kit After extraction, the template is obtained, and 30 ⁇ l of DNA is extracted for each dilution. It was found by PCR that the sensitivity of this method was 10TCID 50 (see Figure 5).
  • the second step of PCR Dilute the stock solution of pseudorabies virus HB98 strain into a virus solution containing 40TCID 50 , 400 TCID 50 , 4 ⁇ 10 3 TCID 50 , 4 ⁇ 10 4 TCID 50 , 4 ⁇ 10 5 TCID 50 per ml, and take 250 ⁇ l After extraction with Omega kit, 30 ⁇ l of template DNA was obtained. It was found through PCR that the sensitivity of this method can reach 10TCID 50 (see Figure 6).
  • the first step of PCR Take the pMD19T-ZJ01/UL44, pMD19T-LA/UL44 recombinant plasmid DNA containing UL44 gene as a template, and perform PCR after 10-fold dilution to determine the sensitivity of the PCR method.
  • the sensitivity can reach 0.01ng/ ml (see Figure 7).
  • the second step of PCR Take the pMD19T-ZJ01/UL36, pMD19T-HB98/UL36 recombinant plasmid DNA containing UL36 gene as a template, and perform PCR after 10-fold dilution to determine the sensitivity of the PCR method.
  • the sensitivity can reach 0.1ng/ ml (see Figure 8).
  • PCR primer sequence is: 3F: 5'-TCCACTCGCAGCTCTTCT-3' 3R: 5'-GCACGTCATCACGAAGGA-3'.
  • PCR reaction system Taqmix 12.5 ⁇ l, distilled water 8.5 ⁇ l, DMSO1 ⁇ l, 10uM upstream primer F (gD), downstream primer R (gD) are 1 ⁇ l, 1 ⁇ l, DNA template 1 ⁇ l.
  • PCR reaction conditions 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30s, 61°C annealing for 30s, 72°C extension for 45s, a total of 35 cycles; finally 72°C for final extension for 10 minutes.
  • PCR primer sequence is: 4F: 5'-CAGGAGGACGAGCTGGGGCT-3' 4R: 5'-GTCCACGCCCCGCTTGAAGCT-3'.
  • PCR reaction system Taqmix 12.5 ⁇ l, distilled water 8.5 ⁇ l, DMSO1 ⁇ l, 10uM upstream primer F (gD), downstream primer R (gD) are 1 ⁇ l, 1 ⁇ l, DNA template 1 ⁇ l.
  • PCR reaction conditions 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30s, 65°C annealing for 30s, 72°C extension for 30s, a total of 35 cycles; finally 72°C for final extension for 10 minutes.
  • the positive rate was 55.17% (32/58), of which 32 were variant strains and 0 were classic strains; the PCR method for pseudorabies virus gE gene detection showed a positive rate of 55.17% (32/58). /58); PRV national standard method detection, the positive rate is 50.0 (29/58). The coincidence rate of this method with the gE gene PCR method and the national standard method reached 100% and 94.8%.
  • the first step PCR amplified bands around 178bp and 467bp, and the second step PCR amplified bands around 211bp. The results of the three times were consistent, indicating that the tested sample was a variant strain of pseudorabies virus.

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Abstract

本发明公开了鉴别猪伪狂犬病毒变异毒株的PCR引物及方法，提取待鉴别病毒DNA，采用本发明设计的引物进行第一步扩增，扩增出两条条带的为变异株或HB98疫苗毒株，扩增出1条条带的为经典毒株；扩增出两条条带的病毒DNA再进行第二步扩增，扩增产物分子量大小为211bp的为变异株,扩增产物分子量大小为293bp的为HB98疫苗毒株。第一步PCR的敏感度可以达到10TCID50病毒量或0.01ng/ml质粒量，而第二步PCR敏感度达到10TCID50病毒量或者0.1ng/ml质粒量。该敏感度可以有效的检测出临床病料中是否有伪狂犬病毒感染，并区分出伪狂犬病毒经典毒株或者变异毒株感染。

Description

鉴别猪伪狂犬病毒变异毒株的PCR引物及方法 技术领域
本发明涉及生物毒株鉴别技术领域，特别涉及鉴别猪伪狂犬病毒变异毒株的PCR引物及检测方法。
背景技术
伪狂犬病毒(Pseudorabies Virus,PRV)是疱疹病毒科，α疱疹病毒亚科的双股DNA病毒，基因组在140kd左右，编码69个开放阅读框。该病毒主要引起母猪流产、死胎及呼吸系统症状，仔猪出现神经症状和腹泻，给养猪业造成了巨大的经济损失。目前，欧美洲很多国家通过免疫接种和净化技术已经根除该病。我国近十多年内通过采用强化免疫和净化技术，该病也得到有效控制。但是，2011年末，我国PRV疫苗免疫猪场再次暴发该病。全基因组分析结果也表明，此次新发的伪狂犬病病毒与之前的经典伪狂犬病毒属于不同的亚群，其抗原性发生了变化，变异毒株位于一个新的基因分支。因此建立PRV变异毒株和经典毒株鉴别方法十分必要。
目前现有的检测伪狂犬病毒感染的PCR方法有很多，其中最常用的两种是国标法与伪狂犬gE基因检测法。国标法主要针对伪狂犬病毒gD基因，可以特异的扩增出220bp的片段。因为gD基因是伪狂犬病毒感染过程中一个重要的囊膜蛋白，包括疫苗毒与野毒在内的各种类型的伪狂犬病毒(gD缺失的伪狂犬病毒除外)都存在该基因，所以该方法可以用于鉴别伪狂犬病毒的感染与否，但不能够区分疫苗毒与野毒株。而伪狂犬病毒gE基因检测法主要用于鉴别疫苗毒与野毒株，野毒株可以扩增出632bp的片段，而疫苗毒由于缺失了gE基因，所以不能扩增出相应的片段。因为现阶段猪场都有Bartha-K61等疫苗免疫，所以国标方法不再适合进行鉴别，而用于区分疫苗毒与野毒的伪狂犬病毒gE基因检测法得到了很好的应用，但是由于伪狂犬病毒变异毒株的出现，使这一方法受到了局限。该方法不能够区分出变异毒株与经典毒株，而变异毒株与经典毒株的区分在免疫与监测上又至关重要，所以需要一种可以有效区分伪狂犬病毒经典毒株与变异毒株的PCR诊断方法。
发明内容
为了有效的预控伪狂犬病的发生，区分出经典毒株与变异毒株的感染及其流行的区域至关重要。本研究通过不同毒力毒株全基因组序列比对，找到了经典毒株与变异毒株的基因差 异区域UL44和UL36，设计PCR引物，建立了两步法PCR，具有较高特异性和敏感性，可用于鉴别PRV经典毒株与变异毒株，为该病诊断提供了有效方法。
本发明的技术方案包括以下内容：
用于鉴别猪伪狂犬病毒变异毒株的PCR引物组，该PCR引物组包括引物1F-C、1F-V、1R、2F和2R，其中各引物的序列如下：
1F-C：5’-ACGCCGACCCCGAGTACTTTGACG-3’，
1F-V：5’-GAGCCCGTCTCGGGGACGAC-3’，
1R：5’-GGCCACGCGCACGGACACC-3’，
2F:5’-GATGCGGTCACCGTCGGGTTT-3’，
2R:5’-CCGCCGCTCAGCCCCCATCGT-3’。
上述的PCR引物组在鉴别猪伪狂犬病毒变异毒株中的应用，包括以下步骤：提取待鉴别病毒DNA，使用所述PCR引物组中的引物1F-C、1F-V、1R进行第一步扩增，扩增出两条条带的为变异株或HB98疫苗毒株，扩增出1条条带(178bp)的为经典毒株；扩增出两条条带(178bp、467bp)的病毒DNA再使用所述PCR引物组中的引物2F和2R进行第二步扩增，扩增产物分子量大小为211bp的为变异株，扩增产物分子量大小为293bp的为HB98疫苗毒株。
通过对中国新流行伪狂犬病毒变异毒株与国内外经典毒株全基因组序列比对发现，在UL44区域内，变异毒株比经典毒株多插入了21个碱基的序列。鉴此，首先设计了3条引物的PCR方法，一条下游引物，两条上游引物。理论上，经典毒株只能与一条上游引物和下游引物结合，产生一条约178bp的条带；而变异毒株(包括HB98毒株，序列近似EA毒株)可以与两条上游引物和下游引物结合，产生一条约467bp的条带和一条178bp左右的条带。由此可以区分出伪狂犬病毒经典毒株与变异毒株。但是，我们在检测疫苗毒株时发现，HB98毒株也出现两条条带，基因序列测定结果显示，HB98毒株UL44也存在着21个碱基的插入。我们在UL36区域内又设计了一对引物，通过第二次PCR，HB98扩增出293bp条带，变异毒株扩增出211bp的条带，从而可以有效区分HB98与变异毒株，结果证明，通过以上两次PCR可以有效地区分伪狂犬变异毒株与经典毒株。
上述的PCR引物组在制备用于鉴别猪伪狂犬病毒变异毒株的试剂盒中的应用。
一种用于鉴别猪伪狂犬病毒变异毒株的试剂盒，该试剂盒中包含上述的PCR引物组。
一种鉴别猪伪狂犬病毒变异毒株的方法，提取待鉴别病毒DNA，使用上述PCR引物组中 的引物1F-C、1F-V、1R进行第一步扩增，扩增出两条条带的为变异株或HB98疫苗毒株，扩增出1条条带的为经典毒株；扩增出两条条带的病毒DNA再使用上述PCR引物组中的引物2F和2R进行第二步扩增，扩增产物分子量大小为211bp的为变异株，扩增产物分子量大小为293bp的为HB98疫苗毒株。
上述的方法，优选的，第一步扩增的反应体系：Taqmix 12.5μl，蒸馏水7.5μl，DMSO1μl，10uM的上游引物1F-C、下游引物1F-V、鉴别引物1R各1μl，DNA模板1μl。
上述的方法，优选的，第一步扩增的反应条件：95℃预变性5min；95℃变性30s，61℃退火30s，72℃延伸30s，30个循环，最后72℃终延伸10min。
上述的方法，优选的，第二步扩增的反应体系：Taqmix 12.5μl，蒸馏水8.5μl，DMSO1μl，10uM上游引物F’、下游引物R’分别为1μl、1μl，DNA模板1μl。
上述的方法，优选的，第二步扩增的反应条件：95℃预变性5min；95℃变性30s，62℃退火30s，72℃延伸30s，共30个循环；最后72℃终延伸10min。
上述的方法，第一次扩增的敏感度达到10TCID 50病毒量或0.01ng/ml质粒量；第二次扩增的敏感度达到10TCID 50病毒量或者0.1ng/ml质粒量。
本发明PCR鉴别方法，准确率达到90％到100％。
区分经典毒株与变异毒株的原则是：第一次PCR，如果扩增产物只有178bp一个条带，则为经典PRV毒株，如果扩增产物含有467bp和178bp两个条带，则为变异毒株或HB98疫苗毒株。如果出现两个条带，则需要进行第二步PCR。如果第二次PCR扩增产物大小为211bp，则为变异毒株，如果扩增产物大小为293bp，则为HB98疫苗毒株。该判断方法简便易行。
本发明的有益效果：
1)第一步PCR的敏感度可以达到10TCID 50病毒量或0.01ng/ml质粒量，而第二步PCR敏感度达到10TCID 50病毒量或者0.1ng/ml质粒量。该敏感度可以有效的检测出临床病料中是否有伪狂犬病毒感染，并区分出伪狂犬病毒经典毒株或者变异毒株感染；
2)通过与国标检测方法和常规的PCR方法进行比较发现，在30份病料中，国标方法可以检测出21份阳性，常规PCR方法可以检测出19份阳性，本方法可以检测出21份阳性，其中20份属于变异毒株，与国标方法符合率为100％，且很好的区分出变异毒株与经典毒株。所以该方法可以广泛用于临床病原的检测、监控与流行病学调查。
附图说明
图1为经典毒株与变异毒株UL44区段的扩增。1F-C1引物：M，DL2000 DNA marker；1，变异毒株ZJ01；2，经典疫苗毒株HB98；3，经典野毒株LA；4，经典疫苗毒株Bartha-K61；5经典疫苗毒株Bucharest；6，阴性对照。1F-C2引物：M，DL2000 DNA marker；1，经典野毒株LA变异毒株ZJ01；2，经典疫苗毒株Bartha-K61；3，变异毒株ZJ01；4，经典疫苗毒株HB98；5，阴性对照；
图2为HB98与伪狂犬变异毒株ZJ01的区分，M，DL2000 DNA marker；1，阴性对照；2，ZJ01株；3，HB98株；
图3为第一步PCR特异性试验，M，DL2000 DNA Marker；1，猪圆环病毒2型；2，猪繁殖与呼吸综合征病毒；3，猪瘟病毒；4，猪日本乙型脑炎病毒；5，猪脑心肌炎病毒；6，猪流行性腹泻病毒；7，猪塞尼卡病毒；8，阴性对照；9，猪伪狂犬病毒ZJ01株；10，猪伪狂犬病毒LA株；
图4第二步PCR特异性试验，M，DL2000 DNA Marker；1，猪圆环病毒2型；2，猪繁殖与呼吸综合征病毒；3，猪瘟病毒；4，猪日本乙型脑炎病毒；5，猪脑心肌炎病毒；6，猪流行性腹泻病毒；7，猪塞尼卡病毒；8.阴性对照；9，猪伪狂犬病毒ZJ01株；10，猪伪狂犬病毒HB98株；
图5第一步PCR鉴别PRV经典毒株(LA株)与变异毒株(ZJ01株)的敏感性试验。M：DL5000 DNA marker；1，100000TCID 50；2，10000TCID 50；3，1000TCID 50；4，100TCID 50；5，10TCID 50；6，阴性对照；
图6第二步PCR方法鉴别PRV变异株和HB98的敏感性试验，M，DL5000；1，100000TCID 50；2，10000TCID 50；3，1000TCID 50；4，100TCID 50；5，10TCID 50；6，阴性对照；
图7含UL44基因的重组质粒DNA敏感性试验，M，DL5000 DNA marker；1，100000ng/ml；2，10000ng/ml；3，1000ng/ml；4，100ng/ml；5，10ng/ml；6，1ng/ml；7，0.1ng/ml；8，0.01ng/ml；9，阴性对照；
图8含UL36基因的重组质粒DNA敏感性试验，M，DL5000 DNA marker；1，100000ng/ml；2，10000ng/ml；3，1000ng/ml；4，100ng/ml；5，10ng/ml；6，1ng/ml；7，0.1ng/ml；8，0.01ng/ml；9，阴性对照。
具体实施方式
下面结合具体实施例对本发明进行进一步说明：
实施例1
1材料和方法
1.1试剂和仪器
TaqMix购自诺唯赞公司，DMSO购自Sigma公司。引物合成自南京金斯瑞公司。PCR仪购自eppendorf公司。病毒DNA提取试剂盒购自Omega公司。
1.2病毒和病料
病毒：PRV变异毒株ZJ01株(本实验室于2011年分离鉴定),PRV传统毒株LA株(中国动物疫病卫生中心范伟兴研究员提供)(Dong et al.,Virology 2018)，HB98疫苗毒株(武汉科前公司PRV商品疫苗)，Bartha-K61毒株(梅里亚公司PRV商品疫苗)，Bucharest毒株(硕腾公司PRV商品疫苗)。
临床病料：来自华东地区发病猪群，脑或肺脏组织悬液共30份，于-20℃保存备用。
1.3引物设计与合成
根据Genebank PRV基因组序列(ZJ01、TJ、Bartha-K61、Becker、Kaplan)与本实验室伪狂犬经典毒株LA基因序列，采用DNAstar和BioEdit软件比对分析，设计5条引物(表1)，第一步PCR，如果采用引物1F-C1、1F-V和1R，变异株和HB98株预期扩增出两条条带(835bp、467bp)，经典毒株预期扩增出1条条带(738bp或813bp)。如果采用引物1F-C2、1F-V和1R，变异株和HB98株预期扩增出两条条带(467bp、178bp)，经典毒株预期扩增出1条条带(178bp)。第二步PCR采用引物2F和2R。变异株和HB98疫苗毒株的扩增产物分子量大小分别为211bp、293bp。引物由南京金斯瑞公司合成。
表1 PCR引物及其预期扩增产物
Table1 PCR primers and expected productions
Figure PCTCN2020100609-appb-000001
注：2R引物与HB98毒株(EA毒株)完全匹配，而与ZJ01毒株5’端有个别碱基错配，不影响扩增效率。
1.4病毒DNA提取
取250μl伪狂犬病毒液，使用Omega公司的病毒DNA试剂盒，按说明书操作，提取病毒基因组，用30μl双蒸水溶解，-20℃保存备用，作为DNA模板。
1.5两步法PCR
第一步PCR：PCR反应体系：Taqmix 12.5μl，蒸馏水7.5μl，DMSOμl，10uM上游引物1F-C、下游引物1F-V、鉴别引物1R各1μl，DNA模板1μl。PCR反应条件：95℃预变性5min；95℃变性30s，61℃退火30s，72℃延伸30s，30个循环，最后72℃终延伸10min。取10ul PCR产物于1.5％琼脂糖凝胶中进行电泳鉴定。
结果见图1，
1F-C1套引物：ZJ01变异毒株扩增出两个条带，分子量大约为835bp和467bp，经典毒株LA、经典疫苗毒株Bucharest和Bartha-K61均只扩增出1条条带，大小约813bp，与预测一致。但经典疫苗毒株HB98中也扩增出两个条带，分子量与ZJ01变异毒株扩增结果一致。PCR产物分别克隆至pMD19-T载体，基因测序结果正确。经典毒株LA扩增条带783bp，经典毒株Bucharest、Bartha-K61疫苗毒株扩增条带813bp，与设计一致。
1F-C2套引物：ZJ01变异毒株扩增出两个条带，分子量大约为178bp和467bp，LA经典毒株、Bartha-K61疫苗毒株均只扩增出1条条带，大小约178bp，与预测一致。但经典疫苗毒株HB98中也扩增出两个条带，分子量与ZJ01变异毒株扩增结果一致。PCR产物分别克隆至pMD19-T载体，基因测序结果正确。
上述结果显示，1F-C2套引物条带更加清晰，变异毒株与经典毒株的区分度更佳。因此，选择1F-C2套引物作为该方法的第一步PCR，用于以下所有试验研究。
第二步PCR：PCR反应体系：Taqmix 12.5μl，蒸馏水8.5μl，DMSO 1μl，10uM上游引物F’、下游引物R’各1μl，DNA模板1μl。PCR反应条件：95℃预变性5min；95℃变性30s，62℃退火30s，72℃延伸30s，共30个循环；最后72℃终延伸10min。取10ul PCR产物于1.5％琼脂糖凝胶中进行电泳鉴定。
取变异毒株ZJ01和经典疫苗毒株HB98，进行第二步PCR，结果见图2，ZJ01毒株和HB98毒株扩增产物大小为211bp和293bp。PCR产物分别克隆至pMD19-T载体，基因测序结果正确。
1.6特异性试验
将ZJ01、LA和HB98毒株的PCR产物分别克隆至pMD19-T载体，挑选阳性重组质粒 送往南京金斯瑞公司测序验证。同时，用该方法检测猪圆环病毒2型(PCV-2)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、脑心肌炎病毒(EMCV)、猪流行性腹泻病毒(PEDV)、流行性乙型脑炎病毒(JEV)，猪塞尼卡病毒(SVV)观察是否能扩增出特异的条带。
以猪圆环病毒2型、猪繁殖与呼吸综合征病毒、猪瘟病毒、日本乙型脑炎病毒、脑心肌炎病毒、塞尼卡病毒和猪流行性腹泻病毒的DNA(或反转录cDNA)为模板，第一步和第二步PCR结果均为阴性，表明该PCR的特异性良好。见图3和4。
1.7敏感性试验
1.7.1检测病毒液
第一步PCR：将PRV ZJ01株、LA株分别接种于BHK21细胞上，待完全出现病变后收取病毒，测定半数组织细胞感染量(TCID 50)。将两种病毒株病毒液稀释成每毫升40TCID 50、400TCID 50、4×10 3TCID 50、4×10 4TCID 50和4×10 5TCID 50病毒，取250μl通过Omega公司的病毒DNA提取试剂盒提取后得到模板，每个稀释度提取得到DNA 30μl。通过PCR发现，本方法敏感度为10TCID 50(见图5)。
第二步PCR：将伪狂犬病毒HB98株病毒原液稀释成每毫升含有40TCID 50、400TCID 50、4×10 3TCID 50、4×10 4TCID 50、4×10 5TCID 50的病毒液，取250μl通过Omega试剂盒提取后得到模板DNA 30μl。通过PCR发现，本方法敏感度可以达到10TCID 50(见图6)。
1.7.2检测重组质粒DNA模板
第一步PCR：取含UL44基因的pMD19T-ZJ01/UL44、pMD19T-LA/UL44重组质粒DNA为模板，10倍梯度稀释后进行PCR，测定PCR方法的敏感性，结果敏感度可以达到0.01ng/ml(见图7)。
第二步PCR：取含UL36基因的pMD19T-ZJ01/UL36、pMD19T-HB98/UL36重组质粒DNA为模板，10倍梯度稀释后进行PCR，测定PCR方法的敏感性，结果敏感度可以达到0.1ng/ml(见图8)。
1.8常规gE-PCR检测方法
按文献报道方法，PCR引物序列为：3F:5’-TCCACTCGCAGCTCTTCT-3’3R：5’-GCACGTCATCACGAAGGA-3’。PCR反应体系：Taqmix 12.5μl，蒸馏水8.5μl，DMSO1μl，10uM上游引物F(gD)、下游引物R(gD)分别为1μl、1μl，DNA模板1μl。PCR反应条件：95℃预变性5min；95℃变性30s，61℃退火30s，72℃延伸45s，共35个循环；最后 72℃终延伸10min。
1.9国家标准gD-PCR检测方法
按国标法进行。PCR引物序列为：4F:5’-CAGGAGGACGAGCTGGGGCT-3’4R:5’-GTCCACGCCCCGCTTGAAGCT-3’。PCR反应体系：Taqmix 12.5μl，蒸馏水8.5μl，DMSO1μl，10uM上游引物F(gD)、下游引物R(gD)分别为1μl、1μl，DNA模板1μl。PCR反应条件：95℃预变性5min；95℃变性30s，65℃退火30s，72℃延伸30s，共35个循环；最后72℃终延伸10min。
1.10临床样品检测结果
取58份临床样品，采用本方法检测PRV，阳性率为55.17％(32/58)，其中变异株32份，经典毒株0份；伪狂犬病毒gE基因PCR方法检测，阳性率55.17％(32/58)；PRV国标方法检测，阳性率50.0(29/58)。本方法与gE基因PCR方法、国标方法的符合率达到了100％和94.8％。
2.4重复性试验
取3份病毒样品，重复检测3次。第一步PCR扩增出178bp与467bp左右的条带，第二步PCR扩增出211bp左右的条带，三次结果一致，表明检测样品为伪狂犬病毒变异毒株。
我们也看到，该方法只是基于全基因组序列比对以及部分参考文献做出的，虽然通过临床检测样品初步的验证了该方法的可靠性，但是基于更多流行毒株分子生物学方面的证据仍然不足，需要进一步进行研究和探索。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受实施例的限制，其它任何未背离本发明的精神实质与原理下所做的改变、修饰、组合、替代、简化均应为等效替换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 用于鉴别猪伪狂犬病毒变异毒株的PCR引物组，其特征在于，该PCR引物组包括引物1F-C、1F-V、1R、2F和2R，其中各引物的序列如下：
    1F-C：5’-ACGCCGACCCCGAGTACTTTGACG-3’，
    1F-V：5’-GAGCCCGTCTCGGGGACGAC-3’，
    1R：5’-GGCCACGCGCACGGACACC-3’，
    2F:5’-GATGCGGTCACCGTCGGGTTT-3’，
    2R:5’-CCGCCGCTCAGCCCCCATCGT-3’。
  2. 权利要求1所述的PCR引物组在鉴别猪伪狂犬病毒变异毒株中的应用，其特征在于，提取待鉴别病毒DNA，使用所述PCR引物组中的引物1F-C、1F-V、1R进行第一步扩增，扩增出两条条带的为变异株或HB98疫苗毒株，扩增出1条条带(178bp)的为经典毒株；扩增出两条条带(178bp、467bp)的病毒DNA再使用所述PCR引物组中的引物2F和2R进行第二步扩增，扩增产物分子量大小为211bp的为变异株，扩增产物分子量大小为293bp的为HB98疫苗毒株。
  3. 权利要求1所述的PCR引物组在制备用于鉴别猪伪狂犬病毒变异毒株的试剂盒中的应用。
  4. 一种用于鉴别猪伪狂犬病毒变异毒株的试剂盒，其特征在于，该试剂盒中包含权利要求1所述的PCR引物组。
  5. 一种鉴别猪伪狂犬病毒变异毒株的方法，其特征在于，提取待鉴别病毒DNA，使用权利要求1所述PCR引物组中的引物1F-C、1F-V、1R进行第一步扩增，扩增出两条条带的为变异株或HB98疫苗毒株，扩增出1条条带的为经典毒株；扩增出两条条带的病毒DNA再使用权利要求1所述PCR引物组中的引物2F和2R进行第二步扩增，扩增产物分子量大小为211bp的为变异株，扩增产物分子量大小为293bp的为HB98疫苗毒株。
  6. 根据权利要求5所述的方法，其特征在于，第一步扩增的反应体系：Taqmix 12.5μl，蒸馏水7.5μl，DMSO 1μl，10uM的上游引物1F-C、下游引物1F-V、鉴别引物1R各1μl，DNA模板1μl。
  7. 根据权利要求5所述的方法，其特征在于，第一步扩增的反应条件：95℃预变性5min；95℃变性30s，61℃退火30s，72℃延伸30s，30个循环，最后72℃终延伸10min。
  8. 根据权利要求5所述的方法，其特征在于，第二步扩增的反应体系：Taqmix 12.5μl，蒸馏水8.5μl，DMSO 1μl，10uM上游引物F’、下游引物R’分别为1μl、1μl，DNA模板1μl。
  9. 根据权利要求5所述的方法，其特征在于，第二步扩增的反应条件：95℃预变性5min；95℃变性30s，62℃退火30s，72℃延伸30s，共30个循环；最后72℃终延伸10min。
  10. 根据权利要求5-9中任一项所述的方法，其特征在于，第一次扩增的敏感度达到10TCID 50病毒量或0.01ng/ml质粒量；第二次扩增的敏感度达到10TCID 50病毒量或者0.1ng/ml质粒量。
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