PRRS、PCVD and CSF病毒多重PCR方法的建立及其应用研究
摘要
猪繁殖与呼吸综合征(PRRS)、猪圆环病毒病(PCVD)和猪瘟(CSF)是危害养猪业发展的一类重要的传染病,这三种疾病每年均给养猪业造成巨大的经济损失。这三种病均能引起猪繁殖障碍和呼吸系统疾病,由于这三种疾病的临床表现非常相似,临床诊断容易引起混淆,因此早期的鉴别诊断对控制这三种疾病的流行显得尤其重要。目前针对这三种病的病原体的常规诊断方法有病原分离鉴定和血清学等方法。病原分离鉴定繁琐、耗时、检出率低;血清学方法虽然简便快速,但存在非特异性反应,并且不能完全区别免疫接种与自然感染阳性等缺点。有学者建立了检测PRRSV、PCV2和CSFV的PCR技术,但同时检测这三种病毒的多重PCR方法还未见报道。本试验建立了一种快速、敏感、特异的检测PRRSV、PCV2和CSFV多重PCR方法,并使用该方法对临床病料进行了检测。
1检测PRRSV、PCV2和CSFV多重PCR的建立
参照GeneBank收录的猪繁殖与呼吸综合征病毒美洲型标准株(ATCCVR-2332)的ORF7保守序列、猪圆环病毒2型(AF381175)的ORF2基因保守序列和猪瘟病毒的E2基因保守序列,设计合成了三对特异性引物。通过优化镁离子浓度、引物浓度和退火温度等条件,建立了检测PRRSV、PCV2和CSFV的多重PCR方法。该方法特异性强,灵敏度高,能检出三种病毒的最低含量分别为4.8pg、5.0pg和14.5pg。
2 PRRSV、PCV2和CSFV多重PCR方法的临床应用
利用建立的PRRSV、PCV2和CSFV多重PCR方法对安徽省合肥、安庆、六安、蚌埠、滁州、巢湖、淮北、宿州、阜阳和池州10个地区的120份临床疑似病料进行检测,除阜阳地区未分离到PRRSVV和CSFV外,PRRSV、PCV2和CSFV总阳性检出率分别为58.33%、49.17%和23.33%。经生物统计分析,PRRSV和PCV2的感染在安徽省猪群中最为严重,且有上升的趋势。此外,PRRSV的感染还存在明显的地区差异,其中安庆、六安和滁州地区感染严重。
对病料进行PRRSV、PCV2和CSFV混合感染的检测,检测结果是PRRSV与PCV2、PRRSV与CSFV、PCV2与CSFV以及PRRSV、PCV2、CSFV三者混合感染的阳性检出率分别为32.50%、17.50%、9.17%、6.67%。经生物统计分析,在安徽省发病猪群中存在着PRRSV、PCV2和CSFV的混合感染,其中以PRRSV与PCV2的混合感染率最高。
3高致病性猪繁殖与呼吸综合征病毒(Nsp2 1594～1680)RT-PCR检测试剂盒和PRRSV美洲型标准株(ATCCVR-2332)PCR方法的比较
应用高致病性猪繁殖与呼吸综合征病毒((Nsp2 1594～1680)RT-PCR检测试剂盒(方法1)和PRRSV美洲型标准株(ATCCVR-2332)PCR方法(方法2)对安徽省合肥、安庆、六安、滁州、巢湖、淮北、宿州和池州8个地区的65份病料进行同步检测,方法1对病料的阳性检出率为73.84%,方法2对病料的阳性检出率为75.38%,两种检测方法的符合率达86.15%。生物统计结果表明,当前安徽省猪群中PRRSV的感染以变异株为主。
以上研究表明,本试验建立的这种快速、敏感、特异的检测PRRSV、PCV2和CSFV多重PCR方法具有重要的临床意义。
The infectious diseases caused by porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus 2 type(PCV2) and classical swine fever virus(CSFV) are becoming the main threat to the development of swine-breeding industry,and leading to tremendous economic losses annually.The three infectious diseases induce reproduction barrier and disease of respiratory system to pigs and The extreme similarities of the three infectious diseases in their clinical manifestations make it more difficult to diagnose.However,the correct diagnosis is critical prior to the effective treatment.At present,the routine diagnosis of these diseases are pathogen isolated and serum technology,etal.But,the former is trouble,time consumed and lowly detective rate.and the latter exists the default of non-specific reaction and can not distinguish between natural infection and immunization positive though it is simple and quick.Some scholars have established a PCR for testing PRRSV,PCV2 and CSFV,but the multiplex PCR for the three viruses is none.Aimed at the early and specific identification of the relevant infection,a multiplex PCR has been designed and tested in the present study.
1 Establishment of multiplex PCR for PRRSV,PCV2 and CSFV
First,three pairs of specific primers were separately designed according to the analysis of the highly conservative DNA sequences within ORF7 of PRRSV American strain(ATCCVR-2332),ORF2 of PCV2(AF381175) strain,and E2 gene of CSFV.The multiplex PCR-working conditions were then optimized in terms of magnesium ion concentration,the combination of primer concentrations,and the annealing temperatures.After a series of trials,we finally established the effective multiplex PCR methodology for the detection of PRRSV,PCV2 and CSFV.It demonstrated that this method is characterized by rapidity,specificity,as well as sensitivity.Using this PCR system,the minimum detectable amounts of DNA for PRRSV,PCV2,and CSFV are respectively 4.8pg,5.0pg,and 14.5pg.
2 The clinical application of the multiplex PCR for the detection of PRRSV,PCV2 and CSFV
One hundred and twenty samples(including lymphocytes,lungs,livers,spleens and kidneys) from sick pigs were collected from the below ten areas within Anhui province:Hefei,Anqing,Liuan,Bengbu,Chuzhou,Chaohu,Huaibei,Suzhou,Fuyang and Chizhou.The multiplex PCR was then conducted in order to determine the pathogenic viruses.The positive ratio of PRRSV,PCV2 and CSFV was 58.33%,49.17%and 23.33%separately,except that PRRSV and CSFV were isolated from Fuyang area.With the statistical analysis of biological,the infections of PRRSV and PCV2 was the most serious and both appeared an increasing trend in Anhui Province.Moreover,the PRRSV infection distributed in a regionally different fashion, since the infection occurred in Anqing,Liuan,and Chuzhou were more serious than in other areas.
The co-infection among PRRSV,PCV2 and CSFV was detected,The results were that,the positive ratio of,PRRSV and CSFV,PCV2 and CSFV,three of PRRSV、PCV2、CSFV was respectively 32.50%、17.50%、9.17%、6.67%.With the statistical analysis of biological,the co-infection among PRRSV,PCV2 and CSFV were prevalent,in which the PCV2 and PRRSV dual infections accounted for the majority.
3 The comparison between HPPRRSV(high pathogenic porcine reproductive and respiratory syndrome virus)(Nsp2 1594～1680 variation)RT-PCR test reagent kit and the PCR of PRRSV
Sixty-five samples collected from sick pigs in eight different areas(Hefei, Anqing,Liuan,Chuzhou,Chaohu,Huaibei,Suzhou,and Chizhou) in Anhui province were tested in parallel with HPPRRSV RT-PCR kit(way 1) and our PCR method(way 2).The positive ratio by way 1 was 73.84%,and way 2 was 75.38%.The coincidence rate of two methods peaked as 86.15%.With the statistical analysis of biological,at present,the variant infections of PRRSV is overwhelming in Anhui province.
Above all,the established multiplex PCR assay is quick,specific,sensitive and has important clinical significance.
1检测PRRSV、PCV2和CSFV多重PCR的建立
参照GeneBank收录的猪繁殖与呼吸综合征病毒美洲型标准株(ATCCVR-2332)的ORF7保守序列、猪圆环病毒2型(AF381175)的ORF2基因保守序列和猪瘟病毒的E2基因保守序列,设计合成了三对特异性引物。通过优化镁离子浓度、引物浓度和退火温度等条件,建立了检测PRRSV、PCV2和CSFV的多重PCR方法。该方法特异性强,灵敏度高,能检出三种病毒的最低含量分别为4.8pg、5.0pg和14.5pg。
2 PRRSV、PCV2和CSFV多重PCR方法的临床应用
利用建立的PRRSV、PCV2和CSFV多重PCR方法对安徽省合肥、安庆、六安、蚌埠、滁州、巢湖、淮北、宿州、阜阳和池州10个地区的120份临床疑似病料进行检测,除阜阳地区未分离到PRRSVV和CSFV外,PRRSV、PCV2和CSFV总阳性检出率分别为58.33%、49.17%和23.33%。经生物统计分析,PRRSV和PCV2的感染在安徽省猪群中最为严重,且有上升的趋势。此外,PRRSV的感染还存在明显的地区差异,其中安庆、六安和滁州地区感染严重。
对病料进行PRRSV、PCV2和CSFV混合感染的检测,检测结果是PRRSV与PCV2、PRRSV与CSFV、PCV2与CSFV以及PRRSV、PCV2、CSFV三者混合感染的阳性检出率分别为32.50%、17.50%、9.17%、6.67%。经生物统计分析,在安徽省发病猪群中存在着PRRSV、PCV2和CSFV的混合感染,其中以PRRSV与PCV2的混合感染率最高。
3高致病性猪繁殖与呼吸综合征病毒(Nsp2 1594～1680)RT-PCR检测试剂盒和PRRSV美洲型标准株(ATCCVR-2332)PCR方法的比较
应用高致病性猪繁殖与呼吸综合征病毒((Nsp2 1594～1680)RT-PCR检测试剂盒(方法1)和PRRSV美洲型标准株(ATCCVR-2332)PCR方法(方法2)对安徽省合肥、安庆、六安、滁州、巢湖、淮北、宿州和池州8个地区的65份病料进行同步检测,方法1对病料的阳性检出率为73.84%,方法2对病料的阳性检出率为75.38%,两种检测方法的符合率达86.15%。生物统计结果表明,当前安徽省猪群中PRRSV的感染以变异株为主。
以上研究表明,本试验建立的这种快速、敏感、特异的检测PRRSV、PCV2和CSFV多重PCR方法具有重要的临床意义。
The infectious diseases caused by porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus 2 type(PCV2) and classical swine fever virus(CSFV) are becoming the main threat to the development of swine-breeding industry,and leading to tremendous economic losses annually.The three infectious diseases induce reproduction barrier and disease of respiratory system to pigs and The extreme similarities of the three infectious diseases in their clinical manifestations make it more difficult to diagnose.However,the correct diagnosis is critical prior to the effective treatment.At present,the routine diagnosis of these diseases are pathogen isolated and serum technology,etal.But,the former is trouble,time consumed and lowly detective rate.and the latter exists the default of non-specific reaction and can not distinguish between natural infection and immunization positive though it is simple and quick.Some scholars have established a PCR for testing PRRSV,PCV2 and CSFV,but the multiplex PCR for the three viruses is none.Aimed at the early and specific identification of the relevant infection,a multiplex PCR has been designed and tested in the present study.
1 Establishment of multiplex PCR for PRRSV,PCV2 and CSFV
First,three pairs of specific primers were separately designed according to the analysis of the highly conservative DNA sequences within ORF7 of PRRSV American strain(ATCCVR-2332),ORF2 of PCV2(AF381175) strain,and E2 gene of CSFV.The multiplex PCR-working conditions were then optimized in terms of magnesium ion concentration,the combination of primer concentrations,and the annealing temperatures.After a series of trials,we finally established the effective multiplex PCR methodology for the detection of PRRSV,PCV2 and CSFV.It demonstrated that this method is characterized by rapidity,specificity,as well as sensitivity.Using this PCR system,the minimum detectable amounts of DNA for PRRSV,PCV2,and CSFV are respectively 4.8pg,5.0pg,and 14.5pg.
2 The clinical application of the multiplex PCR for the detection of PRRSV,PCV2 and CSFV
One hundred and twenty samples(including lymphocytes,lungs,livers,spleens and kidneys) from sick pigs were collected from the below ten areas within Anhui province:Hefei,Anqing,Liuan,Bengbu,Chuzhou,Chaohu,Huaibei,Suzhou,Fuyang and Chizhou.The multiplex PCR was then conducted in order to determine the pathogenic viruses.The positive ratio of PRRSV,PCV2 and CSFV was 58.33%,49.17%and 23.33%separately,except that PRRSV and CSFV were isolated from Fuyang area.With the statistical analysis of biological,the infections of PRRSV and PCV2 was the most serious and both appeared an increasing trend in Anhui Province.Moreover,the PRRSV infection distributed in a regionally different fashion, since the infection occurred in Anqing,Liuan,and Chuzhou were more serious than in other areas.
The co-infection among PRRSV,PCV2 and CSFV was detected,The results were that,the positive ratio of,PRRSV and CSFV,PCV2 and CSFV,three of PRRSV、PCV2、CSFV was respectively 32.50%、17.50%、9.17%、6.67%.With the statistical analysis of biological,the co-infection among PRRSV,PCV2 and CSFV were prevalent,in which the PCV2 and PRRSV dual infections accounted for the majority.
3 The comparison between HPPRRSV(high pathogenic porcine reproductive and respiratory syndrome virus)(Nsp2 1594～1680 variation)RT-PCR test reagent kit and the PCR of PRRSV
Sixty-five samples collected from sick pigs in eight different areas(Hefei, Anqing,Liuan,Chuzhou,Chaohu,Huaibei,Suzhou,and Chizhou) in Anhui province were tested in parallel with HPPRRSV RT-PCR kit(way 1) and our PCR method(way 2).The positive ratio by way 1 was 73.84%,and way 2 was 75.38%.The coincidence rate of two methods peaked as 86.15%.With the statistical analysis of biological,at present,the variant infections of PRRSV is overwhelming in Anhui province.
Above all,the established multiplex PCR assay is quick,specific,sensitive and has important clinical significance.
引文
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