根据猪瘟病毒(CSFV)以及猪繁殖与呼吸综合征病毒(PRRSV)基因组序列特点,设计3对特异性引物和3条TaqMan探针,经过反应条件的优化,建立了能同时检测并区分CSFV以及美洲型、欧洲型PRRSV的多重TaqMan荧光定量RT-PCR (qRT-PCR)方法.该方法具有特异性强、敏感性高、重复性好的特点,可以特异扩增CSFV和PRRSV,而与猪的其它常见病毒无交叉反应;对CSFV及美洲型、欧洲型PRRSV3种重组质粒标准品的检出下限均为2.68拷贝/μL;组内及组间重复试验的变异系数均小于1.5％.应用该方法对253份临床疑似样品进行检测,结果病原阳性106份,其中20份为CSFV阳性,86份为美洲型PRRSV阳性,11份为CSFV和美洲型PRRSV混合感染,未检测到欧洲型PRRSV.试验表明,研究建立的多重qRT-PCR方法可用于CSFV和PRRSV的快速鉴别检测及流行病学调查.

为建立猪繁殖与呼吸综合征病毒(PRRSV)的快速鉴别检测方法,本研究针对PRRSV美洲型经典株、变异株及弱毒活疫苗TJM-F92株的基因序列差异,设计了2对特异性引物和3条TaqMan探针,经过反应条件的优化,建立了能够同时检测并区分PRRSV美洲型经典株、变异株及TJM-F92疫苗株的多重TaqMan荧光定量RT-PCR方法.结果显示,该方法具有特异性强、敏感性高、重复性好的特点,与猪其他常见病毒无交叉反应;经典株、变异株、TJM-F92疫苗株重组质粒标准品的检出下限均为2.68拷贝/μL,比常规PCR敏感100倍;组内及组间重复试验的变异系数均小于1.5％.此外,该方法对324份疑似PPRSV感染的临床样品进行检测,结果显示PRRSV阳性114份,其中美洲型变异株105份、经典株6份、TJM-F92疫苗株3份,变异株和经典株混合感染4份.本研究建立的多重TaqMan荧光定量RT-PCR方法可以为PRRSV美洲型野毒株、疫苗株的快速鉴别检测及流行病学调查提供有效的技术手段.

1发病情况<br>　　2013年3月21日，武鸣县某猪场就猪病来求诊，并送检患病仔猪5头，发病母猪和仔猪的血液样品各4份。该猪场存栏42头母猪，断奶仔猪132头。3月上旬有8头母猪发病，表现为呼吸急促，经用抗菌、退热等药物对症治疗一周后病情没有好转，发病率升高，并且有19头仔猪死亡，妊娠母猪早产、产死胎和弱仔。该猪场之前仅进行过猪瘟、猪伪狂犬病疫苗免疫。

Porcine reproductive and respiratory syndrome(PRRS) caused by PRRS virus(PRRSV) is an acute,fatal infectious disease,which is characterized with reproductive failure in sows and respiratory problems in pigs of all age,especially in neonatal piglets.Since the outbreak of highly pathogenic PRRS(HPPRRS) in China in 2006,the disease has spread all over the country and it is very hard to control and eradicate.HP-PRRS has caused great economic loss to the pig industry in China.It is very important to detect PRRSV promptly and accurately for diagnosis,prevention and control of PRRS.The detection techniques for PRRSV include viral isolation and identification,viral antigen detection,viral nucleic acid detection and so on.Each type of technique also includes several specific detection methods.This review focused on the progress on the main detection techniques of PRRSV and also prospected the research of them in the future.

To investigate the genetic variation of porcine reproductive and respiratory syndrome virus(PRRSV),the full-length genome of PRRSV GX1003 strain from Guangxi province were amplified by RT-PCR,sequenced and analyzed.The results showed that,excluding the poly(A) tail,the genomic sequence of GX1003 strain was 15 320 nucleotides(nt) in length.Sequence analysis revealed that GX1003 strain shared 85.0%-99.4% nucleotide identity and 82.1%-99.1% amino acid identity with North American(NA) type isolates,and shared 60.6% nucleotide identity and 51.3% amino acid identity with European(EU) prototype LV strain.Sequence comparison demonstrated that GX1003 strain had discontinuous 30 aa deletion in 481 aa and 533-561 aa of NSP2 region compared with that of the NA prototype VR-2332,which was considered as the genetic marker of highly pathogenic PRRSV(HP-PRRSV),and had new multiple genomic variations in different regions.Phylogenetic analysis showed that all the NA type isolates could be divided into four subgroups,while GX1003 strain distributed in subgroup 4 represented by the highly pathogenic JXA1 strain.The results indicated that GX1003 strain from Guangxi province belonged to HP-PRRSV with new genetic variations in different regions.