荧光定量RT-PCR法对高致病性PRRSV变异株感染猪体内分布规律的研究

根据GenBank发表的高致病性猪繁殖与呼吸综合征病毒(PRRSV)变异株Nsp2基因序列设计1对特异引物,以重组质粒作为阳性标准品,采用SYBR-Green Ⅰ嵌合荧光染料建立了1种荧光定量RT-PCR方法用于检测该病毒核酸载量,在101~106拷贝/μL范围内具有良好的线性关系,相关系数为R2=0.999 8,扩增效率为E=0.950,对质粒标准品最低检测限为12.8拷贝/μL,重复性检测的变异系数低于2.0%。用高致病性PRRSV变异毒株人工感染25日龄非免疫仔猪,对不同时间采集的血清进行了病毒核酸定量检测,结果表明病毒血症于攻毒后48 h被检测出,于7~10 d达到高峰,其病毒核酸载量能达到106拷贝/μL。试验猪临床表现为体温升高至40.5℃以上,持续7~11 d;出现嗜睡、打喷嚏、眼结膜炎、偶见一过性的耳尖发绀、皮肤苍白或有小疱疹、被毛粗糙、消瘦、便秘、后躯无力等临床症状。感染猪发生高热期与毒血症消长规律有一定相关性。对人工感染猪体内病毒分布检测结果表明,以多种脏器均有病毒存在,如扁桃体、淋巴结、心脏、肾脏中含量较高,肝、脾脏和肺次之,脑组织中也检测到病毒存在。试验表明,该方法可用于PRRSV变异毒株核酸定量检测,为该病毒致病性、致病机理、疫苗免疫及诊断等方面的研究提供了技术手段。

Studies On highly pathogenic porcine reproductive and respiratory syndrome virus distributions in the experimentally infected pigs by real-time quantitative PCR

Based on the Nsp2 gene sequence of porcine reproductive and respiratory syndrome virus (PRRSV) varia-tion strain published in GenBank,a pair of special primers was designed and the recombinant plasmid was used for standard positive template.A real-time quantitative PCR was developed for detection of PRRSV using SYBR-Green Ⅰ fluorescent dye.The assay showed a good linear relation when the plasmid DNA was diluted from 10~1 to 10~6 cop-ies/μL; and showed that dependent coefficient R~2=0.999 8 and the amplified efficiency E=0.950.The lowest de-tection limit number was 12.8 copies/μL; and variation coefficient was less than 2.0%.Twenty-five days old piglets were inoculated by the variation strain of PRRSV.The viremia time course of the infected pig sera was detected by the assay.The virus nuclear acid was detected post-infection (PI) 48 h,and reached a peak at PI 7 to 10 days.The maximum level nuclear acid load gave 10~6 copies/μL in the virus-infected pig sera.There was a positive relativity be-tween the rectal temperature and viremia.The clinical symptoms of the pigs showed high fever,and temperature was more than 40.5℃ for 7-11 days.The pigs displayed asleep,sneezes,rubefaction,temporarily ear cyanopathy,spirit depressed,inappetence,hind leg week and weight loss.The virus antigen was detected in the multi-organs.The higher virus load was found in the tonsil,lymph nodes,kidney and heart; and the next was liver,spleen and lung.The virus antigen was also detected in the brain.This study suggests that the real-time quantitative PCR is a good tool for detection of PRRSV variation strain in vivo.It provides a good basis for further research on pathogenic-ity,genetic variation,vaccination and diagnosis of the virus.