猪场爆发猪繁殖与呼吸综合征的临床诊断及实验检测

The clinical diagnosis and experiment detection of PRRS of one hogpen

关键词:  猪繁殖与呼吸综合征  临床诊断  RT-PCR检测

Key words:  PRRS, clinical diagnosis, RT-PCR detection

摘要:

摘 要: 猪繁殖与呼吸综合征是由猪繁殖与呼吸综合征病毒(PRRSV)引起的猪繁殖和呼吸道接触性传染病。临床特征为母猪流产、死胎和木乃伊胎增多，仔猪出现呼吸道症状和断奶前死亡率增高以及免疫抑制，严重危害种公猪和繁殖母猪及其仔猪。本文参考 GenBank上PRRSV LV和VR-2332毒株核苷酸序列，设计并合成一对特异性引物，以提取来自贵州某猪场猪繁殖与呼吸综合征疑似病料（包括血液、肝、肺、肾、淋巴结、心、脾组织）的总RNA为模版，应用反转录-聚合酶链式反应(RT-PCR)方法，经94℃ 预变性2 min；94℃变性45s，60℃退火 1min，72℃延伸1min进行35个循环；72℃ 再延伸10min进行扩增。扩增产物经0.8%琼脂糖凝胶电泳检测，结果表明所扩增的目的DNA条带大小约750bp，与预期大小相似。扩增产物经回收纯化后，应用pMD18－T 载体进行克隆后测序。测序结果表明，所扩增DNA片段大小为739bp，该核苷酸序列与湖南发现的PRRSV序列同源性为100%。本实验说明临床诊断及实验RT-PCR检测方法作为猪繁殖与呼吸综合征的诊断方法是快速有效的，可为猪繁殖与呼吸综合征诊断提供参考依据。

Abstract:

bstract: PRRS caused by PRRSV is a contact and infective disease of the porcine reproductive and respiratory tract. Clinical features appear, such as sows abortion, stillbirth and fetal mummy, increasing in the mortality rate of pro-weaned piglets and immune suppression, it is seriously harmful to boars, breeding sows and their piglets. A pair of specific primer was designed referring to PRRSVLV and VR-2332 strain nucleotide sequence from Gen Bank to extract he total RNA form porcine reproductive and respiratory syndrome suspected disease material (including blood, liver, lung, kidney, lymph node, heart, spleen) from Guizhou, RT-PCR methods were used as follows, denaturing 94℃ 2 min; 94℃ 45 s, 60℃ 1 min, 72℃ 1 min and 35 cycles; 72℃ 10 min to further extend the amplification. The results showed that the DNA was amplified with the size of about 750 bp, when PCR products was tested by 0.8% agarose gel electrophoresis , with the expected similar size. Purified and recoveried PCR products were tested after cloning by pMD18-T cloning vector. The results showed that the expansion nucleotide sequence of the size was 739 bp the same as Hunan PRRSV Strain of 100%. The results indicated that is fast and effective about clinical diagnosis and RT-PCR method as a porcine reproductive and respiratory syndrome diagnosis method, that can be as reference for the diagnosis of PRRS.