高致病性猪繁殖与呼吸综合征病RT-PCR快速鉴别诊断方法的建立与临床应用

Development and Clinical Application of RT-PCR Differential Diagnosis Method for High Virulent Porcine Reproductive and Respiratory Syndrome

摘要:

根据高致病性猪繁殖与呼吸综合征病毒(PRRSV)Nsp2基因的缺失信息, 设计了3条特异性引物, 以含高致病性PRRSV Nsp2基因的质粒pMDNSP2及普通PRRSV VR2332株RNA为模板, 建立了快速诊断高致病性PRRSV RT-PCR方法。通过对临床组织病料的总RNA进行不同稀释倍数检测, 结果表明该方法能从0.265 pg的总RNA中检测到PRRSV的基因, 说明敏感性高。用该方法对猪瘟病毒(CSFV)、Ⅱ型圆环病毒(PCV-2)、伪狂犬病病毒(PRV)、链球菌(Streptococcus)、副猪嗜血杆菌(Haemophilus parasuis)和大肠杆菌(Escherichia coli)同条件检测, 结果都为阴性。进一步对36份疑似高致病性PRRSV临床组织病料细胞培养物、2株PRRSV商品活疫苗以及52个猪场所送检的184份临床样品进行了检测应用, 结果36份疑似高致病性PRRSV临床组织病料的细胞培养物有5份样品为阳性且都为高致病性PRRSV, 2株PRRSV商品活疫苗为普通PRRSV, 52个猪场中有42个猪场(123份样品)呈阳性, 其中只有1份为普通PRRSV。实验表明该方法能够准确地鉴别诊断高致病性PRRSV和普通PRRSV, 且具有快速、敏感和特异的特点, 具有临床实用性。

Abstract:

Based on the deletion information of high virulent PRRSV genome, 3 oligonucleotide primer were designed and synthesized. Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of high virulent PRRSV. The sensitivity and specificity of RT-PCR assays were evaluated, the results showing that the detection limit of the assay was found to be 0.265 pg of tissue total RNA, and the protocol have no cross-reaction with classical swine fever virus, porcine circovirus type 2, pseudorabies virus, streptococcus, haemophilus parasuis and Escherichia coli. Then 36 cell cultures, two PRRSV live vaccine strains and 184 clinical specimens from 52 farms were tested. Five PRRSV field isolates were the high virulent PRRSV; two PRRSV live vaccine strains from normal PRRSV, and 123 specimens from 42 farmer were positive (only 1 specimen was normal PRRSV). This RT-PCR method proved to be accurate differential diagnosis of the high virulent PRRSV and normal PRRSV with the characteristics of rapidity, sensitivity and specificity, and has a strong clinical relevance.