猪伪狂犬病病毒套式 PCR 检测方法的建立与应用

摘要

To establish a highly sensitive nested PCR methods for detecting porcine pseudorabies virus,ac-cording to published gene sequences of PRV gD,two pairs of primers were designed and synthesized,the nested PCR method was established by optimizing the reaction system and conditions.The method applica-bility was verified by sensitivity test,specificity test,sample comparison tests.The results showed that the detection limit of the method established is 2.6 × 10-7 ng/mL,the sensitivity is 1 000 times higher than that of national standard method,and the objective band was only amplified from the pseudorabies vi-rus reference strain,and could greatly improve the positive detection rate from suspected samples.The nested PCR detection mehod with high sensitivity and good specificity for rapid PRV detection was success-fully established.%对伪狂犬病病毒（PRV）国标 PCR 方法进行优化，建立一种高灵敏度的套式 PCR 方法。根据伪狂犬病病毒 gD 基因序列，设计并合成了2对引物，通过反应体系和条件的优化建立套式 PCR 方法。通过灵敏性试验、特异性试验、病料检测对比试验等验证建立方法的适用性。结果表明，建立的方法检测伪狂犬病病毒 DNA 的极限为2．6×10－7 ng／mL，灵敏度比国家标准方法提高了1000倍，从疑似 PRV 感染组织病料中能大幅度提高阳性检出率。本研究成功建立了一种灵敏度高、特异性好的快速检测猪伪狂犬病病毒的套式 PCR 检测方法。