猪繁殖与呼吸综合征病毒和猪圆环病毒4型双重SYBR GreenⅠ实时荧光定量PCR检测方法的建立

Simultaneous detection of porcine reproductive and respiratory syndrome virus and porcine circovirus 4 by a duplex SYBR GreenⅠ-based quantitative real-time PCR

分别靶向PRRSV ORF7基因和PCV4 ORF2基因的高度保守区,设计了2对特异性引物,建立了同时检测猪繁殖与呼吸综合征病毒(PRRSV)和猪圆环病毒4型(PCV4)的SYBR GreenⅠ-based双重实时荧光定量PCR方法。PRRSV和PCV4在同一样品中的区别在于它们独特的熔解温度(T_(m)),PRRSV为84.5℃,PCV4为79.0℃,而对其他非靶向的猪常见病毒没有显示特定的熔解峰,且检测极限分别为PRRSV的81.5 copies/μL和PCV4的41.1 copies/μL。采用SYBR GreenⅠ实时荧光定量PCR和常规PCR方法对30例有呼吸和繁殖衰竭症状的猪临床样本进行检测,检测结果显示,PRRSV阳性率为20.0%(6/30),PCV4阳性率53.3%(16/30),且PRRSV阳性样品中PCV4检出率为50.0%(3/6),较常规PCR检测更灵敏。该方法可能是检测PRRSV和PCV4共感染的一种快速、灵敏和可靠的方法。

The SYBR GreenⅠ-based duplex real-time PCR assay was developed for simultaneous detection of porcine reproductive and respiratory syndrome virus(PRRSV)and porcine circovirus 4(PCV4)genomes.The distinction between PRRSV and PCV4 in the same sample lay on their unique melting temperatures(T_(m))of 84.5℃for PRRSV and 79.0℃for PCV4,while no specific melting peak was shown for other non-targeted porcine common viruses.The detection limits were 81.5 copies/μL of PRRSV and 41.1 copies/μL of PCV3,respectively.Thirty clinical samples of pigs with respiratory and reproductive failure were detected by qPCR and conventional PCR.The results of qPCR showed that the positive rates of PRRSV and PCV4 were(6/30)20.0%,53.3%(16/30),the detection rate of PCV4 in PRRSV positive samples was 50.0%(3/6),which was more sensitive than conventional PCR.This method may be a fast,sensitive and reliable method to detect the co-infection of PRRSV and PCV4.