摘要
为了建立一种快速鉴别检测猪圆环病毒2型(PCV-2)、猪圆环病毒3型(PCV-3)、猪圆环病毒4型(PCV-4)的方法,试验设计合成PCV-2、PCV-3、PCV-4特异性鉴别检测引物,经一步法三重RT-PCR反应条件的优化,建立了PCV-2、PCV-3、PCV-4三重PCR鉴别检测方法.该方法对PCV-2、PCV-3、PCV-4、PCV-2/PCV-3/PCV-4可分别扩增出216、415、678 bp、216 bp/415 bp/678 bp的特异性条带,检测PRV、PPV、PRRSV、CSFV、PEDV、TGEV均为阴性,对PCV-2、PCV-3、PCV-4的最低检测量分别为1.0×102、1.0×103、1.0×103拷贝/µL,与PCV-2、PCV-3、PCV-4单项PCR方法的符合率均为100%,应用该方法检测85份临床病料样品,PCV-2、PCV-3、PCV-4阳性感染率分别为21.17%、14.12%、4.71%.说明建立的PCV-2、PCV-3、PCV-4三重PCR鉴别检测方法具有良好的特异性、敏感性、准确性和临床适用性,为PCV-2、PCV-3、PCV-4的临床鉴别诊断提供一种简便、快速的分子生物学检测技术.
Abstract
To develop a rapid method for detection and differentiation of PCV-2,PCV-3 and PCV-4,the specific primers of PCV-2,PCV-3 and PCV-4 were designed and synthesized,for a triple PCR method.The specific bands of 216,415,678,216 bp/415 bp/678 bp were amplified from PCV-2,PCV-3,PCV-4 and PCV-2/PCV-3/PCV-4.The triple PCR method did not amplify PRV,PPV,PRRSV,CSFV,PEDV and TGEV.The minimum detection limits were 1.0×102 copies/µL for PCV-2,1.0×103 copies/µL for PCV-3 and 1.0×103 copies/µL for PCV-4.The coincidence rates of the single PCR method for PCV-2,PCV-3 and PCV-4 were all 100%.Then,85 clinical samples were tested by this method and the positive rates of PCV-2,PCV-3 and PCV-4 were 21.17%,14.12%and 4.71%,respectively.These results showed that the developed triple PCR method for differential detection of PCV-2,PCV-3 and PCV-4 had good specificity,sensitivity,accuracy and clinical applicability,thus provided a simple and rapid molecular biological diagnostic technology for PCV-2,PCV-3 and PCV-4.
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