Johnson Donna J, Ostlund Eileen N, Schmitt Beverly J
USDA Animal and Plant Health Inspection Service, Veterinary Services, National Veterinary Services Laboratories, Ames, IA 50010, USA.
J Vet Diagn Invest. 2003 Sep;15(5):488-93. doi: 10.1177/104063870301500518.
A traditional nested reverse transcription-polymerase chain reaction (RT-PCR) assay specific for eastern equine encephalomyelitis (EEE) virus was designed to multiplex with a previously described West Nile (WN) virus nested RT-PCR assay. Differentiation of EEE and WN was based on base pair size of the amplified product. One hundred fifty-seven mammalian and avian brain tissues were tested by EEE/WN nested multiplex RT-PCR, EEE nested RT-PCR, and WN nested RT-PCR, and results were compared with other diagnostic test results from the same animals. Serological and virus isolation testing confirmed the results of the multiplex PCR assay. When compared with cell culture virus isolation, the multiplex assay was shown to be more sensitive in detecting the presence of EEE or WN virus in brain tissues. The multiplex assay was shown to be sensitive and specific for North American EEE and WN and provided a rapid means of identifying both viruses in brain tissues. No apparent sacrifice in sensitivity was observed in the multiplex procedure compared with the individual EEE and WN nested RT-PCR assays. Data collected from an additional 485 multiplex RT-PCR tests conducted during the summer and fall of 2002 further support the validity of the procedure.
设计了一种针对东部马脑炎（EEE）病毒的传统巢式逆转录-聚合酶链反应（RT-PCR）检测方法，使其能与先前描述的西尼罗河（WN）病毒巢式RT-PCR检测方法进行多重检测。EEE和WN的鉴别基于扩增产物的碱基对大小。采用EEE/WN巢式多重RT-PCR、EEE巢式RT-PCR和WN巢式RT-PCR对157份哺乳动物和禽类脑组织进行检测，并将结果与同一动物的其他诊断检测结果进行比较。血清学和病毒分离检测证实了多重PCR检测的结果。与细胞培养病毒分离法相比，多重检测法在检测脑组织中EEE或WN病毒的存在时表现出更高的敏感性。该多重检测法对北美EEE和WN具有敏感性和特异性，为在脑组织中鉴定这两种病毒提供了一种快速方法。与单独的EEE和WN巢式RT-PCR检测相比，在多重检测过程中未观察到明显的敏感性损失。2002年夏秋季节进行的另外485次多重RT-PCR检测收集的数据进一步支持了该方法的有效性。
