Monroy A M, Scott T W, Webb B A
Department of Entomology, Rutgers University, New Brunswick, NJ 08903, USA.
J Med Entomol. 1996 May;33(3):449-57. doi: 10.1093/jmedent/33.3.449.
A reverse transcriptase polymerase chain reaction (RT-PCR) assay was evaluated for the detection of eastern equine encephalomyelitis virus (EEEV). EEEV was detected by amplification of a 416-bp PCR product from within the E2 gene. Internal restriction endonuclease digestion and hybridizations to EEEV RNA demonstrated that the PCR product was amplified from EEEV. PCR amplifications from serial dilutions of an EEEV isolate identified by a neutralization test and titered by an infectious assay in cell culture indicated that this RT-PCR assay detected viral RNA at concentrations below 1 plaque forming unit(PFU) per reaction. The performance of the PCR assay in detection of EEEV was compared with an infectious assay detection procedure (IA/IFA) as part of the New Jersey 1993 vector surveillance program. During 1993, 7,007 field-collected Culiseta melanura (Coquillett) were assayed in 522 pools by both RT-PCR and IA/IFA. EEEV was detected in 95 pools by RT-PCR and 17 pools by IA/IFA; all IA/IFA positive pools were also positive by RT-PCR. During the 1993 field season, RT-PCR consistently detected virus at enzootic foci earlier that IA/IFA and in greater numbers of mosquito pools. The data indicated that viral RNA may be present earlier and in more mosquitoes than indicated by IA/IFA.
对一种用于检测东部马脑炎病毒（EEEV）的逆转录酶聚合酶链反应（RT-PCR）检测方法进行了评估。通过从E2基因内部扩增出一个416碱基对的PCR产物来检测EEEV。内部限制性内切酶消化以及与EEEV RNA的杂交表明，该PCR产物是从EEEV扩增而来的。对通过中和试验鉴定并在细胞培养中通过感染性测定进行滴定的EEEV分离株的系列稀释液进行PCR扩增，结果表明这种RT-PCR检测方法能够检测到每个反应中浓度低于1个空斑形成单位（PFU）的病毒RNA。作为1993年新泽西州媒介监测项目的一部分，将该PCR检测方法在检测EEEV方面的性能与一种感染性检测程序（IA/IFA）进行了比较。1993年期间，对从野外采集的7007只黑尾库蚊（Coquillett）进行了检测，共分成522组，同时采用RT-PCR和IA/IFA两种方法。通过RT-PCR在95组中检测到了EEEV，通过IA/IFA在17组中检测到了EEEV；所有IA/IFA检测呈阳性的组通过RT-PCR检测也呈阳性。在1993年的野外季节，RT-PCR始终比IA/IFA更早地在地方病疫源地检测到病毒，并且检测到病毒的蚊虫组数更多。数据表明，病毒RNA可能比IA/IFA所显示的更早出现且存在于更多的蚊虫中。
