Armstrong P, Borovsky D, Shope R E, Morris C D, Mitchell C J, Karabatsos N, Komar N, Spielman A
Department of Tropical Public Health, Harvard School of Public Health, Harvard University, Boston, MA.
J Med Entomol. 1995 Jan;32(1):42-52. doi: 10.1093/jmedent/32.1.42.
A sensitive and specific colorimetric dot assay following polymerase chain reaction (PCR) method has been developed to detect 0.1 pg of eastern equine encephalomyelitis viral (EEEV) RNA. The assay is 250-fold more sensitive than analysis by electrophoresis and is based on converting a 291-nucleotide sequence of the viral coat protein amino terminus into a double-stranded DNA (dsDNA) and amplifying the DNA using a specific primer pair and PCR. The amplified complementary DNA (cDNA) is denatured adsorbed onto a nylon strip, baked, and detected with a digoxigenin-labeled probe. Dots with viral cDNA are stained dark red, whereas controls do not stain or stain lightly. The assay is very specific and sensitive and detects only EEEV. RNA of Venezuelan equine encephalitis, St. Louis encephalitis, Keystone, Flanders, Tensaw, and western equine encephalitis viruses were not detected. EEEV (Ten Broeck) RNA was detected at the 10-ng level, indicating that the prototype we used may have different nucleotides in the region where the primer pair binds. The PCR amplified EEEV cDNA that was 92% homologous to the consensus sequence of EEEV. The detection of EEEV in the liver of an infected Emu bird and in field-collected mosquitoes from Florida and Massachusetts that were analyzed concurrently as blind samples by tissue culture plaque assay and by PCR dot analysis proved that the assay is sensitive and can be used to detect infected mosquitoes. The assay can detect at least 1 infected mosquito in a pool of 1,000 uninfected mosquitoes.
已开发出一种灵敏且特异的聚合酶链反应（PCR）比色斑点分析法，用于检测0.1 pg的东部马脑脊髓炎病毒（EEEV）RNA。该分析法比电泳分析灵敏250倍，其原理是将病毒衣壳蛋白氨基末端的291个核苷酸序列转化为双链DNA（dsDNA），并使用一对特异性引物通过PCR扩增该DNA。扩增的互补DNA（cDNA）经变性后吸附到尼龙条上，烘烤，并用地高辛标记的探针进行检测。带有病毒cDNA的斑点染成深红色，而对照则不着色或轻度着色。该分析法非常特异且灵敏，仅能检测到EEEV。未检测到委内瑞拉马脑炎病毒、圣路易斯脑炎病毒、基石病毒、佛兰德斯病毒、滕索病毒和西部马脑脊髓炎病毒的RNA。在10 ng水平检测到了EEEV（滕布罗克毒株）RNA，这表明我们使用的原型毒株在引物对结合区域可能具有不同的核苷酸。PCR扩增的EEEV cDNA与EEEV的共有序列具有92%的同源性。通过组织培养空斑试验和PCR斑点分析对作为盲样同时分析的感染鸸鹋肝脏以及从佛罗里达州和马萨诸塞州野外采集的蚊子中检测到EEEV，证明该分析法灵敏，可用于检测受感染的蚊子。该分析法能够在1000只未感染蚊子的混合样本中检测出至少1只受感染的蚊子。
