Henchal E A, Polo S L, Vorndam V, Yaemsiri C, Innis B L, Hoke C H
Department of Virus Diseases, Walter Reed Army Institute of Research, Washington, DC.
Am J Trop Med Hyg. 1991 Oct;45(4):418-28. doi: 10.4269/ajtmh.1991.45.418.
A set of sense and anti-sense oligomeric DNA primers, degenerate in the third "wobble" base position of codons so as to match all known dengue virus sequences, was evaluated as universal primers in a polymerase chain reaction (PCR) assay for the rapid diagnosis of dengue virus infections. Virus-specific complementary DNA (cDNA) was prepared by reverse transcription (RT) of total RNA extracted from serum. Amplified cDNA was identified by nucleic acid hybridization with four serotype-specific, oligomeric DNA probes. Using sera from patients admitted with dengue fever, RT/PCR followed by nucleic acid hybridization using radiolabeled probes was 68% sensitive (50/74; 95% confidence interval [CI] = 57-78%) and 100% specific. Chemiluminescent detection of hybridized products was 62% sensitive (26/42; 95% CI = 46-75%). Using specimens from which a virus isolate had been obtained, RT/PCR followed by nucleic acid hybridization with radiolabeled probes was 80% sensitive (40/50; 95% CI = 69-91%) and 100% specific. The results suggest that RT/PCR using degenerate primers is a sensitive and specific method for the detection of dengue viruses in clinical specimens.
一组有义寡聚脱氧核糖核酸引物和反义寡聚脱氧核糖核酸引物在密码子的第三个“摆动”碱基位置上是简并的，以便与所有已知的登革病毒序列相匹配，这些引物在聚合酶链反应（PCR）检测中被评估为通用引物，用于快速诊断登革病毒感染。通过对从血清中提取的总RNA进行逆转录（RT）来制备病毒特异性互补DNA（cDNA）。通过与四种血清型特异性寡聚脱氧核糖核酸探针进行核酸杂交来鉴定扩增的cDNA。使用登革热患者的血清，RT/PCR随后使用放射性标记探针进行核酸杂交，敏感性为68%（50/74；95%置信区间[CI]=57-78%），特异性为100%。杂交产物的化学发光检测敏感性为62%（26/42；95%CI=46-75%）。使用已获得病毒分离株的标本，RT/PCR随后使用放射性标记探针进行核酸杂交，敏感性为80%（40/50；95%CI=69-91%），特异性为100%。结果表明，使用简并引物的RT/PCR是检测临床标本中登革病毒的一种敏感且特异的方法。
