一种用于检测加利福尼亚血清群病毒和卡奇谷病毒的双重实时逆转录聚合酶链反应检测法。
A duplex real-time reverse transcriptase polymerase chain reaction assay for the detection of California serogroup and Cache Valley viruses.
作者信息
Wang Heng, Nattanmai Seela, Kramer Laura D, Bernard Kristen A, Tavakoli Norma P
机构信息
Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.
出版信息
Diagn Microbiol Infect Dis. 2009 Oct;65(2):150-7. doi: 10.1016/j.diagmicrobio.2009.07.001.
Abstract
A duplex TaqMan real-time reverse transcriptase polymerase chain reaction (PCR) assay was developed for the detection of California (CAL) serogroup viruses and Cache Valley virus (CVV), for use in human surveillance. The targets selected for the assay were the sequences encoding the nucleocapsid protein of CAL and the G1 glycoprotein of CVV. Conserved regions were selected by aligning genetic sequences from various strains available in the GenBank database. Primers and probes were selected in conserved regions. The assay sensitivity was 75 gene copies (gc)/reaction for CAL serogroup viruses and 30 gc/reaction for CVV. The performance of the assay was linear over at least 6 log(10) gc. The assay was specific, given that it did not cross-react with a variety of pathogens. It did, however, detect 11 viruses within the CAL serogroup and 12 CVV isolates. The use of an internal control ensured that possible inefficiency in nucleic acid extraction or PCR inhibition would be detected.
摘要
开发了一种双重TaqMan实时逆转录聚合酶链反应（PCR）检测方法，用于检测加利福尼亚（CAL）血清群病毒和卡奇谷病毒（CVV），以用于人类监测。该检测方法选择的靶标是编码CAL核衣壳蛋白和CVV G1糖蛋白的序列。通过比对GenBank数据库中各种毒株的基因序列来选择保守区域。在保守区域选择引物和探针。该检测方法对CAL血清群病毒的灵敏度为75个基因拷贝（gc）/反应，对CVV为30 gc/反应。该检测方法的性能在至少6个对数（10）gc范围内呈线性。该检测方法具有特异性，因为它不与多种病原体发生交叉反应。然而，它确实检测到了CAL血清群内的11种病毒和12种CVV分离株。使用内部对照可确保检测到核酸提取过程中可能出现的低效情况或PCR抑制。
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