通过竞争性逆转录-PCR并共提取标准RNA对贝类中的甲型肝炎病毒进行定量分析。
Quantification of hepatitis A virus in shellfish by competitive reverse transcription-PCR with coextraction of standard RNA.
作者信息
Arnal C, Ferre-Aubineau V, Mignotte B, Imbert-Marcille B M, Billaudel S
机构信息
Laboratoire de Virologie, Institut de Biologie, Centre Hospitalier Universitaire, 44093 Nantes Cedex, France.
出版信息
Appl Environ Microbiol. 1999 Jan;65(1):322-6. doi: 10.1128/AEM.65.1.322-326.1999.
Abstract
To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay. The quantifiable range with this reproducible method was 10(4) to 10(7) copies of HAV/gram or 400 to 10(6) 50% tissue culture infective doses/ml.
摘要
为了对实验性污染贻贝中的甲型肝炎病毒（HAV）进行定量，我们开发了一种具有7个核苷酸缺失的内标RNA，用于竞争性逆转录（RT）-PCR。该标准品直接加入样品中，用作提取对照和定量工具。经过共提取和竞争性RT-PCR后，通过与特异性探针的差异杂交和DNA酶免疫测定法检测标准品和野生型产物。这种可重复方法的可定量范围为每克HAV 10⁴至10⁷拷贝，或每毫升400至10⁶ 50%组织培养感染剂量。
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