从猪样本中提取 RNA 并通过实时 RT-PCR 检测猪流感病毒。

RNA Extraction from Swine Samples and Detection of Influenza A Virus in Swine by Real-Time RT-PCR.

机构信息

Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA.

出版信息

Methods Mol Biol. 2020;2123:295-310. doi: 10.1007/978-1-0716-0346-8_21.

DOI:10.1007/978-1-0716-0346-8_21

PMID:32170696

Abstract

Real-time reverse-transcription PCR (rRT-PCR) assays are currently the method of choice in many laboratories for the detection and subtyping of influenza A virus (IAV) in swine. Traditionally, nasal swabs and lung tissues (sometimes bronchoalveolar lavage and tracheal tissues) are the primary specimens for IAV testing. However, oral fluids are becoming more common for IAV prognostic profiling. In this chapter, we describe (1) procedures of RNA extraction from the common clinical specimens, (2) two rRT-PCR assays for detection of IAV in swine, and (3) an rRT-PCR assay for subtyping swine IAV. RNA extraction procedures include a magnetic bead method optimized for extraction from nasal swabs and tissue homogenates and a magnetic bead method optimized for extraction from oral fluids. Two rRT-PCR assays for detection of swine IAV include a USDA-validated IAV rRT-PCR targeting the matrix gene and the USDA-licensed VetMAX™-Gold Swine Influenza Virus Detection rRT-PCR kit (Thermo Fisher Scientific) targeting the nucleoprotein and matrix genes. The swine IAV subtyping assay described here is VetMAX™-Gold Swine Influenza Virus Subtyping rRT-PCR kit (Thermo Fisher Scientific) which distinguishes swine IAV H1 from H3 and N1 from N2.

摘要

实时逆转录聚合酶链反应 (rRT-PCR) 检测方法目前是许多实验室检测和鉴定猪流感病毒 (IAV) 的首选方法。传统上，鼻拭子和肺组织（有时是支气管肺泡灌洗液和气管组织）是用于 IAV 检测的主要标本。然而，口腔液越来越常用于 IAV 的预后分析。在本章中，我们描述了（1）从常见临床标本中提取 RNA 的程序，（2）两种用于检测猪 IAV 的 rRT-PCR 检测方法，以及（3）一种用于鉴定猪 IAV 亚型的 rRT-PCR 检测方法。RNA 提取程序包括一种针对鼻拭子和组织匀浆优化的磁珠方法，以及一种针对口腔液优化的磁珠方法。两种用于检测猪 IAV 的 rRT-PCR 检测方法包括美国农业部验证的针对基质基因的 IAV rRT-PCR 和美国农业部许可的 VetMAX™-Gold 猪流感病毒检测 rRT-PCR 试剂盒（Thermo Fisher Scientific），该试剂盒针对核蛋白和基质基因。本文描述的猪 IAV 亚型鉴定检测方法是 VetMAX™-Gold 猪流感病毒亚型鉴定 rRT-PCR 试剂盒（Thermo Fisher Scientific），它可以区分猪 IAV H1 和 H3 以及 N1 和 N2。