Lappan Rachael, Jirapanjawat Thanavit, Williamson Deborah A, Lange Sigrid, Chown Steven L, Greening Chris
Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.
Department of Microbiology and Immunology, University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.
MethodsX. 2022 Apr 20;9:101707. doi: 10.1016/j.mex.2022.101707. eCollection 2022.
Quantitative polymerase chain reaction (qPCR) is a gold standard method for the detection and quantification of pathogenic organisms. Standard qPCR is inexpensive, sensitive and highly specific to the pathogen of interest. While qPCR assays can be multiplexed to allow the detection of multiple organisms in one reaction, it is prohibitively labour intensive to screen large numbers of samples for several pathogens at the same time. The TaqMan Array Card (TAC) is a cost-effective and accurate technique that expands the number of assays that can be simultaneously performed on a sample, with no increase in set-up time and only small reductions in sensitivity. This approach is highly beneficial in settings where there is a need to monitor a large panel of pathogens. We illustrate the application of TAC to the monitoring of gastrointestinal pathogens, which span viral, bacterial, protist and helminth taxa. This protocol outlines the laboratory set-up of a TaqMan Array Card, and some recommended data processing steps to aid in accurate interpretation of the results. A video protocol is additionally provided to assist in the use of the technique.•The TAC is designed primarily for gene expression assays, but has recently been utilised in several studies for pathogen detection in human clinical samples.•We expand the use of TAC for pathogen detection across human, animal and environmental sample types, and have developed a protocol and guidelines for the processing and interpretation of results that circumvents issues with the automated outputs.•This technique is applicable to pathogen or organism detection in any context, if quality nucleic acid extracts can be obtained from the sample type of interest.
定量聚合酶链反应（qPCR）是检测和定量病原生物的金标准方法。标准qPCR成本低廉、灵敏度高且对目标病原体具有高度特异性。虽然qPCR检测可进行多重检测，以便在一个反应中检测多种生物，但同时对大量样本进行多种病原体的筛查，其劳动强度大得令人望而却步。TaqMan阵列卡（TAC）是一种经济高效且准确的技术，它增加了可在一个样本上同时进行的检测数量，设置时间没有增加，灵敏度仅略有降低。这种方法在需要监测大量病原体的情况下非常有益。我们阐述了TAC在监测胃肠道病原体方面的应用，这些病原体涵盖病毒、细菌、原生生物和蠕虫类群。本方案概述了TaqMan阵列卡的实验室设置，以及一些推荐的数据处理步骤，以帮助准确解读结果。此外还提供了一个视频方案，以协助该技术的使用。•TAC主要设计用于基因表达检测，但最近已在多项研究中用于检测人类临床样本中的病原体。•我们将TAC在病原体检测方面的应用扩展到人类、动物和环境样本类型，并制定了一个方案和结果处理与解读指南，以规避自动输出结果时出现的问题。•如果能够从感兴趣的样本类型中获得高质量的核酸提取物，那么这项技术适用于任何情况下的病原体或生物检测。
