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Multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB)—a practical epidemiological and diagnostic tool

Nature Protocols volume 1, pages 2668–2680 (2006)Cite this article

Abstract

Combining multiplex PCR, sequentially, with reverse line blot hybridization (mPCR/RLB) is a convenient, objective way to identify up to 43 targets in 43 individual specimens simultaneously (using a 45-lane membrane format). It is more flexible and less expensive than DNA microarray. The number of targets is adequate for epidemiological and most clinical diagnostic applications; based on the same target (43) and specimen numbers (43), it is much more practical than conventional uniplex PCR (uPCR) and mPCR. We have used the protocol to identify and subtype bacteria, viruses and fungi and identify pathogens in clinical specimens; potentially, it could be used for many other applications, such as detection of mutations in, or identification of alleles of, eukaryotic genes. Development of each assay involves (i) careful primer and probe design, based on literature and sequence database searches, which are critical to success of the assay; and (ii) bench-top evaluation, using known samples, controls and dilution series, to confirm sensitivity, specificity and reproducibility. The assay takes about one and half working days to complete; about 4 h for the mPCR and 6 h for the RLB, including a total of 4 h 'hands-on' time.

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Figure 1: The mPCR/RLB principles relevant to PROCEDURE steps.
Figure 2
Figure 3
Figure 4: Relative locations of primers and probes.
Figure 5: An actual example of mPCR/RLB results, on X-ray film, for S. agalactiae—identification of capsular serotype and detection of nine antibiotic resistance-related genes.

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Acknowledgements

The authors (F.K. and G.L.G.) wish to thank Peter Jelfs, Gregory James and staff of the reference laboratories at CIDM for support and assistance; Ping Zhu for design of the RLB reading template and, with Kathryn Shaw, for contribution to the figures; Mitchell Brown, Kathryn Shaw, Michelle McKechnie, Danny Ko, Fei Zhou, Zhongsheng Tong and Qinning Wang for help with editing and checking the manuscript; Vitali Sintchenko, Heather Gidding, Sharon Chen, Xianyu Zeng, Yongwei Cai, Zutao Zhao, Yajuan Wang, Meng Xiao, Xu Wang, Jon Iredell, Bin Wang and Frank Lin for helpful comments; Hui Wang, Geoffrey Playford, Ying Sun, Archana Sabananthan, Lin Ma, Yonghong Yang, Likuan Xiong and Weixiong He for contributions to development of individual assays. F.K. also would like to thank Zhenfang Ma for her continuous support and encouragement for his research and writing over many years.

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Authors and Affiliations

  1. Centre for Infectious Diseases and Microbiology–Public Health (CIDM–PH), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Sydney West Area Health Service (SWAHS), Westmead, 2145, New South Wales, Australia

    Fanrong Kong & Gwendolyn L Gilbert

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  1. Fanrong Kong
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F.K. performed the experiments and wrote the manuscript; G.L.G. supervised the research and the writing of the manuscript.

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Correspondence to Gwendolyn L Gilbert.

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Supplementary information

Supplementary Fig. 1

We have designed a reading template with the Y-axis for probes; the legend can be modified according to targets of assay. This step makes reading the blot much easier. The template matches the 45 lanes and users can download the template, type in the probe names in the left column and print on A4 paper. X-ray films are placed on the template to match the probe/specimen numbers with hybridization signals. (PDF 416 kb)

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Kong, F., Gilbert, G. Multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB)—a practical epidemiological and diagnostic tool. Nat Protoc 1, 2668–2680 (2006). https://doi.org/10.1038/nprot.2006.404

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