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

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.