Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific. Published applications of mPCR/RLB include detection of antibiotic resistance genes(1,2), typing of methicillin-resistant Staphylococcus aureus(3-5) and Salmonella sp(6), molecular serotyping of Streptococcus pneumoniae(7,8), Streptococcus agalactiae(9) and enteroviruses(10,11), identification of Mycobacterium sp(12), detection of genital(13-15) and respiratory tract(16) and other(17) pathogens and detection and identification of mollicutes(18). However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms. The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.