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 sp⁶, molecular serotyping of Streptococcus pneumoniae^7,8, Streptococcus agalactiae⁹ and enteroviruses^10,11, identification of Mycobacterium sp¹², detection of genital^13-15 and respiratory tract¹⁶ and other¹⁷ pathogens and detection and identification of mollicutes¹⁸. 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.