REAL-TIME MICROCHIP PCR FOR DETECTING SINGLE-BASE DIFFERENCES IN VIRAL AND HUMAN DNA

This report describes real-time 5' nuclease PCR assays to rapidly dist inguish single-base polymorphism using a battery-powered miniature ana lytical thermal cycling instrument (MATCI). Orthopoxviruses and the hu man complement component C6 gene sewed as targets to demonstrate the f easibility of using the MATCI for diagnosis of infectious diseases and genetic disorders. In the Orthopoxvirus assay, consensus Orthopoxviru s PCR primers were designed to amplify 266-281 base-pair (bp) segments of the hemagglutinin (HA) gene in camelpox, cowpox, monkeypox, and va ccinia viruses. A vaccinia virus-specific fluorogenic (TaqMan) probe w as designed to detect a single-base (A/G) substitution within the HA g ene. In the C6 gene assay, a 73-bp segment of the C6 gene was PCR-ampl ified from human genomic DNA, and TaqMan probes were used to detect a single-base (A/C) polymorphism in the second position of codon 98. The MATCI correctly identified the nucleotide differences in both viral D NA and human genomic DNA, In addition, using a rapid DNA preparation m ethod, it was possible to achieve sample, preparation of human genomic DNA, DNA amplification, and real-time detection in less than 1 h.