Modification of the multiplex PCR for unambiguous differentiation of the El Tor & classical biotypes of Vibrio cholerae O1.

摘要: Background & Objectives: Biotyping of Vibrio cholerae O1 using multiplex PCR (ctxA-tcpA) exploits the nucleotide sequence differences the major subunit protein toxin co-regulated pilus (TCP) gene (tcpA) to differentiate between classical and El Tor biotypes. However, presence biotype specific tcpA amplicon with strains often complicates interpretation. The effect variables on amplification biotype in has been investigated. Methods: Reference toxigenic V. O1 belonging biotypes were selected optimize for unambiguous determination by PCR. Results: In assay, a reduction reaction volume from 100 microliters 25 microliters annealing temperature 64 degrees C, strain produced ctxA amplicon (302 bp) along amplicons 618 bp 472 which are respectively. The simplex primer pairs showed either or template. With strain, pair yielded expected size. Lowering 60 C resulted elimination amplification nonspecific strain. Interpretation Conclusion: A comparison theoretical melting (Tm) values reacting primers, their alignment revealed basis biotyping at C.