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

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dc.contributor.authorDe, Ken_US
dc.contributor.authorRamamurthy, Ten_US
dc.contributor.authorGhose, A Cen_US
dc.contributor.authorIslam, M Sen_US
dc.contributor.authorTakeda, Yen_US
dc.contributor.authorNair, G Ben_US
dc.contributor.authorNandy, R Ken_US
dc.date.accessioned2001-09-05en_US
dc.date.accessioned2009-05-27T07:41:17Z
dc.date.available2001-09-05en_US
dc.date.available2009-05-27T07:41:17Z
dc.date.issued2001-09-05en_US
dc.description.abstractBACKGROUND & OBJECTIVES: Biotyping of Vibrio cholerae O1 using multiplex PCR (ctxA-tcpA) exploits the nucleotide sequence differences of the major subunit protein of the toxin co-regulated pilus (TCP) gene (tcpA) to differentiate between the classical and El Tor biotypes. However, the presence of classical biotype specific tcpA amplicon with the El Tor strains often complicates the interpretation. The effect of PCR variables on the amplification of biotype specific tcpA in the multiplex PCR has been investigated. METHODS: Reference strains of toxigenic V. cholerae O1 belonging to classical and El Tor biotypes were selected to optimize the PCR variables for the unambiguous biotype determination by multiplex PCR. RESULTS: In the multiplex PCR assay, a reduction in the reaction volume from 100 microliters to 25 microliters and the annealing temperature of 64 degrees C, the El Tor strain produced ctxA amplicon (302 bp) along with tcpA amplicons of 618 bp and 472 bp which are specific for classical and El Tor tcpA respectively. The simplex PCR with biotype specific tcpA primer pairs showed the amplification of either 472 bp or 618 bp tcpA amplicon with El Tor template. With the classical biotype strain, the specific primer pair yielded tcpA amplicon of the expected size. Lowering of PCR annealing temperature from 64 to 60 degrees C resulted in the elimination of the amplification of the nonspecific tcpA amplicon with El Tor strain. INTERPRETATION & CONCLUSION: A comparison of the theoretical melting temperature (Tm) values of the reacting primers, and their alignment to the biotype specific tcpA revealed the basis of unambiguous biotyping of V. cholerae O1 at a PCR annealing temperature of 60 degrees C.en_US
dc.description.affiliationNational Institute of Cholera & Enteric Diseases (ICMR), Kolkata, India.en_US
dc.identifier.citationDe K, Ramamurthy T, Ghose AC, Islam MS, Takeda Y, Nair GB, Nandy RK. Modification of the multiplex PCR for unambiguous differentiation of the El Tor & classical biotypes of Vibrio cholerae O1. Indian Journal of Medical Research. 2001 Sep; 114(): 77-82en_US
dc.identifier.urihttps://imsear.searo.who.int/handle/123456789/21682
dc.language.isoengen_US
dc.source.urihttps://icmr.nic.in/ijmr/ijmr.htmen_US
dc.subject.meshBacterial Outer Membrane Proteins --chemistryen_US
dc.subject.meshBacterial Typing Techniquesen_US
dc.subject.meshBase Sequenceen_US
dc.subject.meshCholera --microbiologyen_US
dc.subject.meshFimbriae Proteinsen_US
dc.subject.meshFimbriae, Bacterial --metabolismen_US
dc.subject.meshHumansen_US
dc.subject.meshMolecular Sequence Dataen_US
dc.subject.meshPolymerase Chain Reaction --methodsen_US
dc.subject.meshProtein Subunitsen_US
dc.subject.meshTemperatureen_US
dc.subject.meshVibrio cholerae --classificationen_US
dc.titleModification of the multiplex PCR for unambiguous differentiation of the El Tor & classical biotypes of Vibrio cholerae O1.en_US
dc.typeJournal Articleen_US
dc.typeResearch Support, Non-U.S. Gov'ten_US
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