, 2008) and rtxA1 was used to determine the location of CTX prophage in the large chromosome (Colombo et al., 1994; O’Shea et al., 2004). The rtxA gene encodes a presumptive cytotoxin that Ku-0059436 supplier is a part of the RTX (repeats in toxin) gene cluster containing GD-rich repeated motifs, which represent a family of toxin well disseminated in Gram-negative bacteria and has been reported to be present in the large chromosome adjacent to ctx genes (Lin et al., 1999; Sheahan et al., 2004). Vibrio cholerae O1 strains devoid of a CTX prophage in the small chromosome but possessed of the same in the large chromosome without

any direct repeat sequence (RS) element connecting the core downstream of ctx genes will yield an amplicon of nearly 2.4 kb. Another combination of primers zotF and rtxA1 was this website used to determine the presence of CTX prophages lacking the ctxAB operon and lying downstream of the RS1 element adjacent to rtx genes, which will produce an expected amplicons of ∼2.35 kb. Purified genomic DNA was treated with suitable restriction endonuclease enzymes and separated by electrophoresis in 0.8% agarose

gels. DNA fragments were denatured by treatment with alkali and subsequently transferred to a nylon membrane (Hybond-N+; Amersham Pharmacia Biotech), according to the procedure of De et al. (2005), and hybridized with a DNA probe. CTX typing was performed by digesting the genomic Thiamet G DNA with HindIII, PstI, AvaI and BglII (Takara). A 540-bp XbaI–ClaI fragment of ctxA

was ligated with the EcoRI linker and subsequently the ligated product was cloned into the EcoRI site of pKTN901 that served as a probe for ctxA (Kaper et al., 1988). The specific probes of cep (core-encoded pilus) encoding a putative colonization factor present in the core (Pearson et al., 1993), rstRET and rstRcalc, which are cloned in the plasmids pSC01, pSC06 and pSC10, respectively, were obtained by digesting the plasmids individually with EcoRI (Chatterjee et al., 2007). DNA probes were labelled with chemiluminescent dye (Amersham Biosciences) and hybridization reactions were developed following the manufacturer’s protocol and recognition patterns recorded on X-ray film. The results of MAMA PCR showed that all V. cholerae O139 strains isolated up to 1995 yielded amplicons with El Tor allelic primer pair of ctxB only. But 54% and 18% of the V. cholerae O139 strains isolated during 1996 produced amplicons with El Tor and classical specific ctxB primer pairs, respectively, while 28% of the tested strains yielded amplicon with both classical and El Tor primer pairs of ctxB (Table 2). The same trend was continued among V. cholerae O139 strains isolated in 1997. Strains isolated during 1998 did not produce amplicons using only the El Tor ctxB primer pair, but 68% produced amplicon with classical specific ctxB primers and 32% yielded amplicons with both classical and El Tor-specific ctxB primer pairs.