The cell pellet was then resuspended in 300 μL of sterile distilled water and boiled for Selleck MDV3100 10 min in a water bath. The boiled sample was snap-cooled on ice and centrifuged at 13 684 g for 10 min. The supernatant was collected in a sterile microfuge and 5 μL of this supernatant was used as template for PCR analysis. Mismatch amplification mutation assay (MAMA) PCR, which detects sequence polymorphisms between CT genotype 1 (classical type CT) and genotype 3 (El Tor type CT) based on the nucleotide position 203 of the ctxB gene (Morita et al., 2008), has been utilized in this study with O139 strains. The rstR PCR was performed to determine the allele type of rstR (regulatory
region for phage lysogeny) of CTX phages present in the O139 strains of Kolkata (Kimsey et al., 1998; Nusrin et al., 2004). The primers used in this study are given in the Table 1. Vibrio cholerae O1 Everolimus purchase strains O395 and N16961 were used
as standard reference strains for classical and El Tor biotypes, respectively. To determine the nucleotide sequence of the ctxB, PCR amplification of ctxB locus of 22 strains of V. cholerae O139 was performed in a 25-μL reaction mixture using Ex Taq™ polymerase (Takara, Japan) with proofreading activity. The PCR primers and conditions used have been described previously (Olsvik et al., 1993). PCR products were purified with the QIAquick PCR purification kit (Qiagen GmBH, Germany) and both strands were sequenced in an automated sequencer (ABI PRISM 3100 Genetic Analyser, Applied Biosystems). The sequences obtained here were deposited in GenBank with the accession numbers FJ999956–FJ999988. Amplicons of ∼3,
6.3 and 6 kb were obtained by PCR with primer pairs ctxA (F) and rtxA1, rstR2F and ctxB (R), and rstR3F and ctxB (R), respectively, using XT-20 PCR system (Bangalore Genei), and the products were separated by electrophoresis using 1% agarose gel in TAE buffer followed by staining with ethidium bromide. A λ-HindIII molecular size ladder (Takara) was run with the gel. The desired DNA fragments were excised from agarose gels and purified using a Gel Extraction kit (Qiagen GmBH). The purified DNA of ∼3, 6.3 and 6 kb thus obtained were used as template for nested PCR using ctxB (F) and ctxB (R) primers (Olsvik et Osimertinib nmr al., 1993). Nucleotide sequencing of ctxB genes was performed with the resulting 460-bp amplicon. Chromosomal localization of the CTX prophages of V. cholerae O139 strains was performed using two sets of primers followed by Southern blot hybridization. The specific primer pair consisting of CIIF and CIIR, as described earlier (Maiti et al., 2006), was used to confirm the CTX prophage in the small chromosome. Strains that do not have CTX prophage in the small chromosome will give an expected PCR amplicon of 766 bp. The strains that have CTX prophage integrated between these regions in the small chromosome will not yield any amplicon in the assay due to the large size (around 8 kb) of the target gene.