Culture-independent real-time PCR reveals extensive polymicrobial infections in hospitalized diarrhoea cases in Kolkata, India A. Sinha, S. SenGupta,

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Culture-independent real-time PCR reveals extensive polymicrobial infections in hospitalized diarrhoea cases in Kolkata, India A. Sinha, S. SenGupta, S. Guin, S. Dutta, S. Ghosh, P. Mukherjee, A.K. Mukhopadhyay, T. Ramamurthy, Y. Takeda, T. Kurakawa, K. Nomoto, G.B. Nair, R.K. Nandy Clinical Microbiology and Infection Volume 19, Issue 2, Pages 173-180 (February 2013) DOI: 10.1111/j.1469-0691.2011.03746.x Copyright © 2013 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 1. (Comparative analysis for detecting pathogens by culture-dependent and culture-independent methods. Clinical Microbiology and Infection 2013 19, 173-180DOI: (10.1111/j.1469-0691.2011.03746.x) Copyright © 2013 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 2. Melting curve analysis of real-time SYBR Green PCR amplicons of (a) wb of Vibrio cholerae Ol, (b) 23S rDNA of Vibrio parahaemolyticus, (c) It of enterotoxigenic Escherichia coli (ETEC), (d) st of ETEC, (e) aagR of enteroaggregative E. coli, (EAEC), (f) eaeA of enteropathogenic E. coli (EPEC) (g) 16S rDNA of Campylobacter spp. and (h) ipaH of Shigella spp. Temperature of melting (Tm) of the amplicons specific to seven different pathogens are indicated. In all the panels bold line (—) represents analysis of PCR amplicons obtained against DNA from each of the above-mentioned pathogens used individually with respective primers whereas broken line (—) represents analysis of PCR amplicons obtained against mixture of 1:100 times diluted DNA (all above-mentioned seven pathogens) with pathogen-specific primers. Clinical Microbiology and Infection 2013 19, 173-180DOI: (10.1111/j.1469-0691.2011.03746.x) Copyright © 2013 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 3. Standard curve generated by plotting varying number or Vibrio cholerae viable cells against corresponding Ct value obtained in a real-time PCR assay carried out in 20-μL reaction volume with SYBR Green as detecting dye. Estimation of actual counts of viable cells was made by dilution plating method using ten-fold serial dilution of the cells. The average Ct value for duplicate samples obtained per dilution was used for plotting. Clinical Microbiology and Infection 2013 19, 173-180DOI: (10.1111/j.1469-0691.2011.03746.x) Copyright © 2013 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 4. Detection of pathogens (a) in 68 diarrhoeal stool specimens with aetiology by culture-dependent and culture-independent real-time PCR assays and (b) in 54 diarrhoeal stool specimens with ‘no known pathogen’ by culture-dependent assay but with aetiology when analysed by real-time PCR assays. White bar (□) and black bar (▪) represent detection of pathogens by culture-based assays and culture-independent real-time PCR assays, respectively. Number on the side of each bar represents specimens with respective aetiology. Abbreviations: n, total number of specimens analysed; Vc, Vibrio cholerae; Vp, Vibrio parahaemolyticus; Cj, Campylobacter spp.; and Shig, Shigella spp. Clinical Microbiology and Infection 2013 19, 173-180DOI: (10.1111/j.1469-0691.2011.03746.x) Copyright © 2013 European Society of Clinical Infectious Diseases Terms and Conditions