Indian Journal of Experimental Biology Vol. 55, December 2017, pp. 876-879 Note Real-Time PCR assay and melting curve analysis for identification of diarrheagenic Escherichia coli in paediatric population Taru Singh 1 *, Shukla Das 1 , VG Ramachandran 1 , Khan Amir Maroof 2 & Arvind Rai 3 1 Department of Microbiology; 2 Department of Community Medicine, UCMS & Guru Teg Bahadur Hospital, New Delhi, India; 3 Divisioin of Biotechnology & Molecular Biology National Centre for Disease Control, New Delhi, India Received 29 August 2015; revised 12 June 2016 In developing countries, including India, diarrhea causes considerable death among infants and children. Early diagnosis plays a vital role in preventing the fatality rate. Conventional methods for early detection of diarrheagenic E. coli (DEC) are complicated and inaccurate. Hence, in this study we proposed a syber green based Real-Time PCR to determine the prevalence of DEC. Detection of diarrheagenic E. coli using Real-Time Syber Green based PCR assay was performed in 120 isolates including 80 isolates from healthy children. Virulent genes were detected in 106 (88.3%) samples [39 (97.5%) and 67 (83.7%)] in cases and controls, respectively. EPEC was found (typical, atypical and bfp) in 87 (72.5%) specimens followed by EAEC ( eagg) in 76 (63.3%), ETEC (elt and est) in 55 (45.8%) and EHEC (stx and hyla) in 11 (9.2%) specimens. Melting curve analysis showed distinct peaks for all target genes. Keywords: Diarrhoea, E. coli infections, Real-Time PCR In India, diarrhea remains one of the five important causes of death among infants and under five children 1 . On the basis of distinct epidemiological and clinical features, specific virulence determinants and association with certain serotypes, the pathogenesis of E. coli has been known into six categories 2 . Only few studies have shown the prevalence of all DEC types together in paediatric population. Since conventional methods for early detection of diarrheagenic E. coli (DEC) remains complicated and imprecise 3 . Here, we performed Syber green based Real-Time PCR to determine the prevalence of DEC in children under five years of age suffering from diarrhea and their “presence” as commensal in non-diarrheal stool samples of healthy children. Materials and Methods This study had two groups: Group I included 40 children suffering from acute diarrhoea (of less than 72 h duration) and attending the Out Patient Department services of a tertiary care hospital; and Group II included 80 healthy children below five years of age. The study protocol was approved by the institutional ethical committee. Fresh stool samples were collected and inoculated on media as per standard laboratory methods and E. coli was identified based on biochemical reactions 4 . DNA extraction was done using commercial kit. (Cat no. YGB100 Hi Yield Real Biotech Corporation, Taiwan). Primers used for amplifying the sequences (Table 1) 5-10 , were based on previously published literature. Standard bacterial control strains were obtained from the National Institute of Cholera and Enteric Diseases (NICED, Kolkata, India). Initially, conventional multiplex PCR was also performed following previously published literature 8,10 . Real- Time PCR was carried out in a Roche Light Cycler 480-II. Melting peak for each gene was shown and average Tm was calculated by software. Statistical analysis was done using Statistical Package for Social Sciences (SPSS) package (SPSS; Version 20.0). The chi square test and Fisher's exact test were used to determine statistical significance of data. P value <0.05 was considered significant. Multivariable logistic regression was also done to determine the risk of predominant DEC infection with increase in age in cases and controls. Results and Discussion Despite the existence of National Guidelines for management of diarrhea at the community level, diarrheagenic E. coli has been identified as an important cause of infantile and young childhood diarrhea in all the developing countries 11 . The role of these pathogens remains uncertain due to inappropriate diagnostic methods. The pathogenic strains of E. coli may carry several virulence factors that directly involved in pathogenesis of these bacteria, although commensal strains may also cause —————— *Correspondence: Phone: +91 2692 271605, 271606 Ext. 209; Fax: +91 2692 271601 E-mail: taru9458@gmail.com Supplementary data available online at NOPR with respective file.