Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 55 issue 8 (august 2021) : 956-959

Development of Isothermal LAMP Assay for Detection of Intimin Gene (eae) in E. coli Associated with Piglet Diarrhea

Sanjeev Kumar, Jagan Mohanarao Gali, T.K. Dutta, P. Roychoudhury, P.K. Subudhi
1Department of Veterinary Microbiology, Central Agricultural University-Imphal, Selesih, Aizawl-796 015, Mizoram, India.
Cite article:- Kumar Sanjeev, Gali Mohanarao Jagan, Dutta T.K., Roychoudhury P., Subudhi P.K. (2021). Development of Isothermal LAMP Assay for Detection of Intimin Gene (eae) in E. coli Associated with Piglet Diarrhea. Indian Journal of Animal Research. 55(8): 956-959. doi: 10.18805/IJAR.B-4173.

ABSTRACT

Background: Diarroeagenic Escherichia coli (DEC) including enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) is associated with acute diarrhoea in children and young animals. The virulence is associated with attaching and effacing lesions encoded by eaeA gene is considered as marker for EPEC and EHEC. Laboratory diagnosis of such infections is carried out by traditional bacteriological techniques and by conventional PCR assays. Those techniques often provide false negative result and at the same time are costly as well as difficult to perform in the field level. The loop-mediated isothermal amplification (LAMP) is a new generation DNA amplification assay is developed for detection of eaeA gene in E. coli isolated from diarrhoeic piglets.
Methods: Samples were collected from diarrhoeic piglets for isolation and identification of E. coli. eaeA gene was detected by conventional PCR using specific primers in all the isolates. LAMP assay was standardized for detection of eaeA gene. Analytical sensitivity of LAMP was evaluated using 10 fold serially diluted E. coli genomic DNA. The specificity of the LAMP assay was determined by evaluating the cross reactivity with 19 other enteric and non-enteric bacterial species. Standardized LAMP was applied for detection of eaeA gene in the field isolates.
Result: A total of 37 (24.67%) isolates were recorded as positive for eaeA gene by conventional PCR, while 49 (32.67%) isolates were recorded as positive for eaeA gene by LAMP assay. The LAMP assay was 10 times more sensitive than conventional PCR. LAMP assay was found to be more sensitive, specific, cost effective, user friendly and reliable technique over conventional PCR, which can be applied for screening of the clinical isolates for confirmation of EPEC and/or EHEC.

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