Simultaneous Identification of Diarrheagenic Escherichia coli Using Thermostabilized Multiplex PCR Formulation

Shruthi Aradhya¹ , Shylaja Ramlal² , Radhika Madalgi³ , Harishchandra S. Murali⁴ , Bhairab Mondal⁵ , Ajantha G.S.⁶ , Raghavendra D. Kulkarni⁷ , Harshvardhan V. Batra⁸

Section:Research Paper, Product Type: Journal-Paper
Vol.7 , Issue.5 , pp.38-46, Oct-2020

Online published on Oct 31, 2020

Copyright © Shruthi Aradhya, Shylaja Ramlal, Radhika Madalgi, Harishchandra S. Murali, Bhairab Mondal, Ajantha G.S., Raghavendra D. Kulkarni, Harshvardhan V. Batra . This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
 

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Abstract :
The study was aimed to develop a multiplex PCR (mPCR) coupled with the thermostabilization process for the simultaneous detection of major genes of diarrheagenic Escherichia coli (DEC) with internal amplification control (IAC) to avoid false-negative results. The most important virulence-associated genes of major pathotypes are targeted in the mPCR assay. The genes include aggR, eltA, bfpA, vt2 and ipaH along with an IAC. Further, to enhance the application of mPCR, the master mix containing all reagents except template DNA was lyophilized with an appropriate lyoprotectant and storage stabilization studies were carried out to evaluate the performance of the lyophilized master mix. The assay was able to detect 103 CFU/mL for the simultaneous detection of targeted genes and 104 CFU/g bacterial load in spiked stool samples. The shelf-life of lyophilized master mix with a suitable lyoprotectant was found to be six months at 4 oC and 1.5 months at ambient temperature and could be stored at ambient temperature without a cold-chain facility. Considering the importance of DEC in the field of diagnostics, rapid, robust, user-friendly detection platforms are essential to prevent any outbreaks. The results obtained in the present study supported that it could be used as a highly reliable diagnostic tool in point-of-care laboratories and also field-based investigations. Besides, the developed assay can also cut down costs in terms of storage and transportation suggesting a potential field application format.

Key-Words / Index Term :
Diarrheagenic Escherichia coli; Multiplex PCR; Thermo stabilization; E. coli pathotypes; lyophilization

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