Development of a new method for diagnosis of Group B Coxsackie genome by reverse transcription loop-mediated isothermal amplification

Jaianand, K.; Saravanan, N.; Gunasekaran, P.; Sheriff, A. K.
April 2011
Indian Journal of Medical Microbiology;Apr2011, Vol. 29 Issue 2, p110
Academic Journal
Background: Coxsackie B viruses (genus, Enterovirus; family, Picornaviridae) can cause aseptic meningitis, encephalitis, pleurodynia, and fatal myocarditis, and are implicated in the pathogenesis of dilated cardiomyopathy. The differentiation of the group B Coxsackieviruses into their subtypes has potential clinical and epidemiological implications. Objective: In this study, we developed a one-step, single-tube genogroup-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of group B Coxsackie genomes targeting 5′ UTR region. Materials and Methods: The amplification can be obtained in less than 1 hour by incubating all the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63°C. Detection of gene amplification could be accomplished by agarose gel electrophoresis and the monitoring of gene amplifi cation can also be visualised with the naked eye by using SYBR green I fluorescent dye. Results: A total of 40 samples comprising 31 positive samples and 9 negative samples were used in this study for comparative evaluation. The results were compared with those from Real-Time Polymerase Chain Reaction (RT-PCR). None of the RT-PCR-positive samples were missed by RT-LAMP, thereby indicating a higher sensitivity of the RT-LAMP assay. Conclusion: Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is extremely rapid, cost-effective, highly sensitive, and specifi c and has potential usefulness for rapid detection of non-polio enterovirus (NPEV) not only by well-equipped laboratories but also by peripheral diagnostic laboratories with limited fi nancial resources in developing countries.


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