Development of a simple restriction fragment length polymorphism assay for subtyping of coxsackie B viruses.
Coxsackie B viruses (genus, Enterovirus; family, Picornaviridae) can cause aseptic meningitis, encephalitis, pleurodynia, 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. In the present study, a simple restriction fragment length polymorphism (RFLP) assay was developed for typing of group B coxsackieviruses into subtypes 1-6. It is a two step process, first, virus isolation and identification by virus neutralization assay, using pools of polyclonal antisera, second, the reverse transcription polymerase chain reaction (RT-PCR) using a single primer pair selected from the conserved 5'-untranslated region (5'-UTR) of enterovirus genome followed by RFLP. A 440 bp product was amplified from the reference strains of each subtype of group B coxsackievirus and 29 clinical isolates (positive for group B coxsackieviruses by neutralization assay). The amplified products were subjected to restriction endonuclease digestion by enzyme BsaJI. The assay was able to distinguish all six serotypes of coxsackie B viruses. The results were comparable to serotyping and showed that due to the relatively conserved nature of 5'-UTR in enterovirus genome, this region can be used for subgeneric molecular identification of enteroviruses.
Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical sciences (S.G.P.G.I.M.S.), Lucknow 226 014, India.
D D
DD
Patel
A
A
Kapoor
A
A
Ayyagari
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TN
Dhole
D020121:5' Untranslated Regions / Q000235:genetics; D017931:DNA Primers; D029822:Enterovirus B, Human / Q000145:classification* / Q000235:genetics; D004769:Enterovirus Infections / Q000175:diagnosis / Q000821:virology*; D005838:Genotype; D006801:Humans; D012150:Polymorphism, Restriction Fragment Length; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods
D020121:5' Untranslated Regions; D017931:DNA Primers