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.
Patel
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DD
Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical sciences (S.G.P.G.I.M.S.), Lucknow 226 014, India.
Kapoor
A
A
Ayyagari
A
A
Dhole
T N
TN
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