Comparison of specific and random priming in the reverse transcriptase polymerase chain reaction for genotyping group A rotaviruses

• Published in: Journal of virological methods 1999-03, Vol.78 (1), p.93-103
• Main Authors: Iturriza-Gomara, M., Green, J., Brown, D.W.G., Desselberger, U., Gray, J.J.
• Format: Article
• Language: English
• Subjects: Aged; Biological and medical sciences; Child; Diarrhea - virology; DNA Primers; DNA, Complementary; Enzyme-Linked Immunosorbent Assay; Feces - virology; Fundamental and applied biological sciences. Psychology; Genotype; Genotyping; Humans; Microbiology; Random priming; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - isolation & purification; Rotavirus - classification; Rotavirus - genetics; Rotavirus - isolation & purification; Rotavirus Infections - virology; Rotaviruses; RT-PCR; Serotyping; Techniques used in virology; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/S0166-0934(98)00168-2

This study describes an approach to the molecular typing of rotaviruses which requires only a single RNA extraction and reverse transcription (RT) reaction using random primers. Random-primed RT provides complementary DNA (cDNA) which can be used not only for G- and P-typing polymerase chain reactions (PCR), but also for the detection of other RNA viruses which may act as enteric pathogens. It is a sensitive and specific method that can detect 10 virus particles/ml of 10% faecal suspension provided the cDNA is amplified in a nested typing-PCR. Of 121 specimens positive for rotavirus by EM and analysed using this method, only 8% could not be G- or P-genotyped. The untyped samples were tested again performing the RT reaction with G- and P-specific primers, achieving a 5% increase in sensitivity. Comparing G-genotyping against G-serotyping, 92% were genotyped through random priming RT-PCR whereas only 64% were serotyped using G-serotype specific monoclonal antibodies (MAbs).