Comparison of specific and random priming in the reverse transcriptase polymerase chain reaction for genotyping group A rotaviruses.
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).
Iturriza-Gomara
M
M
Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge, UK.
Green
J
J
Brown
D W
DW
Desselberger
U
U
Gray
J J
JJ
D000368:Aged; D002648:Child; D017931:DNA Primers; D018076:DNA, Complementary; D003967:Diarrhea / Q000821:virology*; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000821:virology; D005838:Genotype; D006801:Humans; D012367:RNA, Viral / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D012400:Rotavirus Infections / Q000821:virology*; D012703:Serotyping
D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral