Evaluation of a reverse line blot assay for genotyping common human rotaviruses.
To allow high-throughput genotyping of group A rotaviruses (RVA) in a routine surveillance setting, we developed a reverse line blotting method for the determination of the most common human RVA G- and P-genotypes: G1-G4, G9, G12, P[4], P[6] and P[8]. Using the reverse line blotting method on 951 clinical RVA positive feces samples, in 905 (95%) of the samples the G-genotyping yielded a result while in 945 (99%) of the samples the P-genotyping was successful. Comparison of the reverse line blotting-method as it is used currently to a sequence based method for genotyping RVAs showed an agreement of 96% for single strain infections (75 out of 78) but only 48% for mixed infections (10 out of 21). The reverse line blotting method is successful in genotyping common RVA strains in surveillance settings. For genotyping of rare strains, the number of probes on the blot can be expanded or a sequence-based method can be performed as a complementary approach on the samples that are positive in the detection PCR but negative in the reverse line blotting genotyping assay. The complete reverse line blotting protocol takes 8h to complete for 36 samples, with 3h hands-on-time. In conclusion, a RVA genotyping method that is accurate, cheap and fast is presented.
Division for Virology, Centre for Infectious Diseases Control, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlands. Electronic address: Erwin.Duizer@RIVM.nl.
Erwin
E
Duizer
Bas
B
van der Veer
Erwin
E
de Bruin
Mark
M
Zeller
Elisabeth
E
Heylen
Marc
M
van Ranst
Jelle
J
Matthijnssens
Marion
M
Koopmans
Harry
H
Vennema
D005243:Feces / Q000821:virology*; D005838:Genotype; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D012401:Rotavirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D012400:Rotavirus Infections / Q000821:virology*; D013997:Time Factors; D014773:Virology / Q000379:methods*
Genotyping; Reverse line blot; Rotavirus