Application of new assays for rapid confirmation and genotyping of isolates of rubella virus.
Rubella virus (RV) isolation is recommended by the WHO Measles and Rubella Labnet for studying the etiology and epidemiology of rubella. However, the absence of cytopathologic effects (CPE) in many of the cell lines used commonly makes it difficult to confirm RV growth. In this study, two assays amplifying RV cDNA were developed and validated in order to confirm and genotype RV isolates after cell culture. A SYBR Green I-based real-time PCR (Rtime-SGE317) was established for initial rapid detection of RV in Vero cells and a nested PCR (PCR-E860) was used for amplifying further the 739 nt window of the E1 gene for the identification of RV genotype as recommended by the WHO. Sensitivities of the two assays were evaluated using eight RV isolates, two from infants with the congenital rubella syndrome (CRS) and six from patients with acute rubella. All the isolates had cycle threshold (C(t)) values <37 after the third passage, which is recommended as the cut-off for the confirmation of a viable RV isolate. Phylogenetic analysis based on the 739 nt window generated by the PCR-E860 showed that the eight RV isolates belonged to genotypes 1E, 1G, and 2B. The Rtime-SGE317 assay can be carried out in local public health laboratories, which would extend the molecular surveillance of rubella and contribute to the WHO goal of eradicating rubella worldwide.
Centre for Disease Control and Prevention of Zhejiang Province, Institute of Viral Diseases, Hangzhou, China.
Yan
Y
Feng
Sabine
S
Santibanez
Hazel
H
Appleton
Yiyu
Y
Lu
Li
L
Jin
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002522:Chlorocebus aethiops; D005838:Genotype; D006801:Humans; D007223:Infant; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics; D012409:Rubella / Q000175:diagnosis* / Q000821:virology; D012412:Rubella virus / Q000145:classification* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014709:Vero Cells; D014773:Virology / Q000379:methods*
D012367:RNA, Viral