Genotype analysis for measles vaccine strain and wild-type viruses of H1 genotype by RT-PCR-RFLP / 中华微生物学和免疫学杂志

ABSTRACT

Objective To establish RT-PCR-RFLP method for studying the genotype of wild mea-sles virus strains isolated from Tianjin area from 2002 to 2008. Methods Isolations of measles virus were carried out by tissue culture method from urine and throat swab specimens collected from suspected cases. RNA were extracted from the virus specimens. The 594 bp fragment of C terminal of the N (nucleoprotein) gene was amplified by one-step RT-PCR, then the PCR products were digested with Bcn I , separated on agarose gel electrophoresis and then analyzed by the method of RFLP (restriction fragment length polymor-phism). In addition, above results were compared with DNA sequencing. Phylogenetic tree was plotted based on the results for the genetic relationship and distance analysis. Results Sixty-nine measles virus strains were isolated from 189 specimens from 2002 to 2008, of which the C terminals of N gene were all de-tected positive. Among the 69 strains of measles virus isolates, 98.55% (68/69) belonged to Hla sub-geno-type which was the predominant sub-genotype, and only one strain (1.45%) belonged to H1b sub-genotype by RFLP analysis which was in accordance with the results by DNA sequencing method. Phylogenetic tree analysis indicated the H1a sub-genotype measles virus strains should be further divided into 2 clades, and the variation fluctuated between 0.2% and 3.8%. There were transmission chains caused by different virus strains co-cireulation. Conclusion A genotype, H1a and H1b sub-genotype can be identified by RT-PCR-RFLP assay specically based on the restriction enzyme Bcn I .The RT-PCR-RFLP assay can be a rapid, simple, accurate and efficient method for large-scale surveillance of measles virus strains in China.