Rapid and sensitive genotyping of Epstein-Barr virus using single-strand conformation polymorphism analysis of polymerase chain reaction products.
Two distinct wild-type Epstein-Barr virus (EBV) strains (A and B) that have significantly diverged at the two small RNA-encoding region (EBER) have been identified (Arrand et al., 1989). In order to test whether single-strand conformation polymorphism analysis (SSCP) would correlate with these sequence variations, we designed primer pairs specific for EBER-encoding regions for amplification of divergent sequences by polymerase chain reaction (PCR). The PCR-amplified products from six EBV-positive cell lines were analyzed by SSCP method, and the results were compared with the prototype strains B95-8 (type A) and AG876 (type B). Type-specific point mutations were detected as demonstrated by shifts in mobility due to conformational changes of DNA sequences. The locations of point mutations were identified by direct sequencing of the PCR amplified DNA. Of the three primer pairs designed, the pair that amplified a 190 bp fragment spanning six type-specific point mutations gave the best resolution in SSCP analysis. This pair is now preferred for initial genotyping of EBV-infected tumor biopsies. Thus, SSCP is a simple, fast and efficient technique for genotyping of EBV-associated diseases.
Molecular Biology Section, Centers for Disease Control, Atlanta, GA 30333.
J C
JC
Lin
B K
BK
De
S C
SC
Lin
D000956:Antigens, Viral / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004277:DNA, Single-Stranded / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D019309:Epstein-Barr Virus Nuclear Antigens; D005838:Genotype; D004854:Herpesvirus 4, Human / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D000956:Antigens, Viral; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D015345:Oligonucleotide Probes