Genotyping of human cytomegalovirus using non-radioactive single-strand conformation polymorphism (SSCP) analysis.
Genetic variation in the glycoprotein B (gB) gene may play a role in human cytomegaloviruses (HCMVs) pathogenesis. Using restriction analysis of the gB gene product (PCR-RFLP), amplified by the nested polymerase chain reaction, the HCMV strains can be compared and classified into at least four HCMV groups. PCR single-strand conformation polymorphism (PCR-SSCP) is one of the techniques used to identify a mutant sequence or a polymorphism in a known gene. SSCP analysis has the advantage over RFLP analysis on detection of DNA polymorphisms and point mutations at a variety of positions in DNA fragments. However, the original SSCP protocols using the incorporation of radioactive label and polyacrylamide gel electrophoresis for detection are labour intensive and time-consuming. A simplified SSCP protocol is described to identify HCMV strains and the gB genotype, allowing the detection of sequence variations not residing in the endonuclease recognition sites.
de Albuquerque
Dulcinéia Martins
DM
Department of Pharmacology, College of Medicine, State University of Campinas, Distrito de Barão Geraldo, SP 13083-970, Campinas, Brazil. dulmal@unicamp.br <dulmal@unicamp.br>
Costa
Sandra Cecília Botelho
SC
D016026:Bone Marrow Transplantation / Q000009:adverse effects; D003587:Cytomegalovirus / Q000145:classification* / Q000235:genetics*; D003586:Cytomegalovirus Infections / Q000821:virology*; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014759:Viral Envelope Proteins / Q000235:genetics*
D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus