A novel multiplex RT-PCR for identification of VP6 subgroups of human and porcine rotaviruses.
VP6 protein antigens allow classification of rotaviruses into at least four subgroups, depending on the presence or absence of SG-specific epitopes: SG I, SG II, SG (I+II), and SG non-(I+II). However, MAbs against epitopes on the VP6 protein of human and porcine rotaviruses, sometimes, do not recognize SG-specific epitopes or recognize irrelevant-SG epitopes and therefore result in the incorrect assignment of subgroups. In order to solve this problem, a novel multiplex RT-PCR was developed as an alternative tool to identify VP6 genogroups using newly designed primers which are specific for genogroup I or II. The sensitivity and specificity of the newly developed multiplex RT-PCR method was evaluated by testing with human and porcine rotaviruses of known SG I, SG II, SG (I+II), and SG non-(I+II) strains in comparison with monoplex RT-PCR and VP6 sequence analysis. The results show that the genogroups of both human and porcine rotaviruses as determined by the new multiplex RT-PCR method were in 100% agreement with those determined by monoplex RT-PCR and VP6 sequence analysis. The method was shown to be specific, sensitive, less-time consuming, and successful in genogrouping clinical isolates of rotaviruses circulating in children and piglets with acute diarrhea.
Thongprachum
Aksara
A
Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.
Chaimongkol
Natthawan
N
Khamrin
Pattara
P
Pantip
Chansom
C
Mizuguchi
Masashi
M
Ushijima
Hiroshi
H
Maneekarn
Niwat
N
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics*; D036022:Capsid Proteins / Q000235:genetics*; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000821:virology; D006801:Humans; D017720:Molecular Epidemiology / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D012400:Rotavirus Infections / Q000175:diagnosis* / Q000662:veterinary* / Q000821:virology; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D014773:Virology / Q000379:methods*
D000956:Antigens, Viral; D036022:Capsid Proteins; D017931:DNA Primers; C061332:VP6 protein, Rotavirus