Design of degenerate primers for multiplex nested-PCR detection of human lymphotropic herpesviruses.
To develop the rapid diagnosis and typing of human lymphotropic herpesviruses by using multiplex nested-PCR, the primary PCR (1(o) PCR) primers were redesigned as degenerate primers based on a highly conserved sequences of each DNA polymerase gene of EBV, CMV, HHV-6, HHV-7 and HHV-8. The forward degenerate primer (HHV/1+) contained 12 different sequences, whereas there were 8 different sequences in the reverse degenerate primer (HHV/ 1-). Optimization of multiplex nested-PCR assay conditions were performed to search for the appropriate amount of degenerate primers, dNTP, Taq DNA polymerase, template of secondary PCR (2(o) PCR) and annealing temperature used in 1(o) PCR reaction. Detection sensitivity was the same as described in previous report (approximately 10-100 genome copies). To ensure a true negative result, PCR detection of hepatitis B virus genome was used as internal control. Our presented results, the designed degenerate primers could be used to detect various types of HHV by multiplex nested-PCR.
Nopponpunth
Vanida
V
Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
Changrad
Supawee
S
Rakyuu
Apiwan
A
Nertsawange
Janjuree
J
Chansupit
Weerapa
W
Poovorawan
Yong
Y
D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D006564:Herpesviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006566:Herpesviridae Infections / Q000821:virology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D004279:DNA, Viral