Detection and discrimination of WU/KI polyomaviruses by real-time PCR with melting curve analysis.
WU and KI polyomaviruses are novel viruses of the Polyomaviridae family, which have been identified recently in respiratory secretions from patients with acute respiratory tract infection. Their potential role in respiratory disease is still unclear and requires additional investigation. To facilitate further studies and diagnosis, a real-time PCR with melting curve analysis was optimized and evaluated to detect WU and KI polyomaviruses. Primers specific for the VP1 gene were designed from regions conserved among WU and KI polyomaviruses which provided amplification products of 198 and 231bp corresponding to WU and KI, respectively and thus yielded a difference in melting temperature (Tm) between WU and KI polyomaviruses. The assay proved highly specific for WU and KI polyomaviruses as no cross amplification was detected with other respiratory viruses or human genomic DNA. The assay was also highly sensitive with a detection limit as low as 10copies/microL for both WU and KI polyomaviruses. The performance of the real-time PCR assay was evaluated in terms of amplification efficiency (92%). Finally, the assay was validated using DNA extracted from clinical respiratory specimens for WU and KI polyomaviruses and the results were confirmed by direct nucleotide sequencing. The results obtained by melting curve analysis were in perfect agreement with nucleotide sequencing. In conclusion, this method is advantageous because it is rapid, specific, sensitive, reproducible, accurate, cost-effective and thus, would be feasible and attractive for large-scale analysis aimed at investigating the clinical role of WU and KI polyomaviruses.
Payungporn
Sunchai
S
Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Chieochansin
Thaweesak
T
Thongmee
Chittima
C
Panjaworayan
Nattanan
N
Samransamruajkit
Rujipat
R
Theamboolers
Apiradee
A
Poovorawan
Yong
Y
D036022:Capsid Proteins / Q000235:genetics; D003362:Cost-Benefit Analysis; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011120:Polyomavirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D027601:Polyomavirus Infections / Q000175:diagnosis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
D036022:Capsid Proteins; D017931:DNA Primers; D004279:DNA, Viral; C062481:VP1 protein, polyomavirus