Rapid detection and differentiation of Newcastle disease virus by real-time PCR with melting-curve analysis.
In order to rapidly detect and differentiate Newcastle disease virus (NDV) isolates, a method based on real-time PCR SYBR Green I melting-curve analysis of the fusion (F) protein gene was developed. The detection limit of real-time PCR was 9 x 10(2) plasmid copies and was 100 times more sensitive than conventional PCR. Thirty eight reference NDV strains were rapidly identified by their distinctive melting temperatures (T(m)s): 89.23 +/- 0.27 degrees C for velogenic strains, 90.17 +/- 0.35 degrees C for pigeon mesogenic strains, 91.25 +/- 0.14 degrees C for two lentogenic strains (B1 and Ishii). No amplification was detected from unrelated RNA samples by this method. This real-time PCR directly detected NDV from infected tissues and eliminated the gel electrophoretic step for analyzing PCR product using ethidium bromide. The total time for a PCR run was less than 1 hour. The results obtained in this study showed that the real-time PCR presented here is a good screening test for the identification of NDV.
Pham
H M
HM
Department of Disease Control, Laboratory of Infectious Diseases, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Konnai
S
S
Usui
T
T
Chang
K S
KS
Murata
S
S
Mase
M
M
Ohashi
K
K
Onuma
M
M
D000818:Animals; D052160:Benzothiazoles; D002645:Chickens; D003959:Diamines; D009521:Newcastle Disease / Q000821:virology*; D009522:Newcastle disease virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D044366:Transition Temperature
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I