Development of a quantitative Light Cycler real-time RT-PCR for detection of avian reovirus.
A robust, ultrasensitive, and accurate quantitative assay was developed for avian reovirus (ARV) with the Light Cycler SYBR Green-based real-time reverse transcription-PCR (real-time LC RT-PCR). The assay exhibited high specificity as all negative controls and other avian pathogens, such as Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), avian influenza virus (AIV), and mycoplasma synovia (MS), failed to show any positive detection. A minimum of 39 copies/microl of ARV genomic RNA could be detected by the assay. By dilution analysis, the real-time LC RT-PCR developed in this study was 3-log more sensitive than the conventional RT-PCR for the detection of ARV. The vaccine and field isolates of ARV were detected by the real-time LC RT-PCR. As a result of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time LC RT-PCR will be useful as a routine assay for the clinical diagnosis of ARV infection.
Ke
Guan M
GM
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.
Cheng
Hsueh L
HL
Ke
Liang Y
LY
Ji
Wen T
WT
Chulu
Julius L C
JL
Liao
Ming H
MH
Chang
Tien J
TJ
Liu
Hung J
HJ
D000818:Animals; D052160:Benzothiazoles; D003959:Diamines; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D005456:Fluorescent Dyes; D018628:Gene Dosage; D007201:Indicator Dilution Techniques; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals; D030101:Orthoreovirus, Avian / Q000145:classification / Q000302:isolation & purification*; D011804:Quinolines; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D012088:Reoviridae Infections / Q000175:diagnosis* / Q000662:veterinary* / Q000821:virology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000662:veterinary*; D012680:Sensitivity and Specificity; D012703:Serotyping; D013696:Temperature
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I