Development of a quantitative Light Cycler real-time RT-PCR for detection of avian reovirus

• Published in: Journal of virological methods 2006-04, Vol.133 (1), p.6-13
• Main Authors: Ke, Guan M., Cheng, Hsueh L., Ke, Liang Y., Ji, Wen T., Chulu, Julius L.C., Liao, Ming H., Chang, Tien J., Liu, Hung J.
• Format: Article
• Language: English
• Subjects: Animals; Avian influenza virus; Avian reovirus; Biological and medical sciences; Electrophoresis, Polyacrylamide Gel; Fluorescence; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Gene Dosage; Indicator Dilution Techniques; Infectious bronchitis virus; Infectious bursal disease virus; Light Cyler; Microbiology; Mycoplasma; Newcastle disease virus; Nucleic Acid Denaturation; Organic Chemicals; Orthoreovirus, Avian - classification; Orthoreovirus, Avian - isolation & purification; Polymerase chain reaction; Reoviridae Infections - diagnosis; Reoviridae Infections - veterinary; Reoviridae Infections - virology; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction - instrumentation; Reverse Transcriptase Polymerase Chain Reaction - veterinary; RNA, Viral - analysis; RNA, Viral - genetics; Sensitivity and Specificity; Serotyping; Techniques used in virology; Temperature; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2005.09.011

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/μl 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.