Development and application of multiplex PCR assays for detection of virus-induced respiratory disease complex in dogs

PIEWBANG, Chutchai; RUNGSIPIPAT, Anudep; POOVORAWAN, Yong; TECHANGAMSUWAN, Somporn

Viral nucleic acid extraction, quantification and reverse transcription: Viral nucleic acid from the positive controls and specimens was extracted using the Viral Nucleic Acid Extraction Kit II (GeneAid, Taipei, Taiwan) according to manufacturer's recommendation. Nucleic acid was quantified and qualified using Nanodrop ® Lite (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) at an absorbance of 260 and 280 nm to derive the A 260 /A 280 ratio. The extracted nucleic acid was divided into two aliquots, one for reverse transcription (RT) for detection of the RNA viruses (CIV, CPIV, CDV and CRCoV) and the other for a direct PCR as-say for detection of the DNA viruses (CAdV-2 and CaHV-1). The RT was performed using 100 ng RNA as the template for complementary DNA (cDNA) synthesis using the Omniscript ® Reverse Transcription Kit (Qiagen GmbH, Hilden, Germany). The cDNA and DNA were stored at −20°C until used for further PCR amplification.