<PublisherName>Pakistan Society for Gene and Cell Therapy</PublisherName>
<JournalTitle>Journal of Genes and Cells</JournalTitle>
<ArticleTitle>Development of a Nested Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Assay to Detect Infectious Bronchitis Virus</ArticleTitle>
<Affiliation>Hakim Sabzevari University, Iran. M.momeni@hsu.ac.ir</Affiliation>
<Abstract>Infectious Bronchitis Virus (IBV) is a highly infectious and contagious viral pathogen of chickens worldwide. IBV is a positive sense, single-stranded RNA virus that primarily targets the respiratory tract. IBV belongs to group III of the genus Coronavirus of the Coronaviridae family. In this study, viral RNA was extracted using phenol-chloroform based method. A pair of specific primers for RT-PCR and a pair of specific internal primers for Nested-PCR of the S1 region in the spike protein gene was designed and the tests were optimized for the detection of IBV. To determine the sensitivity of the test, 10 fold dilutions of virus stock (3 × 105 EID50 to 3 × 10-2 EID50/ml) were prepared in distilled water. A 531 bp fragment and a 284 bp of the spike protein gene (S1) were amplified in the RT-PCR and the Nested-PCR test, respectively. The PCR product was cloned in pTZ57R/T vector and sequenced.  The sequence data confirmed the specificity of the test. The sensitivity of RT- PCR was determined to be 3×103 EID50 per ml. Nested-PCR was also carried out and sensitivity was increased to 3×10 EID50 per ml. Due to the high sensitivity of this test, this technique can be used as a robust and rapid diagnostic method for the detection of IBV.</Abstract>
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