An Improved Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Specific Detection of Infectious Bronchitis Virus

摘要

A highly conserved region of 1a gene of Infectious Bronchitis Virus (IBV) was chosen to design the Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) degenerate primers. The developed RT-LAMP assay is a highly sensitive, specific, rapid method to detect IBV in allantoid and tissue. Optimal temperature was around 62°C and duration was 45 min. Sensitivity analysis showed that RT-LAMP assay was 10 fold more sensitive than RT-PCR. Specificity analysis showed that 3 IBV strains omitted by the previous reported RT-LAMP assay could be detected by the improved RT-LAMP assay. In field trials, the improved RT-LAMP assay obtained 98.4% sensitivity in 65 clinical samples while 95.4% of RT-PCR and 93.8% of the previous reported RT-LAMP which indicates it is a powerful tool in the practical application.