Validation of specific quantitative real-time RT-PCR assay panel for Infectious Bronchitis using synthetic DNA standards and clinical specimens
| Autor: | Valerie Leathers, Michael Angelichio, Jongseo Mo, Mark W. Jackwood, Lisa Gow |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Serotype animal structures 030106 microbiology Infectious bronchitis virus Quantitative RT-PCR Biology Real-Time Polymerase Chain Reaction Sensitivity and Specificity Virus Article IBV Birds 03 medical and health sciences Synthetic DNA Limit of Detection Virology Animals Gene DNA Primers Retrospective Studies Detection limit qRT-PCR Hypervariable region 030104 developmental biology Real-time polymerase chain reaction DNA Viral Coronavirus Infections DNA Probes |
| Zdroj: | Journal of Virological Methods |
| ISSN: | 1879-0984 0166-0934 |
| Popis: | Highlights • Real-time quantitative PCR assays were developed for six different IBV types. • Rapid detection of IBV type is important for control. • Analytical sensitivity was evaluated using synthetic DNA standards. • Specificity was determined using clinical and biological specimens. • Linearity over a 5 log10 dynamic range and a limit of detection of ≤10 target copies was realized. Infectious bronchitis (IB) is a highly contagious upper respiratory tract disease of chickens caused by infectious bronchitis virus (IBV), which has various serotypes that do not cross-protect. Vaccine control strategies for this virus are only effective when designed around the currently circulating serotypes. It is essential to not only rapidly detect IBV but also to identify the type of virus causing disease. Six TaqMan™-based quantitative real-time RT-PCR assays (Universal, Ark, Mass, DE/GA98, GA07, GA08) were developed and examined the sensitivity and specificity for each assay. Assays were developed targeting the hypervariable region in the S1 gene subunit. The analytical sensitivity of TaqMan™-based quantitative real-time RT-PCR assays (qRT-PCR) assays was evaluated using synthetic DNA standards that were identical with the target sequence and specificity was further validated using clinical and biological specimens. All developed assays performed equivalently when using synthetic DNA templates as standard material, as it achieved linearity over a 5 log10 dynamic range with a reproducible limit of detection of ≤10 target copies per reaction, with high calculated amplification efficiencies ranging between 90%–115%. Further validation of specificity using clinical and biological specimens was also successful. |
| Databáze: | OpenAIRE |
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