Development and validation of an insulated isothermal polymerase chain reaction assay for the rapid detection of Mycoplasma synoviae

• Published in: Veterinary research forum Vol. 15; no. 1; pp. 7 - 12
• Main Authors: Wang, Lucai, Liu, Lijia, Zhang, Huanrong
• Format: Journal Article
• Language: English
• Published: Iran Veterinary Research Forum 2024
• Subjects: Acids; Arthritis; Cloning; Disease; E coli; Enzymes; Experiments; Influenza; Laboratories; Methods; Pathogens; Polymerase chain reaction; Poultry; Proteins; Salmonella; Vectors (Biology); China; Insulated isothermal PCR; Mycoplasma synoviae; On-site detection
• ISSN: 2008-8140; 2322-3618
• DOI: 10.30466/vrf.2022.554037.3474

, which causes the disease known as chicken synovitis, causes serious immunosuppression. We developed a rapid insulated isothermal polymerase chain reaction (iiPCR) assay for on-site detection of using a primer and probe set targeting the ( ) gene. In addition, the specificity, sensitivity, repeatability, and clinical detection of this method were evaluated. Our iiPCR assay detected clinical isolates and samples successfully and produced negative results on avian viral arthritis, , , and , indicating that the PCR reactions were specific. Additionally, our iiPCR assay detected the prepared positive standard plasmid diluted 10 times (1.00 × 10 - 1.00 × 10 ) as a template. The undiluted positive plasmid was positive and double distilled water was negative indicating that the PCR reactions were sensitive, respectively. Finally, the positive standard plasmid with dilution multiple of 1.00 × 10 - 1.00 × 10 was repeatedly detected three times to evaluate the repeatability of the iiPCR method established in this experiment showing that the iiPCR of is repeatable. The established iiPCR was also used to detect 50 chicken joint enlargement samples. The thermostatic detection PCR established in this experiment was comparable to a reference real-time PCR (qPCR).