Genus specific PCR-RFLP and multiplex PCR for diagnosis of avian mycoplasmosis

Abstract

Diagnosis of avian mycoplasmosis remains a serious problem, due to unusual nature of etiological agent. Although, many protocols for PCR are set with the published primers; they do not always yield vis-à-vis results under different existing conditions of laboratories. Therefore, the present research was aimed towards the optimization and evaluation of genus specific and multiplex PCR for molecular diagnosis of avian mycoplasmosis. The research was carried out systematically where in first DNA extraction protocol was standardized followed by optimization of PCR. Using optimized protocols, screening of 165 clinical specimens from mycoplasmosis suspected birds was carried out employing genus specific PCR-RFLP, followed by the confirmation of positive samples by multiplex PCR assay and sequencing of unidentified PCR products. Phenol chloroform isoamyl method was found superior to rapid heat freeze method for extraction of better quality and quantity of DNA. Genus specific 16S rRNA based PCR-RFLP was found sensitive and convenient method for screening large number of clinical specimens for detection of mycoplasma infection.

Keywords

• Avian mycoplasmosis
• Mycoplasma gallinarum
• Mycoplasma gallisepticum
• Mycoplasma synoviae
• PCR
• RFLP