Development of two novel SYBR green-based quantitative PCR assays for detection and quantification of Mycoplasma gallisepticum and Mycoplasma synoviae

Authors: MOHSEN BASHASHATI, FERESHTEH SABOURI

Abstract: Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are considered the most pathogenic and economically devastating avian mycoplasmas. A definite MG and MS diagnosis depends on three main approaches, including isolation and identification, demonstration of specific antibodies, and detection of genetic materials. A fast and precise diagnosis technique is an indispensable prerequisite to prevent and control avian pathogens more effectively. This study aimed to develop and validate convenient SYBR green-based quantitative PCR (qPCR) assays using genome specific primers to rapidly detect and quantify MG and MS. Two sets of primers were designed based on the highly conserved regions of mgc2 and vlhA genes in MG and MS, respectively. The efficiencies of both reactions were calculated around 96% with a detection limit of as few as 10 copy numbers of DNA/μL in a sample. The standard curves showed linearity over a dynamic detection range of 6 log units from 10$^{1}$ to 10$^{6}$ DNA copy numbers/μL without any significant variations in inter- and intra-assays. The melting temperatures of the amplicons for MG and MS were determined to be 79.90 °C and 80.79 °C, respectively. The results showed that only MG and MS were detected as a single melt peak without cross reaction with other non-targeted pathogenic agents. The developed assays were further applied to the 60 swab specimens from commercial poultry with unknown MG and MS statuses and compared with the conventional PCR assays. The current assays detected 1 and 11 samples as MG and MS, respectively. However, five MG and three MS positive samples were tested positive in gel-based assays. The protocols developed here have the potential for use as a diagnostic research tool to detect and quantify MG and MS in clinical samples. Furthermore, the proposed qPCR techniques can provide an alternative approach for diagnosis and surveillance in epidemiological studies.

Keywords: Mycoplasma gallisepticum, Mycoplasma synoviae, qPCR assay, SYBR green

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