Table_5_Comparative Genomics of Mycoplasma synoviae and New Targets for Molecular Diagnostics.DOCX

Mycoplasma synoviae is an important pathogen of poultry, causing significant economic losses in this industry. Analysis of the unique genes and shared genes among different M. synoviae strains and among related species is helpful for studying the molecular pathogenesis of M. synoviae and provides valuable molecular diagnostic targets to facilitate the identification of M. synoviae species. We selected a total of 46 strains, including six M. synoviae strains, from 25 major animal (including avian) Mycoplasma species/subspecies that had complete genome sequences and annotation information published in GenBank, and used them for comparative genomic analysis. After analysis, 16 common genes were found in the 46 strains. Thirteen single-copy core genes and the 16s rRNA genes were used for genetic evolutionary analysis. M. synoviae was found to have a distant evolutionary relationship not only with other arthritis-causing mycoplasmas, but also with another major avian pathogen, Mycoplasma gallisepticum, that shares the major virulence factor vlhA with M. synoviae. Subsequently, six unique coding genes were identified as shared among these M. synoviae strains that are absent in other species with published genome sequences. Two of the genes were found to be located in the genetically stable regions of the genomes of M. synoviae and were determined to be present in all M. synoviae isolated strains (n = 20) and M. synoviae-positive clinical samples (n = 48) preserved in our laboratory. These two genes were used as molecular diagnostic targets for which SYBR green quantitative PCR detection methods were designed. The two quantitative PCR methods exhibited good reproducibility and high specificity when tested on positive plasmid controls and genomic DNA extracted from different M. synoviae strains, other major avian pathogenic bacteria/mycoplasmas, and low pathogenic Mycoplasma species. The detection limit for the two genes was 10 copies or less per reaction. The clinical sensitivity and specificity of the quantitative PCR methods were both 100% based on testing chicken hock joint samples with positive or negative M. synoviae infection. This research provides a foundation for the study of species-specific differences and molecular diagnosis of M. synoviae.

滑液支原体（Mycoplasma synoviae）是禽类的重要致病菌，给该养殖业造成了显著的经济损失。对不同滑液支原体菌株以及相关物种间的特有基因与共有基因进行分析，有助于探究滑液支原体的分子致病机制，同时可为滑液支原体的物种鉴定提供极具价值的分子诊断靶点。本研究从基因库（GenBank）中已发布完整基因组序列及注释信息的25种主要动物（包括禽类）支原体物种及亚种中，共选取46株菌株（其中包含6株滑液支原体菌株）用于比较基因组学分析。经分析，46株菌株中共存在16个共有基因。选取13个单拷贝核心基因以及16S核糖体RNA（16S rRNA）基因开展遗传进化分析。结果显示，滑液支原体不仅与其他致关节炎支原体的进化关系较远，同时与另一种主要禽类致病菌——鸡毒支原体（Mycoplasma gallisepticum）的进化关系也较远，二者共享主要毒力因子vlhA。随后，本研究在滑液支原体菌株中鉴定出6个特有编码基因，这些基因在其他已发布基因组序列的物种中均不存在。其中2个基因位于滑液支原体基因组的遗传稳定区域，经检测，其存在于本实验室保藏的所有滑液支原体分离菌株（n=20）以及滑液支原体阳性临床样本（n=48）中。以这两个基因为分子诊断靶点，设计了SYBR Green定量聚合酶链反应（SYBR Green quantitative PCR）检测方法。在对阳性质粒对照、不同滑液支原体菌株、其他主要禽类致病菌/支原体以及低致病性支原体的基因组DNA进行检测时，这两种定量PCR方法均表现出良好的重复性与较高的特异性。两种基因的检测限均达到每反应体系10拷贝甚至更低。基于对滑液支原体感染阳性及阴性的鸡跗关节样本的检测，该定量PCR方法的临床灵敏度与特异性均为100%。本研究为滑液支原体的物种特异性差异研究以及分子诊断工作奠定了基础。