Department of Veterinary Public Health and Epidemiology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar 125004, India.
Department of Veterinary Public Health and Epidemiology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar 125004, India.
J Microbiol Methods. 2022 Jan;192:106365. doi: 10.1016/j.mimet.2021.106365. Epub 2021 Nov 10.
Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically important disease of poultry industry. The present study was aimed to develop duplex PCR as a rapid, specific and economical method for accurate detection of MG and MS in poultry and its comparison with single (monoplex) MG/MS PCR. During present investigation, a total of 146 poultry flocks having clinical history of respiratory disease were screened. Pooled tissue samples (trachea, lungs and air sacs) from 4-5 birds of each flock were collected during necropsy at disease investigation laboratories, Hisar, Haryana, India. The single and duplex PCR assays were standardized using primers of intergenic spacer region (IGSR; 16S-23S rRNA) for MG and hemagglutinin vlhA gene for MS, with expected amplicon size of 812 bp and 1200 bp products, respectively. In single PCR, 6.85%, 2.74% and 2.74% tissue samples were found positive for MG, MS and both MG and MS, respectively. However, duplex PCR showed, 7.53%, 2.74% and 1.37% positivity for MG, MS and both MG and MS, respectively. Taking the results of monoplex PCR as a gold standard, sensitivity and specificity of the developed duplex PCR was found to be 94.44% and 100%, respectively. Moreover, Cohen's kappa statistic (k = 0.97) measured a 'perfect' agreement between monoplex and duplex PCR assays. The positive and negative predictive values of duplex PCR was found to be 1.0 and 0.9922, respectively at 95% confidence interval (CI), as compared to monoplex PCR. The simultaneous use of two genes in a duplex PCR was more rapid and economical than two separate single PCR reactions.
禽支原体病主要由鸡毒支原体（MG）和滑液支原体（MS）引起，是家禽养殖业中一种具有重要经济意义的疾病。本研究旨在开发一种快速、特异和经济的双重 PCR 方法，用于准确检测家禽中的 MG 和 MS，并将其与单重（单plex）MG/MS PCR 进行比较。在本研究中，共筛选了 146 个具有呼吸道疾病临床病史的家禽群。在印度哈里亚纳邦希萨尔疾病调查实验室剖检时，从每个禽群的 4-5 只禽中采集混合组织样本（气管、肺和气囊）。使用 MG 的基因间 spacer 区（IGSR；16S-23S rRNA）和 MS 的血凝素 vlhA 基因的引物，对单重和双重 PCR 检测方法进行了标准化，预期扩增产物大小分别为 812 bp 和 1200 bp。在单重 PCR 中，分别有 6.85%、2.74%和 2.74%的组织样本检测出 MG、MS 和 MG/MS 阳性。然而，双重 PCR 显示 MG、MS 和 MG/MS 的阳性率分别为 7.53%、2.74%和 1.37%。以单重 PCR 的结果为金标准，所开发的双重 PCR 的灵敏度和特异性分别为 94.44%和 100%。此外，Cohen's kappa 统计量（k=0.97）表明单重和双重 PCR 检测方法之间存在“完美”的一致性。与单重 PCR 相比，双重 PCR 的阳性和阴性预测值分别在 95%置信区间（CI）为 1.0 和 0.9922。在双重 PCR 中同时使用两个基因比两个单独的单重 PCR 反应更快速和经济。
Zotero 插件