Group Biological Products R & D Center, Shandong Sinder Technology Co., Ltd. Qingdao, Shandong 266100, China; College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei 071000, China.
Group Biological Products R & D Center, Shandong Sinder Technology Co., Ltd. Qingdao, Shandong 266100, China.
Poult Sci. 2024 Aug;103(8):103907. doi: 10.1016/j.psj.2024.103907. Epub 2024 May 28.
An attenuated vaccine against the Mycoplasma gallisepticum ts-11 strain has become an effective prevention and control method against MG infection. However, the ts-11 strain is usually difficult to distinguish from the non-ts-11 strain (including field isolates and other vaccine strains (F and 6/85)). Therefore, it is critical to establish a rapid and effective method to distinguish ts-11 strains from non-ts-11 strains. The gene sequences of the ts-11 strain (CP044225.1) and the non-ts-11 strain (including the wild-type (CP006916.3), 6/85 (CP044224.1), and F strains (NC_017503.1) were used to construct a conserved region containing a single point mutation in the potC gene in the ts-11 strain, after which a primer-probe combination method was designed. The primer-probe method was able to accurately and efficiently identify the ts-11 and non-ts-11 strains with minimum detection limits of 2.43 copies/μL and 1.65 copies/μL, respectively. Moreover, it could simultaneously distinguish the ts-11 strain from a non-ts-11 strain, and amplifications of avian influenza virus, infectious bronchitis virus, Newcastle disease virus, fowl adenovirus, infectious laryngotracheitis virus, infectious bursal disease virus, chicken anemia virus, Marek's disease virus, Mycoplasma synoviae, and Ornithobacter rhinotracheale were negative. The detection of clinical samples revealed that the established dual-probe fluorescence quantitative PCR method could be used to screen for mixed and single infections of the ts-11 strain and non-ts-11 strains effectively, with lower variation coefficients for intra- and interbatch repetition. The established cycleave dual-probe fluorescence quantitative PCR method showed good specificity, sensitivity, and repeatability and provides powerful technical support for the rapid and efficient differential diagnosis of the MG ts-11 strain from non-ts-11 strains.
一株减毒鸡毒支原体 ts-11 株疫苗已成为防治 MG 感染的有效方法。然而，ts-11 株通常难以与非 ts-11 株（包括野毒株和其他疫苗株（F 和 6/85））区分。因此，建立一种快速有效的方法来区分 ts-11 株和非 ts-11 株至关重要。使用 ts-11 株（CP044225.1）和非 ts-11 株（包括野毒株（CP006916.3）、6/85 株（CP044224.1）和 F 株（NC_017503.1））的基因序列，构建了 ts-11 株中 potC 基因单碱基点突变的保守区，设计了引物-探针组合方法。该引物-探针方法能够准确、高效地鉴定 ts-11 株和非 ts-11 株，最低检测限分别为 2.43 拷贝/μL 和 1.65 拷贝/μL。此外，它还可以同时区分 ts-11 株和非 ts-11 株，并且对禽流感病毒、传染性支气管炎病毒、新城疫病毒、禽腺病毒、传染性喉气管炎病毒、传染性法氏囊病病毒、鸡贫血病毒、马立克氏病病毒、鸡滑液囊支原体和鼻气管鸟杆菌的扩增均为阴性。对临床样本的检测表明，建立的双探针荧光定量 PCR 方法可用于有效筛选 ts-11 株和非 ts-11 株的混合和单一感染，其批内和批间重复的变异系数较低。所建立的循环依赖的双探针荧光定量 PCR 方法具有良好的特异性、敏感性和重复性，为快速高效地鉴别鸡毒支原体 ts-11 株和非 ts-11 株提供了有力的技术支持。
