National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Key Laboratory of Zoonoses, Ministry of Agriculture, Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Key Laboratory of Animal Vaccine Development, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.
Guangdong AIB Polytechnic, Tropical agriculture and Forestry College, Guangzhou, Guangdong 510507, China.
Poult Sci. 2024 Aug;103(8):103874. doi: 10.1016/j.psj.2024.103874. Epub 2024 May 16.
Mycoplasma synoviae (MS) is a contagious pathogen that poses a significant threat to the poultry industry. Detection plays an important role in the prevention and control of MS, particularly in differentiating between wild-type MS and live attenuated vaccine strains for vaccination selection and culling of animals with wild-type only. The live attenuated ts+ vaccine strain MS-H is recognized as the most effective and widely used vaccine. In this study, we have developed a method called double enzyme-activated differentiation probes PCR (DEA-probes PCR) for the differentiation of MS-H vaccine strain from wild-type strain by targeting the single nucleotide polymorphism (SNP) of the 367th nucleotide in the Obg gene sequence. We developed 2 modified probes with the ribonucleotide insert. When the probe perfectly complements with the target, the ribonuclease H2 (RNase H2) will cleave the ribonucleotide, resulting in the generation of fluorescent signal. With a detection limit of 5.8 copies/µL, the DEA-probes PCR method demonstrates 100% specificity in distinguishing wild-type MS from MS-H strains in 1 h. The method demonstrated great performance in real application of 100 superior palate cleft swab samples from chickens in poultry farms. Twenty-eight samples were detected as MS positive, consistent with the results of the Chinese industry standard method. Additionally, our method was able to distinguish 19 wild-type MS strains from 9 MS-H vaccine strains. The DEA-probes PCR method is rapid, specific and sensitive for SNP detection, overcoming the misidentification in MS detection and differentiation. It can be also applied to the differentiation of infected from vaccinated animals (DIVA) for other pathogens.
滑液支原体（MS）是一种具有传染性的病原体，对家禽业构成重大威胁。检测在 MS 的预防和控制中起着重要作用，特别是在区分野生型 MS 和活减毒疫苗株方面，用于疫苗接种选择和对仅携带野生型的动物进行淘汰。活减毒 ts+疫苗株 MS-H 被认为是最有效和广泛使用的疫苗。在这项研究中，我们针对 Obg 基因序列第 367 个核苷酸的单核苷酸多态性（SNP），开发了一种称为双酶激活区分探针 PCR（DEA-probes PCR）的方法，用于区分 MS-H 疫苗株和野生型株。我们开发了 2 个带有核糖核苷酸插入的改良探针。当探针与靶标完全互补时，核糖核酸酶 H2（RNase H2）将切割核糖核苷酸，从而产生荧光信号。DEAP-robes PCR 方法的检测限为 5.8 拷贝/µL，在 1 小时内能够 100%特异性地区分野生型 MS 和 MS-H 株。该方法在对来自家禽养殖场的 100 个优质腭裂拭子样本的实际应用中表现出色。28 个样本被检测为 MS 阳性，与中国行业标准方法的结果一致。此外，我们的方法能够区分 19 个野生型 MS 株和 9 个 MS-H 疫苗株。DEA-probes PCR 方法快速、特异、灵敏，用于 SNP 检测，克服了 MS 检测和区分中的误识别。它还可以应用于其他病原体的感染后免疫动物区分（DIVA）。
