Kreizinger Zsuzsa, Sulyok Kinga Mária, Pásztor Alexandra, Erdélyi Károly, Felde Orsolya, Povazsán János, Kőrösi László, Gyuranecz Miklós
Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Pest, Hungary.
Veterinary Diagnostic Directorate, National Food Chain Safety Office, Budapest, Hungary.
PLoS One. 2015 Jul 24;10(7):e0133554. doi: 10.1371/journal.pone.0133554. eCollection 2015.
Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.
鸡和火鸡感染滑膜支原体可导致呼吸道疾病、感染性滑膜炎和蛋壳顶端异常；因此，它是一种具有重要经济意义的病原体。控制滑膜支原体感染包括根除、药物治疗或疫苗接种。在疫苗接种计划中，区分温度敏感（ts+）MS-H疫苗株与田间分离株至关重要。本研究提供了基于熔解曲线和琼脂糖凝胶的错配扩增突变分析（MAMA），以根据obg基因nt367和nt629处的单核苷酸多态性区分ts+ MS-H疫苗株、其非温度敏感的重新分离株和野生型滑膜支原体分离株。两种熔解-MAMA和两种琼脂糖-MAMA能够清楚地区分ts+ MS-H疫苗株基因型与其非温度敏感的重新分离株基因型以及野生型滑膜支原体分离株基因型，并且与感染禽类的其他支原体物种无交叉反应。熔解-MAMA和琼脂糖-MAMA的灵敏度分别为103和104拷贝数。这些分析可直接在临床样本上进行，并且可以在相同的退火温度下同时进行。这些分析可以在设施有限的实验室中进行，使用基本的实时PCR仪或与琼脂糖凝胶电泳联用的传统热循环仪。与先前使用的方法相比，所描述的分析方法具有简单、灵敏度足够、省时、成本效益高和特异性强的优点。
