检测支原体属特异性的5种PCR方法的评估和应用

Assessment of five PCR methods for routine detection of Mycoplasmas SPP.

为了筛选一种可靠的PCR方法作为实验室日常快速检测支原体的手段,利用实验室保存的17种55株支原体和13种16株细菌,对5种已发表的支原体属特异性PCR方法进行了评估和筛选。结果显示,方法 4(使用引物对RNA5和MGSO扩增1 021 bp的16S rRNA基因片段)具有良好的特异性,能够完全区分支原体与非支原体细菌,以牛支原体为扩增对象,最低能够检出10 pg牛支原体基因组DNA以及浓度为100 ccu/m L的牛支原体培养物。利用该PCR方法和支原体分离方法检测人工感染牛支原体样品和临床采集样品73份,二者符合率为93.15%。结果表明方法4可作为实验室支原体常规快速检测方法。

To screen a reliable genus-specific PCR for detection of Mycoplasmas in routine laboratory diagnostic use, five published PCR tests were assessed with 55 strains of 17 Mycoplasmas species and 16 strains of 13 bacterial speices. The PCR method showed with primers RNA5 and MGSO used for the amplification of 1021bp 16S rRNA gene fragment showed good specificity. And the detection limits of this method could be up to 10 pg of genome DNA or 100 ccu/mL culture of M. bovis. A total of 73 samples from bovines experimentally infected with M. boris as well as clinical cases suspected being infected with Mycoplasmas, were detected by PCR. The PCR method gave an average agreement rate of 93.15% with the Mycoplasmas isolation method. These results indicated that the screened PCR method is reliable for rapid detection of Mycoplasma in routine laboratory diagnostic use.