含有内部阳性对照的支原体荧光PCR检测方法的建立及验证

Establishment and validation of a fluorescence PCR with internal positive control for Mycoplasma detection

目的建立含有内部阳性对照的支原体荧光PCR检测方法并进行验证,以期用于支原体的快速检测。方法建立含有内部阳性对照的支原体荧光PCR检测方法并进行专属性、检测限和耐用性评价。应用建立的方法检测发热伴血小板减少综合征毒种是否污染支原体,并与培养法和指示细胞法结果相比较。结果建立的支原体荧光PCR检测方法特异度好,可以扩增11种含有支原体16S rRNA基因的质粒,扩增效率较高。与生孢梭菌、丙酮丁醇梭菌、嗜酸乳杆菌、肺炎链球菌、枯草芽孢杆菌、金黄葡萄球菌、肠炎沙门菌、大肠埃希菌、铜绿假单胞菌、黑曲霉、白念珠菌、Vero细胞、RD细胞、SF9细胞基因组DNA均无交叉反应。内部阳性对照扩增效率与支原体目的基因扩增效率基本一致,可用于检测是否存在PCR抑制物。检测敏感度高,用7500 Fast实时荧光定量PCR系统可检测到10菌落形成单位(CFU)/ml的发酵支原体,5 CFU/ml的精氨酸支原体、鸡毒支原体、猪鼻支原体、莱氏无胆甾原体、口腔支原体、肺炎支原体、滑液支原体,以及1 CFU/ml的螺原体。耐用性好,在每种支原体检测限水平上,不同型号荧光定量PCR仪阳性检出率差异无统计学意义。应用该方法检测发热伴血小板减少综合征毒种为支原体阳性,与培养法和指示细胞法结果一致。结论本研究建立的含有内部阳性对照的支原体荧光PCR检测方法特异度好、敏感度高、耐用性好,可作为支原体检测的替代方法用于支原体的快速检测。

ObjectiveTo establish and validate a fluorescence PCR with internal positive control for rapid Mycoplasma detection.MethodsA fluorescence PCR with internal positive control for Mycoplasma detection was developed and verified for its specificity,limit of detection,and robustness.A sample of fever with thrombocytopenia syndrome(SFTSV)virus strains was tested with this method,and the result was compared with those of culture method and indicator cell culture method.ResultsThe established fluorescence PCR had good specificity and could amplify 11 kinds of plasmids containing Mycoplasma 16S rRNA gene with high efficiency.There was no cross reaction with the genomic DNAs of Clostridium sporogenes,Clostridium acetobutylicum,Lactobacillus acidophilus,Streptococcus pneumoniae,Bacillus subtilis,Staphylococcus aureus,Salmonella enteritidis,Escherichia coli,Pseudomonas aeruginosa,Aspergillus niger,Candida albicans,Vero cells,RD cells,and SF9 cells.The amplification efficiency of the internal positive control was basically consistent with that of the target gene of Mycoplasma,suggesting that the internal positive control could be used to detect the presence of PCR inhibitors.The sensitivity of the established method was high,and the detection limit was 10 colony-forming unit(CFU)/ml for Mycoplasma fermentans,5 CFU/ml for Mycoplasma arginine,5 CFU/ml for Mycoplasma gallisepticum,5 CFU/ml for Mycoplasma hyorhinis,5 CFU/ml for Acholeplasma laidlawii,5 CFU/ml for Mycoplasma orale,5 CFU/ml for Mycoplasma pneumoniae,5 CFU/ml for Mycoplasma synoviae,and 1 CFU/ml for Spiroplasma citri by 7500 Fast real-time PCR system.At the detection limit of each species,there was no significant difference in the positive detection rate using different thermal cycler types.The established fluorescence PCR,culture method,and indicator cell culture were performed to detect Mycoplasma in the sample of SFTSV virus strains,and the results all showed Mycoplasma contamination.ConclusionsThe established fluorescence PCR has high specificity,sensitivity,and robustness,and can be used as an alternative method for rapid detection of Mycoplasma.