【摘要】 目的建立实时荧光环介导等温扩增(LAMP)法检测肺炎支原体。方法在LAMP体系中添加适量荧光染料SYTO-9,通过实时定量PCR仪监测LAMP反应结果。以肺炎支原体P1基因重组质粒制备一系列浓度标准品,分别使用实时荧光LAMP法和SYBR GreenⅠ染料LAMP法检测,比较2种方法的检出限。肺炎支原体M129标准株菌液以10倍梯度稀释,分别以实时荧光LAMP法和肺炎支原体荧光PCR试剂盒检测,比较2种方法灵敏度。以实时荧光LAMP法检测常见呼吸道感染病原体肺炎链球菌、金黄色葡萄球菌、表皮葡萄球菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌和大肠埃希菌基因组DNA,评估该方法特异性。采集疑似肺炎支原体感染患儿咽拭子标本,分别以实时荧光LAMP法和荧光PCR法检测,比较2种方法检出率差异。结果实时荧光LAMP法可以检出P1基因最低限为10~3 copies/μL,当基因拷贝数大于10~4 copies/μL时,反应30 min样本组即可出现明显荧光信号。SYBR GreenⅠ染料LAMP法可以检出P1基因最低限为10~4 copies/μL,反应需1 h才可获得足够产物用于显色。实时荧光LAMP法灵敏度高于实时定量PCR法100倍,且对常见呼吸道病原体DNA无扩增。实时荧光LAMP法和荧光PCR法检测疑似肺炎支原体感染咽拭子标本的阳性率差异无统计学意义(P>0.05)。结论实时荧光LAMP法可以有效避免因开盖检测造成的气溶胶污染,反应时间短,灵敏度高,适于在基层医院中推广使用。更多还原

【Abstract】 Objective To establish a real-time fluorescence loop-mediated isothermal amplification(LAMP) assay for Mycoplasma pneumoniae(M. pneumoniae) detection. Methods An appropriate amount of fluorescent dye SYTO-9 was added into the LAMP system. The results of LAMP reaction were monitored by real-time quantitative PCR. The standards with a series of concentrations were prepared by the recombinant plasmid of M. pneumoniae P1 gene. Both real-time fluorescence LAMP and fluorescent dye SYBR GreenⅠLAMP assay were used to detect M. pneumoniae P1 gene, and the detection limits of the two methods were compared. The standard strain of M. pneumonia M129 was diluted with 10-fold gradient and detected by real-time fluorescence LAMP and real-time fluorescent PCR simultaneously to compare the sensitivities of the both methods. The genomic DNA of common respiratory infection pathogens, e.g., Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli, were detected by real-time fluorescence LAMP to evaluate its specificity. The throat swabs of children with suspected mycoplasma pneumoniae infection were collected and detected by real-time fluorescence LAMP and real-time PCR simultaneously, to compare the detection rates of the both methods. Results The lowest limit of P1 gene detected by real-time fluorescent LAMP method was 10~3 copies/μL. When the copy number of P1 gene was more than 10~4 copies/μL, the sample group showed obvious fluorescent signal after 30 min of reaction, while the minimum limit of SYBR GreenⅠLAMP assay for the detection of P1 gene was 10~4 copies/μL, which took 1 hour of the reaction to obtain enough products for color development. The real-time fluorescence LAMP method was 100 times more sensitive than the real-time PCR, and no amplification for the other common respiratory pathogens DNA was found. There was no significant difference between real-time fluorescent LAMP and fluorescent PCR in the positive rate of throat swabs specimens from suspected infection with Mycoplasma pneumoniae(P>0.05). Conclusion The real-time fluorescence LAMP developed in this study may effectively avoid aerosol pollution caused by open cap in detection with short reaction time and high sensitivity, which is very suitable for promotion and application in primary hospitals.更多还原