抗菌肽裂解法联合环介导等温扩增技术检测尿沙眼衣原体的研究

Combining antimicrobial peptide lysis and loop-mediated isothermal amplification technology to detect Chlamydia trachomatis

目的将环介导等温扩增(LAMP)技术与抗菌肽裂解法相结合,实现人尿样沙眼衣原体的快速且灵敏的检测。方法选取盘锦市中心医院2019年12月至2020年06月接受尿液衣原体检测的患者100例,分别使用PCR荧光法及LAMP法检测,并进行特异度、灵敏度及临床验证实验。结果与未接受抗菌肽裂解处理的尿液样本相比,裂解后样本反应效率大幅提高,10循环左右即可达到平台期;特异性实验中所用6种常见病原菌(大肠埃希菌、屎肠球菌、表皮葡萄球菌、肺炎克雷伯菌、金黄色葡萄球菌、副溶血链球菌)在LAMP扩增实验中均未出现特异性曲线,仅沙眼衣原体样本出现了特异性扩增;灵敏性实验中随着模板浓度降低,到达平台期的循环数增加,但在4.46×10^(-11)ng/μL模板浓度下仍能在25循环左右到达平台期完成LAMP扩增;100例受试者中,经PCR荧光法测定沙眼衣原体阳性42例,未经裂解肽处理的尿样LAMP实验敏感度、特异度、准确度及一致率均低于抗菌肽裂解联合LAMP实验法。结论联合抗菌肽裂解法的环介导等温扩增技术能实现尿液沙眼衣原体的快速检测,且特异度、灵敏度及临床验证结果较好,具有一定临床应用前景。

Objective Combining the loop-mediated isothermal amplification(LAMP)technique with the antimicrobial peptide lysis to realize the rapid and sensitive detection of Chlamydia trachomatis in human urine.Methods A total of 100 patients who were tested for urinary chlamydia from December 2019 to June 2020 in our hospital were selected,and the PCR fluorescence method and LAMP were used to detect respectively and conduct the specificity,sensitivity and clinical validation experiments.Results Compared with urine samples that did not receive antimicrobial peptide lysis treatment,the reaction efficiency of the samples after lysis was greatly improved,and the plateau period was reached in about 10 cycles;the 6 common pathogens(E.coli,Enterococcus faecium,epidermal Staphylococcus,Klebsiella pneumoniae,Staphylococcus aureus,and Streptococcus parahaemolyticus)showed no specificity curve in the LAMP amplification experiment.Only the Chlamydia trachomatis sample showed specific amplification;in the sensitivity experiment,as the template concentration decreases,the number of cycles that reach the plateau phase increases,but at a template concentration of 4.46×10^(-11) ng/μL,it can still reach the plateau phase to complete LAMP amplification at about 25 cycles;100 subjects were determined by the PCR fluorescence method and 42 cases were positive for Chlamydia trachomatis.The sensitivity,specificity,accuracy and consistency rate of LAMP test in urine samples not treated with cleavage peptide were lower than that of antimicrobial peptide cleavage combined with LAMP test.Conclusion The loop-mediated isothermal amplification technique combined with the antimicrobial peptide lysis method can realize the rapid detection of urinary Chlamydia trachomatis,with good specificity,sensitivity and clinical validation,and has certain clinical application prospects.