改良环介导等温扩增技术在疟原虫耐药基因SNP检测中的价值及可行性

目的 探讨改良环介导等温扩增技术(LAMP)对常见药物抗性基因SNP的检测价值及可能性.方法 采集非洲赤道几内亚马拉博地区医院提供的500例当地疟疾患者的外周血液样本,以巢式PCR为金标准,比较改良环介导等温扩增技术与其在恶性疟原虫耐药基因SNP检测中的反应效率、敏感性及特异性.结果 对照金标准巢式PCR方法,对500例患者进行检测,两种方法的检测符合率为96.4%,差异无统计学意义(P>0.05).检测效率比较显示,通过与金标准进行比较,改良LAMP的初反应时间较快,在10 min内即起跳并迅速达到阳性反应阈值,而巢式PCR扩增效率相对较低,明显落后于改良LAMP(q浑浊度=6.492,P<0.001;q反应速率=0.931,P=0.079);敏感性比较显示,改良LAMP扩增的阳性反应阈值较巢式PCR出现得早,并且随着时间推移,反应扩增量逐渐增加,在反应的20 min内,改良LAMP反应起跳时间较早(q浑浊度=20.171,P<0.001;q反应速率=0.326,P=0.674);特异性比较显示,改良LAMP对四种疟原虫均有较好的特异性,无论是起跳时间、扩增产物速率均优于巢式PCR(q浑浊度=18.619,P<0.001;q反应速率=1.927,P=0.073).结论 改良环介导等温扩增技术具有较好的反应速率、特异性及敏感性,操作简便、诊断效率高,值得推广.

Value and feasibility of improved loop-mediated isothermal amplification technique in the detection of SNPs of Plasmodium falciparum resistance gene

Objective To investigate the value and possibility of the SNP detection of the common drug resis-tance gene by the improved loop-mediated isothermal amplification (LAMP) technique. Methods A total of 500 pe-ripheral blood samples collected from the local malaria patients in the hospitals of Malabo area of African Equatorial Guinea, using nested PCR as the gold standard, was used to evaluate the reaction efficiency, sensitivity and specificity of the improved loop mediated isothermal amplification technology in the detection of SNPs of Plasmodium falciparum re-sistance gene. Results The coincidence rates of the two methods in 500 patients were 96.4%, and the difference was not statistically significant (P>0.05). The comparison of the detection efficiency showed that the initial reaction time of the modified LAMP was faster than that of the nested PCR amplification and reaches the positive reaction threshold quickly in 10 mins, and the nested PCR amplification efficiency was relatively low, significantly behind the improved LAMP (q Turbidity=6.492, P<0.001;q Reaction rate=0.931, P=0.079). The sensitivity comparison showed that the positive response threshold of modified LAMP amplification appeared earlier than nested PCR, and the reaction amplification gradually in-creased with time. The take-off time of the improved LAMP was early in 20 min (q Turbidity=20.171, P<0.001, q Response rate=0.326, P=0.674). The specificity results showed that the modified LAMP had good specificity for the four kinds of malar-ia parasites, both the take-off time and the rate of the amplified products were better than the nested PCR (q Turbidity=18.619, P<0.001;q Reaction rate=1.927, P=0.073). Conclusion The improved LAMP technology has the advantages of better reaction rate, specificity and sensitivity, easy operation and high diagnostic efficiency, which is worthy of promotion.