基于环介导等温技术检测人甲流H1N1病毒的响应面优化分析

Response surface methodology analysis for detecting human H1N1 influenza virus based on loop-mediated isothermal amplification technology

目的为了建立人甲型流感H1N1病毒的环介导等温扩增(LAMP)体系,本研究以H1N1病毒核酸扩增效率为指标,通过响应面分析优化探讨LAMP检测H1N1流感病毒的最佳反应条件。方法根据Box-Benhnken响应设计原理,对影响LAMP反应关键因素(甜菜碱浓度、引物浓度、反应温度)进行三因素三水平响应面法优化。同时选取60例确诊甲流临床核酸样本和60例健康受试者核酸样本,分别采用聚合酶链反应(PCR)技术和LAMP检测技术进行比对评估(评估指标包括灵敏度、特异性、准确度)。结果响应面回归模型能较好地预测LAMP检测效果与甜菜碱浓度、引物浓度和反应温度之间的关系,经过优化LAMP反应体系提高检测灵敏度和缩短反应时间。同时与PCR检测结果相比,LAMP检测实现100%的灵敏度、98.9%的特异性和99.2%的准确度,检测下限可达100 copies/ml。结论本研究建立的H1N1甲流LAMP检测是一种高效、灵敏、特异性高且不需要复杂设备的检测方法,适用于基层医疗卫生机构或居家自测,对季节性甲流病毒监测及暴发时应急检测具有重要意义。

Objective In order to establish a ring-mediated isothermal amplification detection system for human H1N1 influenza virus,the nucleic acid amplification efficiency of H1N1 virus was used as an indicator,and to explore optimal reaction conditions for loop mediated isothermal amplification(LAMP)detection of H1N1 influenza virus by response surface analysis optimization.Methods Based on the principle of Box-Benhnken response design,the key factors affecting LAMP reaction(betaine concentration,primer concentration,and reaction temperature)were optimized by the three factors level response surface method.At the same time,60 clinical nucleic acid specimens of confirmed influenza A and 60 nucleic acid samples of healthy people were selected and evaluated by polymerase chain reaction(PCR)assay and LAMP assay comparatively(the evaluation indexes included sensitivity,specificity and accuracy).Results The response surface regression model was able to better predict the relationship between the LAMP detection effect and betaine concentration,primer concentration and reaction temperature,and the optimized LAMP improved the detection sensitivity and shortened the reaction time.Compared with PCR results,the LAMP assay achieved 100%sensitivity,98.9%specificity and 99.2%accuracy,and the lower limit of detection was up to 100 copies/ml.Conclusion The LAMP assay for influenza A(H1N1)established in the present study is a highly efficient,sensitive,and specific assay that does not require complex equipment,and it is suitable for the detection of H1N1 in basic community units or at home,which is useful for the monitoring of H1N1 and the post-outbreak epidemic.It is of great significance for the monitoring of influenza A disease and the emergency detection of outbreaks.