侧流层析–重组酶聚合酶扩增技术快速检测土拉弗朗西斯菌

彭遥; 阚飙; 夏连续; 李伟; 张雯; 卢金星; 秦爱平

doi:10.3784/j.issn.1003-9961.2019.05.018

侧流层析–重组酶聚合酶扩增技术快速检测土拉弗朗西斯菌

Establishment of a recombinase polymerase amplification combining with lateral flow strip assay for rapid visual detection of Francisella tularensis

摘要

摘要:
目的利用侧流层析–重组酶聚合酶扩增（LF-RPA）技术建立快速、敏感、特异的土拉弗朗西斯菌（土拉菌）检测方法。
方法基于土拉菌特异性基因（tul4）设计重组聚合酶恒温扩增方法的引物及探针，优化反应时间和温度，同时评价该方法的灵敏度及特异性，并通过模拟血液样品评估此方法的实用性。
结果该方法最佳反应条件是40 ℃，20 min，可检测核酸浓度最低为20 fg/μl，与实时荧光定量PCR（qPCR）方法相近，且不与7种非土拉菌发生交叉反应。土拉菌模拟样本最低检测浓度为460 CFU/ml。
结论该检测方法操作简单，特异性良好，敏感度高且无需大型精密实验设备，在临床及现场检测土拉菌中具有应用前景。

Abstract:
ObjectiveTo establish a rapid, sensitive and specific assay for detection of Francisella tularensis by recombinase polymerase amplification and lateral flow (LF-RPA) strip techniques.
MethodsA primer-probe set targeting specific gene tul4 of F. tularensis for LF-RPA assay was designed and the parameters (temperature and time) for the assay were optimized. The sensitivity and specificity of the assay were evaluated.Field feasibility was explored by running the assay with Francisella -spiked blood samples.
ResultsThe optimal temperature and time of the assay were at 40 ℃, and for 20 min. The limit of detection (LOD) of the LF-RPA assay on genomic DNA of F.tularensis was 20 fg/μl, similar to qPCR method targeting the same gene. No cross-reactions with 7 other non-F. tularensis bacteria species was observed,indicating the specificity of the LF-RPA assay. The LOD of the LF-RPA assay for detecting F. tularensis in simulated blood samples was about 460 CFU/ml.
ConclusionThe newly developed assay is a simple, rapid, sensitive and specific method for the detection of F. tularensis with a potentiality of clinical diagnosis of tularemia and detection of Francisella at an outbreak field.

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