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首页> 外文期刊>Parasitology >Development of a recombinase polymerase amplification (RPA-EXO) and lateral flow assay (RPA-LFA) based on the ITS1 gene for the detection of Angiostrongylus cantonensis in gastropod intermediate hosts
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Development of a recombinase polymerase amplification (RPA-EXO) and lateral flow assay (RPA-LFA) based on the ITS1 gene for the detection of Angiostrongylus cantonensis in gastropod intermediate hosts

机译：基于ITS1基因的重组酶聚合酶扩增（RPA-EXO）和横向流动测定（RPA-LFA）的研制用于检测胃肠杆菌中间体宿主中的Angiostrongylus Cantonensis

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摘要

Angiostrongylus cantonensis is a parasitic nematode known to infect humans through the ingestion of third stage larvae which can cause inflammation and damage to the central nervous system. Currently, polymerase chain reaction (PCR) is one of the most reliable diagnostic methods for detecting A. cantonensis in humans as well as in gastropod hosts, but requires expensive and specialized equipment. Here, we compare the sensitivity and accuracy of a recombinase polymerase amplification Exo (RPA-EXO) assay, and a recombinase polymerase amplification lateral flow assay (RPA-LFA) with a traditional quantitative PCR (qPCR) assay currently available. The three assays were used to test 35 slugs from Hawai‘i for the presence of A. cantonensis DNA. Consistent results among the three tests were shown in 23/35 samples (65.7%), while 7/35 (20%) were discordant in low infection level samples (<0.01 larvae per mg tissue), and 5/35 (14.3%) were equivocal. To evaluate sensitivity, a partial ITS1 gene was cloned, and serial plasmid dilutions were created ranging from 100 copies μL?1 to ~1 copy μL?1. All three assays consistently detected 50–100 copies μL?1 in triplicate and qPCR was able to detect ~13 copies μL?1 in triplicate. RPA-EXO was able to detect 25 copies μL?1 in triplicate and RPA-LFA was not able to amplify consistently below 50 copies μL?1. Thus, our RPA-EXO and RPA-LFA assays do not appear as sensitive as the current qPCR assay at low DNA concentrations; however, these tests have numerous advantages that may make them useful alternatives to qPCR.
机译：广州管圆线虫是一种寄生线虫，已知通过摄入第三阶段幼虫感染人类，可引起炎症和中枢神经系统损伤。目前，聚合酶链反应（PCR）是检测人类和腹足动物宿主中广州管圆线虫最可靠的诊断方法之一，但需要昂贵的专用设备。在这里，我们比较了重组酶聚合酶扩增外显子（RPA-Exo）分析和重组酶聚合酶扩增横向流动分析（RPA-LFA）与目前可用的传统定量PCR（qPCR）分析的灵敏度和准确性。这三项检测用于检测来自夏威夷的35只蛞蝓是否存在广州管圆线虫DNA。三项测试中，23/35样本（65.7%）的结果一致，而7/35（20%）的结果在低感染水平样本（每毫克组织中的幼虫数<0.01）中不一致，5/35（14.3%）的结果模棱两可。为了评估敏感性，克隆了一部分ITS1基因，并建立了一系列质粒稀释液，范围为100μL？1到1拷贝μL？1.所有三种分析均一致检测到50-100个拷贝μL？1份一式三份，qPCR能够检测到约13份μL？一式三份。RPA-EXO能够检测到25个拷贝μL？1份，RPA-LFA不能在50μL以下持续扩增？1.因此，在低DNA浓度下，我们的RPA-EXO和RPA-LFA分析似乎不如当前的qPCR分析敏感；然而，这些测试有许多优点，可能使它们成为qPCR的有用替代品。

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