基于荧光重组酶聚合酶扩增技术快速检测美洲钩虫方法研究

Development and preliminary evaluation of a fluorescence RPA assay for the rapid detection of Necator americanus

目的 建立基于荧光重组酶聚合酶核酸扩增(recombinase polymerase amplification, RPA)技术快速检测粪样中美洲钩虫卵的方法,并评价其效能,为快速检测粪样中美洲钩虫卵提供技术支撑。方法 根据美洲钩虫的cox1基因序列设计荧光RPA引物及探针并进行筛选,建立荧光RPA检测方法。将美洲钩虫卵基因组DNA稀释至100 pg/μL、10 pg/μL、1 pg/μL、100 fg/μL、10 fg/μL、1 fg/μL、0.1 fg/μL等7个浓度梯度以测定荧光RPA法的最低检出值,利用荧光RPA法检测日本血吸虫、蛔虫、华支睾吸虫、肝片形吸虫DNA样本进行交叉反应实验。采集44例人粪样并进行DNA提取,运用改良加藤厚涂片法(Kato-Katz)、半巢式PCR法、荧光RPA法同时检测,评价荧光RPA法的灵敏度和特异度。结果 建立的荧光RPA法可在20 min内特异性扩增出美洲钩虫cox1基因长度为194 bp的片段,最低检出值为10 fg/μL,与日本血吸虫、蛔虫、华支睾吸虫、肝片形吸虫均无交叉反应。通过对44份人粪便样本进行检测,Kato-Katz法和半巢式PCR法均检出26例阳性样本,荧光RPA法检出27例阳性样本,其中1号样本荧光曲线在反应后期略高于阴性对照,但未出现与阳性对照相似反应趋势,因此判定为疑似阴性样本。荧光RPA法与Kato-Katz法和半巢式PCR法相比,其灵敏度均为100.00%,特异度均为94.44%,检测结果一致性较好(Kappa=0.953>0.75)。结论 荧光RPA法可在短时间内实现对美洲钩虫高效、敏感、特异的检测,有望应用于美洲钩虫感染的监测及预警。

Objective To establish a rapid detection assay based on fluorescence recombinase polymerase amplification(RPA)targeting Necator americanus eggs,and to evaluate its efficacy,providing technical support for rapid detection of Necator americanus in fecal samples.Methods The fluorescence RPA primers and probe were designed based on the cox1 gene of Ne⁃cator americanus and then screened the optimal combination to develop the assay.The genomic DNA of Necator americanus eggs was diluted to 7 concentration gradients including 100 pg/µL,10 pg/µL,1 pg/µL,100 fg/µL,10 fg/µL,1 fg/µL,0.1 fg/µL,to determine the detection limit of the assay.The specificity of the assay was demonstrated by detected genomic DNA from Schistosoma japonicum,Ascaris lumbricoides,Clonorchis sinensis and Fasciola hepatica.A total of 44 fecal samples were collected and DNA extraction was performed,and the modified Kato-Katz method,semi-nest PCR method,and fluorescent RPA method were simultaneously used for detection to evaluate the sensitivity and specificity.Results The established fluo‑rescence RPA assay can specifically amplify a fragment of 194 bp of the Necator americanus cox1 gene within 20 min,with a de‑tection limit of 10 fg/µL.There was no cross-reactivity with Schistosoma japonicum,Ascaris lumbricoides,Clonorchis sinensis,Fasciola hepatica after specificity validation.In 44 fecal samples,27 positive samples were detected by the fluorescence RPA assay,and 26 positive samples were detected by both the Kato-Katz and the semi-nested PCR.The fluorescence curve of sam‑ple number 1 was slightly higher than the negative control in the later stage of the reaction,but did not show a similar trend to the positive control,and was therefore judged to be a suspected negative sample.Compared with the Kato-Katz method and the semi-nest PCR method,The sensitivity of the fluorescent RPA method were 100.00%and the specificity were 94.44%,and the consistency of the detection results was good(Kappa=0.953>0.75).Conclusions The assay based on the fluorescence RPA is an efficient,sensitive and specific technique for detecting Necator americanus and it can be applied for surveillance and early warning of hookworm infection.