重组酶介导核酸等温扩增荧光法用于日本血吸虫感染性钉螺早期检测的研究

Early detection of Oncomelania snails infected with Schistosoma japonicum by recombinase aided amplification

目的探索重组酶介导的核酸等温扩增(recombinase aided amplification,RAA)荧光法用于日本血吸虫感染性钉螺早期检测的可行性。方法用日本血吸虫毛蚴人工感染200只湖北钉螺,随机分为阳性对照组和实验组各100只,另取100只未感染钉螺作为阴性对照组。将阳性对照组钉螺饲养至70 d,通过逸蚴法观察感染情况并计算阳性率。实验组钉螺分别在3 h以及5、20、40、70 d随机取20只,压碎镜检,同时提取钉螺软体DNA,进行荧光RAA法检测,获得不同时间点的阳性率,并与阳性对照组相比较。通过二代测序法对RAA检测阳性的特异性扩增产物进行测序鉴定。结果毛蚴感染后3 h、5 d、20 d、40 d、70 d实验组钉螺RAA法检测阳性率分别为60%、60%、65%、60%、70%,压碎镜检的阳性率分别为0、0、0、15%、65%,除70 d外其余时间点两种检测方法的阳性率差异均有统计学(χ^2=17.14,χ^2=17.14,χ^220 d=19.26,χ^2=8.64,均P<0.01);各时间点间阳性率差异无统计学意义(χ^2=0.68,P>0.05),各时间点阳性率与阳性对照组阳性率64.6%比较差异均无统计学意义。RAA扩增产物经二代测序后与数据库比对,确认为血吸虫特异性基因片段。结论 RAA荧光法敏感、特异,可用于日本血吸虫感染性钉螺的早期检测。

Objective To test whether the recombinase-aided amplification(RAA)of DNA is suitable for identification of infected Oncomelania snails in the incubation period.Methods Two hundred cultured Oncomelania snails were infected with the cercariae of S.japonicum and then kept at 25℃in the laboratory.Twenty snails were randomly selected each time at different developmental stages after infection.Snails were dissected under a microscope and then their DNA was extracted to determine if they were infected.Snails were examined with the naked eye or tested with RAA.Differences in the rate of infection were determined in each stage to analyze the practical utility of RAA.Moreover,the products of RAA were sequenced to confirm the existence of schistosomiasis.Differences between testing methods or groups were determined using 2×2 contingency tables,and testing methods or groups compared using the McNemar’s chi-squared test.Results RAA indicated that 60%of snails were infected 3 h after infection,60%were infected 5 d after infection,65%were infected 20 d after infection,60%were infected 40 d after infection,and 70%were infected 70 d after infection.Microscopic dissection indicated that 0%of snails were infected 3 h after infection,0%were infected 5 d after infection,0%were infected 20 d after infection,15%were infected 40 d after infection,and 65%were infected 70 d after infection.Compared to microscopic dissection,RAA detected infected snails at a higher rate(χ^2=46.22,P=0.00),and there were no significant differences between RAA and observation of cercariae shedding(χ^2=0.03,P=0.96).The products of RAA were specific gene fragments of Schistosoma japonicum(max score≥279,expected value≤2×10^-71).Conclusion RAA is sensitive and specific;it is reliable and can distinguish infected Oncomelania snails from normal ones in the early stages.