2022年于卫氏并殖吸虫流行区湖北兴山县和保康县分别采集34只和27只溪蟹,捣碎后用NaOH消化法提取DNA.根据卫氏并殖吸虫5.8S核糖体RNA序列,使用Primer Explorer V5软件设计卫氏并殖吸虫环介导等温扩增(LAMP)特异引物,建立卫氏并殖吸虫LAMP检测方法,评价该方法的特异性、灵敏度、扩增效率及现场应用效果,并与沉淀镜检法比较.结果显示,建立的LAMP检出限可达1个卫氏并殖吸虫囊蚴,且与日本血吸虫、华支睾吸虫、肝片形吸虫、肝毛细线虫、钩虫等虫种无交叉反应.检测4份卫氏并殖吸虫囊蚴DNA样品,出现浊度时间(Tt值)和浊度速率峰值(Df)均值为29.0 min和0.237.LAMP检出兴山和保康县卫氏并殖吸虫阳性溪蟹分别为24只和0只,阳性率分别为70.6％(24/34)和0(0/27);沉淀镜检法检出囊蚴阳性溪蟹分别为23只和0只,阳性率分别为67.6％(23/34)和0(0/27),两种方法结果差异无统计学意义(x2=0.2,P＞0.05).建立的卫氏并殖吸虫LAMP检测方法操作简便、特异性强、灵敏度高,可用于淡水蟹卫氏并殖吸虫流行区现场调查或筛查.
Establishment of loop-mediated isothermal amplification detection for Paragonimus westermani
In 2022,a total of 34 and 27 brook crabs were collected from Xingshan and Baokang Counties,respectively,in Hubei Province,a region where Paragonimus westermani is endemic.The DNA was extracted by NaOH digestion method after crushing from crab.Based on the 5.8S ribosomal RNA sequence of P.westermani,Primer Explorer V5 software was used to design specific primers for loop-mediated isothermal amplification(LAMP)of P.westermani,and a LAMP detection method for P.westermani was established.The specificity,sensitivity,amplifica-tion efficiency,and field application effect of the method were evaluated,and compared with precipitation microscopy.The results indicated that the LAMP method displayed a detection limit of a single P.westermani metacercaria with-out any cross-reactivity with five non-target species,including Schistosoma japonicum,Clonorchis sinensis,Fasciola he-patica,Capillaria hepatica,and hookworm.The assay's performance was quantified using the average turbidity time(Tt value)of 29.0 min and the peak turbidity rate(Df)of 0.237 min for four metacercariae samples.The LAMP method revealed 24 and 0 positive stream crabs infceted with P.westermani in Xingshan County and Baokang County,with positive rates of 70.6％(24/34)and 0(0/27),respectively;the precipitation microscopy method revealed 23 and 0 positive stream crabsin Xingshan County and Baokang County,with positive rates of 67.6％(23/34)and 0(0/27),respectively.There is no statistically significant difference in the results between the two detection methods(x2=0.2,P＞0.05).Conclusively,the LAMP method developed in this study is a straightforward,specific,and highly sensitive diagnostic tool for the detection of P.westermani in freshwater crabs.Its proven efficacy suggests that it is well-suited for in-field surveys or as a screening mechanism in endemic regions.
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