环介导等温扩增法检测粪样中日本血吸虫虫卵DNA的效果评估

发布时间：2018-04-24 01:40

本文选题：日本血吸虫 + 虫卵　；参考：《中国寄生虫学与寄生虫病杂志》2017年03期

【摘要】：目的评估环介导等温扩增(loop mediated isothermal amplification,LAMP)法检测粪样中日本血吸虫(Schistosoma japonicum)虫卵DNA的效果,并评价其检测流行区现场牛野外粪样的效果。方法取1 g新鲜的日本血吸虫虫卵阴性牛粪,分别加入5个新鲜日本血吸虫虫卵、50μl日本血吸虫虫卵排泄分泌产物(egg secretion product,ESP)制备人工模拟阳性粪样。用粪DNA提取试剂盒分别提取加入虫卵的未经研磨或研磨2 min后的人工模拟阳性粪样、加入虫卵ESP的粪样和阴性粪样的DNA,以日本血吸虫28S核糖体DNA(r DNA)为检测靶基因,用LAMP法、PCR法检测,评估LAMP法检测粪样中日本血吸虫虫卵DNA的效果。收集2012-2014年湖南、湖北、江西、安徽等4省日本血吸虫病流行区的牛野外粪样221份,研磨后同法提取DNA,用LAMP法检测,并与PCR法、孵化法的检测结果进行比较,评价其检测流行区现场野外粪样的效果。结果LAMP法检测加入虫卵粪样并经研磨后提取的DNA、加入虫卵ESP粪样提取的DNA均为阳性反应,呈绿色;而加入虫卵粪样未经研磨提取的DNA、阴性粪样DNA则为阴性反应,呈棕色;PCR检测的结果与LAMP法检测结果相近。LAMP法、PCR法和孵化法检测血吸虫病流行区牛野外粪样的阳性率分别为5.43%(12/221)、4.52%(10/221)、0.90%(2/221)。LAMP法的阳性率明显高于孵化法(P0.05),LAMP法与PCR法阳性率差异无统计学意义(P0.05)。结论 LAMP法可用于检测流行区现场野外粪样的日本血吸虫虫卵DNA,其现场应用价值有待进一步验证。
[Abstract]:Objective to evaluate the effect of loop mediated isothermal amplification (LAMP) on the determination of the egg DNA of Schistosoma japonicum (Schistosoma japonicum) in the feces and to evaluate the effect of the detection of the field feces in the field of the epidemic area. Methods 1 g fresh Japanese Schistosoma japonicum egg negative cow dung was taken into 5 fresh Japanese blood respectively. An artificial simulated fecal sample was prepared by the egg excretory secretory product (egg secretion product, ESP) of 50 L Schistosoma japonicum. Using the fecal DNA extraction kit to extract the artificial simulated fecal samples that were added to the eggs without grinding or grinding 2 min, and adding the fecal and negative fecal samples of the eggs ESP to 28S ribosomes of Schistosoma japonicum. DNA (R DNA) was used to detect the target gene, LAMP method and PCR method to evaluate the effect of LAMP method for detecting the egg DNA of Schistosoma japonicum in the feces. 221 cattle field samples from 4 provinces of Japan schistosomiasis, Hunan, Jiangxi, Anhui and other provinces, were collected for 2012-2014 years, and DNA was extracted with the method of LAMP after grinding, and the detection results of the hatching method were with the PCR method. The results were compared. Results the LAMP method was used to detect the field feces in the field of the epidemic area. Results the DNA extracted from the eggs of the eggs was detected by the LAMP method, and the DNA of the fecal sample extracted from the eggs of the insect eggs was positive and green, and the negative fecal DNA was negative, and the negative fecal sample was brown; the PCR detection knot was detected by adding the eggs of the eggs ESP. The results were similar to that of LAMP method. The positive rate of PCR method and hatching method was 5.43% (12/221), 4.52% (10/221), and 0.90% (2/221).LAMP method was significantly higher than that of hatching (P0.05), and there was no statistical difference between LAMP method and PCR method (P0.05). Conclusion LAMP method can be used for detection flow. The field application value of field fecal DNA of Schistosoma japonicum remains to be further verified.

【作者单位】：中国疾病预防控制中心寄生虫病预防控制所世界卫生组织热带病合作中心科技部国家级热带病国际联合研究中心卫生部寄生虫病原与媒介生物学重点实验室;湖南省血吸虫病防治所;
【基金】：上海市卫生和计划生育委员会科研课题(No.201440498) 湖南省科技厅项目(No.2012FJ4103) 国家卫生计生委疾控局委托项目(2015-2016) 中国疾控中心寄生虫病所防治技术储备科研基金(No.CB-15005)~~
【分类号】：R383.24

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