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

中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (3): 230-234.

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

冯婷¹, 秦志强^1,^*(), 许静¹, 周杰², 钱颖骏¹, 祝红庆¹, 吕山¹, 曹淳力¹, 李石柱¹

1 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,上海 200025
2 湖南省血吸虫病防治所,岳阳 414000

收稿日期:2017-03-05 出版日期:2017-03-30 发布日期:2017-09-07
通讯作者: 秦志强
基金资助:
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）

Efficacy evaluation of a loop mediated isothermal amplification technique in detection of DNA of Schistosoma japonicum eggs in fecal samples

Ting FENG¹, Zhi-qiang QIN^1,^*(), Jing XU¹, Jie ZHOU², Yin-jun QIAN¹, Hong-qing ZHU¹, Shan LV¹, Chun-li CAO¹, Shi-zhu LI¹

1 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; WHO Collaborating Centre for Tropic Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology; Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai 200025, China
2 Hunan Institute of Schistosomiasis Control, Yueyang 414000, China

Received:2017-03-05 Online:2017-03-30 Published:2017-09-07
Contact: Zhi-qiang QIN

摘要/Abstract

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、50 μl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 (rDNA)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（P < 0.05）,LAMP法与PCR法阳性率差异无统计学意义 (P > 0.05)。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.

关键词: 日本血吸虫, 虫卵, 核酸, 环介导等温扩增, 效果评估

Abstract: Objective To evaluate the efficiency of loop mediated isothermal amplification (LAMP) method in detecting DNA of Schistosoma japonicum eggs in fecal samples and its performance in field trial. Methods One gram of fresh fecal sample of an egg negative water buffalo was mixed with five S. japonicum eggs or 50 μl of egg secretion product (ESP) to simulate S. japonicum infection. DNA was extracted with a DNA kit from the fecal samples with addition of eggs, with or without a 2-min homogenization procedure, those with addition of ESP, and the negative fecal sample. LAMP was performed to amplify the 28S ribosomal DNA (rDNA) of S. japonicum to detect the DNA of eggs in fecal sample, and the efficacy was compared to that of PCR. In addition, a total of 221 wild fecal samples were collected from endemic areas in Hunan, Hubei, Jiangxi and Anhui provinces, and the DNA of field samples was extracted with a 2-min homogenization procedure, followed by LAMP and PCR respectively as above. The efficacy of LAMP in field application was compared with that of PCR and miracidia hatching test. Results The LAMP method showed the presence of S. japonicum eggs in the fecal samples added with eggs followed by a homogenization procedure, and the fecal sample with addition of ESP, as indicated by green color. Similar results were obtained by PCR. The positive rates of S. japonicum eggs in the wild fecal samples by LAMP, hatching method and PCR were 5.43% (12/221), 0.90% (2/221) and 4.52% (10/221), respectively. The sensitivity of LAMP was significantly higher than the hatching method (P < 0.05), and was comparable to that of the PCR method (P > 0.05). Conclusion LAMP method may be used as a sensitive testing tool in field detection of wild fecal in schistosomiasis endemic area.

Key words: Schistosoma japonicum, Egg, Nucleotide acid, Loop mediated isothermal amplification, Efficiency evaluation

中图分类号:

R383.24

冯婷, 秦志强, 许静, 周杰, 钱颖骏, 祝红庆, 吕山, 曹淳力, 李石柱. 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[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(3): 230-234.

Ting FENG, Zhi-qiang QIN, Jing XU, Jie ZHOU, Yin-jun QIAN, Hong-qing ZHU, Shan LV, Chun-li CAO, Shi-zhu LI. Efficacy evaluation of a loop mediated isothermal amplification technique in detection of DNA of Schistosoma japonicum eggs in fecal samples[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(3): 230-234.

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检测结果 Outcomes	阳性 Positive	阴性 Negative	阳性率/% Positive rate%
环介导等温扩增LAMP	12	209	5.43^a
聚合酶链反应 PCR	10	211	4.52^a
集卵孵化法 Miracidia-hatching test	2	219	0.90

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