横川后殖吸虫与扇棘单睾吸虫双重PCR鉴别方法的建立

中国寄生虫学与寄生虫病杂志

横川后殖吸虫与扇棘单睾吸虫双重PCR鉴别方法的建立

梅雪芳¹，李树清²，康羊群¹，石云良^1，3，黄腾飞⁴，陈志飞²，黄维义^{1，3 *}

1 广西大学动物科技学院，南宁 530004；2 上海出入境检验检疫局，上海 200135；3 广西大学食品安全研究中心，南宁 530004；4 罗氏诊断产品（上海）有限公司，上海 200335

出版日期:2015-06-30 发布日期:2015-09-08

Establishment of Duplex PCR for Identifying Metagonimus yokogawai and Haplorchis taichui

MEI Xue-fang¹，LI Shu-qing²，KANG Yang-qun¹，SHI Yun-liang ^1，3，

1 College of Animal Science and Technology，Guangxi University，Nanning 530004，China；2 Shanghai Entry-Exit Inspection and Quarantine Bureau of the People’s Republic of China，Shanghai 200135，China；3 Research Center of Food Quality and Safety，Guangxi University，Nanning 530004，China；4 Roche Diagnostics（Shanghai） Limited，Shanghai 200335，China

Online:2015-06-30 Published:2015-09-08

摘要/Abstract

摘要：

【摘要】目的建立一种能快速鉴定横川后殖吸虫（Metagonimus yokogawai）和扇棘单睾吸虫（Haplorchis taichui）的双重PCR方法。方法从GenBank中获取横川后殖吸虫和扇棘单睾吸虫以及与其同源性较高的虫种的核糖体DNA第一内转录间隔区（ITS1）序列，应用Primer premier 5.0软件设计2对特异性引物，并优化PCR反应体系和反应条件，建立双重PCR法。将横川后殖吸虫和扇棘单睾吸虫与17个相关虫种一起进行PCR扩增，检测方法的特异性。横川后殖吸虫和扇棘单睾吸虫的ITS1扩增产物经pMD19-T载体进行TA克隆获得质粒，并将质粒进行梯度稀释，检测其敏感性。应用新建的双重PCR法鉴定从47副猫内脏和40副犬内脏中收集的吸虫，检测该方法的准确性和实用性。结果新建的双重PCR法能扩增出横川后殖吸虫和扇棘单睾吸虫的目的片段，大小分别为648 bp和279 bp，不与钩棘单睾吸虫、华支睾吸虫、心形咽口吸虫、次睾属吸虫囊蚴、外睾属吸虫囊蚴、藐小棘隙吸虫、抱茎棘隙吸虫、弗氏棘口吸虫、似锥低颈吸虫、全冠属吸虫、重盘属吸虫、异尖属线虫、东方次睾吸虫、卫氏并殖吸虫、瓦氏瓦生吸虫、背孔属吸虫和宫脂属线虫的DNA发生交叉扩增，具有较好的特异性。敏感性试验表明，应用该双重PCR法，2类吸虫 DNA的最低检测值分别为1.49×10-1 pg和1.14×10-1 pg。对来自猫内脏和犬内脏的吸虫检测表明，该方法能够区分横川后殖吸虫和扇棘单睾吸虫，并且不与猫、狗中其他的吸虫DNA发生交叉扩增。结论本研究所建立的双重PCR法可用于快速鉴定横川后殖吸虫和扇棘单睾吸虫。

关键词: 横川后殖吸虫, 扇棘单睾吸虫, 双重PCR, 鉴别

Abstract:

【Abstract】 Objective To develop a duplex PCR method for identifying Metagonimus yokogawai and Haplorchis taichui. Methods ITS1 sequences of M. yokogawai and H. taichui， as well as those of their homologous species were obtained from GenBank， and two sets of specific primer pairs for M. yokogawai and H. taichui were designed accordingly using Primer Premier 5.0 software. PCR reaction system and conditions were optimized. The established duplex PCR method was applied in a pool of M. yokogawai, H. taichui, and 17 related species to examine its specificity. Sensitivity was evaluated through serial dilutions of plasmids containing their specific sequences. Finally， the duplex PCR was applied to identify M. yokogawai and H. taichui among trematodes collected from the viscera of 47 cats and 40 dogs to test its practicality. Results The duplex PCR method amplified target sequences of M. yokogawai and H. taichui， generating 648 bp and 279 bp products, respectively. No cross reaction was found with the following 17 related species: Haplorchis pumilio, Clonorchis sinensis, Pharyngostomum cordatum, the metacercaria of Metorchis sp. and Exorchis sp., Echinochasmus liliputanus, Echinochasmus perfoliatus, Echinostoma friedi, Hypoderaeum conoideum, Holostephanus sp., Diplodiscus sp., Anisakis sp., Metorchis orientalis, Paragonimus westermani, Watsonius watsoni, Notocotylus sp. and Hysterothylacium sp, indicating a high specificity of this method. The detection limits for DNAs of M. yokogawai and H. taichui were 1.49×10-1 pg and 1.14×10-1 pg, suggesting a good sensitivity for this method. Further, the duplex PCR successfully identified M. yokogawai and , H. taichui from cat and dog viscera, with no cross amplification of other trematodes. Conclusion The duplex PCR is effective in identifying Metagonimus yokogawai and Haplorchis taichui.

Key words: Metagonimus yokogawai, Haplorchis taichui, Duplex PCR, Identify

梅雪芳，李树清，康羊群，石云良，黄腾飞，陈志飞，黄维义*. 横川后殖吸虫与扇棘单睾吸虫双重PCR鉴别方法的建立[J]. 中国寄生虫学与寄生虫病杂志.

MEI Xue-fang，LI Shu-qing，KANG Yang-qun，SHI Yun-liang,. Establishment of Duplex PCR for Identifying Metagonimus yokogawai and Haplorchis taichui[J]. Chinese Journal of Parasitology and Parasitic Diseases.

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[6]	李彦, 刘航, 杨毅梅. 分子生物学技术在带绦虫鉴别中的应用[J]. 中国寄生虫学与寄生虫病杂志, 2011, 29(5): 12-385-388.
[7]	桂希恩;管立人. 内脏利什曼病、播散型组织胞浆菌病及马内菲青霉菌病的诊断和鉴别[J]. 中国寄生虫学与寄生虫病杂志, 2007, 25(1): 16-72.
[8]	郑彬;汤林华;马雅军;王学忠;周水森;施文琦. 微小按蚊A、C的PCR和同工酶鉴别比较研究[J]. 中国寄生虫学与寄生虫病杂志, 2005, 23(2): 4-81.
[9]	黎学铭;杨益超;蓝春庚;谭裕光;黄铿凌;许洪波;林睿. 广西发现扇棘单睾吸虫[J]. 中国寄生虫学与寄生虫病杂志, 2004, 22(1): 16-61.
[10]	马雅军;瞿逢伊;董学书;周红宁. 我国多斑按蚊复合体成员种的分子鉴别研究[J]. 中国寄生虫学与寄生虫病杂志, 2002, 20(6): 1-324.
[11]	马雅军;瞿逢伊;曹毓存;杨宝金. 我国雷氏按蚊和嗜人按蚊的分子鉴别和分类地位的探讨[J]. 中国寄生虫学与寄生虫病杂志, 2000, 18(6): 2-328.
[12]	江莉;温浩;李雄;伊斯拉因·乌斯满. 18kDa抗原诊断泡型包虫病的评价[J]. 中国寄生虫学与寄生虫病杂志, 1999, 17(2): 4-80.
[13]	索爱琴,段瑜,周本立. 脑囊虫病伴发脑部其它器质性病变的鉴别诊断[J]. 中国寄生虫学与寄生虫病杂志, 1996, 14(3): 250-251.
[14]	陈明中,袁树斌. 脑内裂头蚴病一例[J]. 中国寄生虫学与寄生虫病杂志, 1995, 13(1): 64-64.
[15]	雷昌球,韩晓军,李安定,黄学敏,石国祥. 浙江省人体寄生虫感染情况的调查[J]. 中国寄生虫学与寄生虫病杂志, 1994, 12(S1): 222-222.