[用于检测福寿螺中广州管圆线虫的聚合酶链反应检测方法的建立]
[Development of PCR assay for detection of Angiostrongylus cantonensis in Pomacea canaliculata].
作者信息
Zhang Yi, Zhou Xiao-nong, Liu He-xiang, Lv Shan, Li Li-sha, Lin Jin-xiang, Li You-song
机构信息
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, WHO Collaborating Center for Malaria, Schistosomiasis and Filariasis, Shanghai 200025, China.
出版信息
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2006 Oct;24(5):353-5.
OBJECTIVE
To establish a PCR assay for detecting the third-stage larvae of Angiostrongylus cantonensis in Pomacea canaliculata.
METHODS
Polymerase chain reaction primers were designed by the software Lasergene, based on the specific cDNA of the third-stage larvae of A.cantonensis in Genbank. The total RNA was prepared from the third-stage larvae of A.cantonensis and of the snails by TRIzol one-step protocol. Amplification by RT-PCR was carried out following the kit protocol.
RESULTS
RT-PCR assay revealed a clear differentiation between infected and negative snails. When a mixture of the total RNA from the negative snails and the third-stage larvae of A.cantonensis was tested by the PCR assay, the detectable level was 128 pg RNA, a concentration close to one third-stage larva of A.cantonensis, minimum concentration that could be found by naked eyes. The minimum detected total RNA concentration of the third-stage larvae of A.cantonensis was 105 pg by PCR assay.
CONCLUSION
A PCR assay has been developed for detecting A.cantonensis larva in Pomacea canaliculata.
目的
建立一种用于检测福寿螺体内广州管圆线虫三期幼虫的聚合酶链反应（PCR）检测方法。
方法
基于Genbank中广州管圆线虫三期幼虫的特异性cDNA，利用Lasergene软件设计聚合酶链反应引物。采用TRIzol一步法从广州管圆线虫三期幼虫和螺中提取总RNA。按照试剂盒说明书进行逆转录聚合酶链反应（RT-PCR）扩增。
结果
RT-PCR检测显示感染螺和阴性螺之间有明显区分。当用PCR检测法检测阴性螺总RNA与广州管圆线虫三期幼虫的混合物时，可检测水平为128 pg RNA，该浓度接近一条广州管圆线虫三期幼虫，是肉眼可发现的最低浓度。通过PCR检测法，广州管圆线虫三期幼虫的最低检测总RNA浓度为105 pg。
结论
已建立一种用于检测福寿螺体内广州管圆线虫幼虫的PCR检测方法。
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