A specific PCR assay for the identification and differentiation of Schistosoma japonicum geographical isolates in mainland China based on analysis of mitochondrial genome sequences
A specific PCR assay for the identification and differentiation of Schistosoma japonicum geographical isolates in mainland China based on analysis of mitochondrial genome sequences
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基于线粒体基因组序列分析的中国大陆日本血吸虫地理分离株特异性PCR检测方法
DOI：
10.1016/j.meegid.2012.02.020
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发表时间：
2012-07-01
影响因子：
3.2
通讯作者：
Zhu, Xing-Quan
中科院分区：
文献类型：
--
作者：
Zhao, Guang-Hui;Li, Juan;Zhu, Xing-Quan
本研究对中国大陆7个血吸虫病流行省份的8个代表性样本的近全mt基因组序列进行了分析。8个mt基因组之间的序列差异。日本血吸虫样品的含量为0.20- 2.51%。蛋白质编码基因的变异大于rRNA基因的变异。对S.中国西南地区（SW）的日本血吸虫（Japonicum）与长江下游/浙江地区的日本血吸虫（Japonicum）相比，在tRNA-Cys的11727-11728位，微卫星（AG）indel上长2bp。代表性的DNA测序证实这种（AG）插入缺失可用于S的鉴定和区分。中国西南部的云南和四川省的日本血吸虫种群，其与所有其余的沿长江沿着三峡地区或靠近中国东海岸的地方病省份的种群具有两个（AG）重复（例如，浙江），其中只有一个（AG）重复。基于12个蛋白质编码基因的串联氨基酸的系统发育分析也表明，来自中国西南部（四川和云南省）的样本，三峡大坝以上，形成了一个独特的集群。根据这一indel多态性，设计了一对特异性引物，建立了特异性PCR聚丙烯酰胺凝胶检测方法。结果表明，不同菌株的PCR扩增产物长度存在明显差异。两种特有类型的日本血吸虫样品。特异性PCR方法可特异性鉴定S.日本血吸虫DNA的最低检出量为0.05ng，其它近缘吸虫DNA均未检出。该方法成本低廉，操作简便，整个检测过程可在4 h内完成。81 S的测试对云南、四川、湖北、江苏、江西、安徽、湖南等长江下游省份和浙江的264份样品进行PCR检测，验证了该方法的可靠性。结果表明，特异性PCR检测方法可用于沙门氏菌的流行病学监测和溯源。日本血吸虫在中国人和动物中的感染。(C)2012爱思唯尔有限公司版权所有。
In the present study, near-complete mt genome sequences for eight representative Schistosoma japonicum samples from seven endemic provinces in mainland China were analyzed. Sequence differences among the eight mt genomes of S. japonicum samples were 0.20-2.51%. Variation in protein-coding genes was greater than that in rRNA genes. The mt DNA sequences of S. japonicum samples from south-western (SW) China were 2 bp [position 11727-11728 within tRNA-Cys, microsatellite (AG) indel] longer than those of the parasites from the lower Yangtze/Zhejiang areas. Representative DNA sequencing confirmed that such (AG) indel could be exploited for identification and differentiation of S. japonicum populations in SW China's Yunnan and Sichuan province which have two (AG) repeats from those in all remaining endemic provinces along the Yangtze River below the Three Gorges regions or close to the east coast of China (e.g., Zhejiang) which have only one (AG) repeat. Phylogenetic analyses based on the concatenated amino acids of 12 protein-coding genes also showed that samples from SW China (Sichuan and Yunnan provinces), above the Three Gorges Dam, formed a distinct cluster. Based on this indel polymorphism, a pair of specific primers was designed and used to develop a specific-PCR polyacrylamide gel detection assay. There was an obvious length difference in the amplified PCR products between S. japonicum samples from the two endemic types. The specific-PCR assay allowed the specific identification of S. japonicum, with no amplicons being amplified from other closely related trematodes, and the minimum amount of DNA detectable was 0.05 ng. This approach is inexpensive, easy to perform and the whole detection process can be completed within 4 h. Examination of 81 S. japonicum samples from SW China's Yunnan and Sichuan provinces, and 264 samples from the lower Yangtze provinces (Hubei, Jiangsu, Jiangxi, Anhui and Hunan) and from Zhejiang validated the value of the specific PCR assay and proved its reliability. These findings indicate that the specific PCR assay would provide a useful tool for the epidemiological surveillance and for tracing the source of S. japonicum infection in humans and animals in China. (C) 2012 Elsevier B.V. All rights reserved.