A specific PCR assay for the identification and differentiation of Schistosoma japonicum geographical isolates in mainland China based on analysis of mitochondrial genome sequences

Abstract

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 2bp [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 0.05ng. This approach is inexpensive, easy to perform and the whole detection process can be completed within 4h. 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.