A cleaved amplified polymorphic sequence (CAPS) method for the identification of geographical isolates of Schistosoma japonicum in China

Schistosomiasis japonica, caused by the blood fluke Schistosoma japonicum, is a severe parasitic zoonosis, causing significant morbidity, mortality and considerable economic losses in south-east Asia, particularly in mainland China (Zhou et al., 2005; Bergquist et al., 2008; Zhou et al., 2008; Li et al., 2010). Although there have been significant efforts to control schistosomiasis in China, this disease appears to be re-emerging due to climate changes and human activities (Wang et al., 2009). Thus far, schistosomiasis is still endemic in seven provinces (Yunnan, Sichuan, Anhui, Hubei, Jiangxi, Jiangsu and Hunan provinces) of China (Ross et al., 2001), and the impact of this communicable disease in China is almost equal to that of HIV/AIDS and tuberculosis (Engels et al., 2005). In previous studies, genetic variation among S. japonicum populations from different endemic provinces in China has been detected using a variety of genetic markers (Bogh et al., 1999; Sorensen et al., 1999; Zhu et al., 1999; Shrivastava et al., 2005; Zhao et al., 2009a; Zhao et al., 2009b; Zhao et al., 2009c). However, these markers and the detected genetic variability have not been utilized for the practical identification and differentiation of S. japonicum geographical isolates in China. The economic reform and the deterioration of ecological environment, flash floods, landslides and the mobility of human population appear to be having an impact on the transmission of S. japonicum (Li et al., 2000; Seto et al., 2008). Consequently, it is important to be able to identify different genetic variants of S. japonicum in China. Various methods for the analysis of genetic variation have been developed, including single-strand conformation polymorphism (Simoes et al., 2007), denaturing high-performance liquid chromatography (Nickerson et al., 2000), gene chips (Schmalzing et al., 2000), TaqMan probe (Shi et al., 1999) and pyrosequencing technology (Ahmadian et al., 2000). However, such techniques can be time-consuming and/or require detection instruments or labelled oligonucleotides. In contrast, the cleaved amplified polymorphism sequence (CAPS) technique is a simple and effective method to allow the rapid detection of single nucleotide polymorphisms (SNPs) (Neff et al., 1998; Komori and Nitta, 2005). CAPS is based on the PCR-based amplification of targeted genes and subsequent digestion of amplicons using restriction enzymes. The digested PCR products are separated by agarose gel electrophoresis. CAPS generates the same type of data as traditional restriction fragment length polymorphism analysis, but significantly reduces the amount of purified DNA required for analysis and time-consuming steps. This method can detect both homozygous and heterozygous individuals (Weiland and Yu, 2003; Komori and Nitta, 2005). CAPS has been successfully used to study genetic diversity in plants, such as Arabidopsis thaliana (Hardtke et al., 1996; Barth et al., 2002), Cryptomeria japonica (Tsumura and Tomaru, 1999; Tsumura et al., 1999) and Pisum sativum L. (Konovalov et al., 2009), but has seldom been used to study variation in parasites (Gandhi et al., 2009). The aims of the present study were to identify SNP sites specific for geographical isolates of S. japonicum based on results of previous studies. Using these SNPs, the CAPS technique was developed and used to identify and differentiate S. japonicum isolates from Yunnan province and those from other endemic provinces.