Study On Detection Of Schistosoma Japonicum Infection By PCR Technique

At present, schistosomiasis was still a serious threat to public health and hindered social-economic development of epidemic areas. This parasitic disease influences many countries and regions in Asia, Africa and Latin America. In China, with the extablishment of more efficient disease control system and mass treatment and prevention, the epidemiological features of schistosomiasis are changing from high to low prevalence. However, there are more than one million people still suffering from this zoonosis. As known to all, high-sensitive diagnosis method leads to earlier treatment and better life quality, especially for schistosomiasis. Therefore, a sensitive diagnosis method of schistosomiasis is always critical in the control programs. Diagnosis methods with high specificity and sensitivity are urgently demanded and have become the development direction of schistosomiasis diagnosis.Diagnosis techniques could be divided into two categories, the direct and the indirect diagnosis methods. Recommended by WHO as a pathogenic diagnosis method applied to field study, the modified Kato-Katz is money-saving and semi-quantitative, which mainly leads to final diagnosis. However, the previous studies have shown that Kato-Katz considerably underestimated the true prevalence of infection, especially in the regions with relatively low prevalence rate. As the main approach of the indirect diagnosis methods of schistosomiasis, ELISA has been widely used in both clinical and epidemiological settings to detect schistosome infection. However, this immunoassay technique can not distinguish ongoing and previous individual infection. Accordingly, diagnosis method with high sensitivity and specificity should be required for definite individual diagnosis.In recent years, PCR technique was used to detect schistosoma infection. Many comparative studies showed PCR share a higher sensitivity and specificity than other routine diagnosis methods of schistosomiasis, eg. Kato-Katz method and ELISA. But in previous investigations, the choices of target genes were limited, and there was no precise evaluation of the sensitivity of the PCR diagnosis method. Moreover, there was not much comparative research to compare the fecal sample DNA extraction method.SYBR Green Real-time copro-PCR diagnosis method in the present study is with high sensitivity. By mixed fecal samples of health buffalos with counted schistosomal eggs, the sensitivity of SYBR Green Real-time copro-PCR diagnosis method was assessed precisely. On this basis, four distinguished fecal sample DNA extraction methods (ROSE method, IRS-ROSE method, UltraCleanTM Fecal DNA Sample Kit and QIAamp DNA Stool Kit method) were used to extract DNA from artificial positive fecal samples with different egg counts, and studied comparatively accordingly. Finally, in order to establish a copro-PCR aimed on field study, the current study also used conventional PCR technique to explore the usage of Schistosoma japonicum specific and repeated sequences as PCR target sequences. The main results are as follows:1. Sensitivity of SYBR Green Real-time Copro-PCR on Detection of Schistosoma japonicum infection: The artificial positive fecal samples were prepared by mixing counted schistosomal eggs with fecal samples of healthy buffalos. A modified protocol of QIAamp DNA Stool Kit was used to extract DNA from the artificial positive fecal samples as the templates for SYBR Green real-time PCR reaction. One schistosomal egg in 200mg fecal samples was identified by this method. A linear relationship (r2 = 0.9406) between the logarithmic transformated egg counts of artificial positive fecal samples and Ct values was studied. Strict quality control was performed throughout the procedure in order to achieve persuasive results.2. The comparative research of ROSE method, IRS-ROSE method, UltraCleanTM Fecal DNA Sample Kit method (MoBio Kit method): in this study, ROSE method, IRS-ROSE method, UltraCleanTM Fecal DNA Sample Kit method were used to extract DNA from artificial positive samples with different levers of counted schistosomal eggs, then testify the DNA templates with SYBR Green Real-time PCR. Compared with the other two methods, the MoBio Kit method shared a higher sensitivity. Even DNA template of artificial positive fecal sample with only one schistosomal egg achieved positive result. While DNA templates artificial positive fecal sample with five or ten schistosomal eggs were detectable using IRS-ROSE method or ROSE method respectively.3. Conventional PCR using Schistosoma japonicum specific and repeated sequences as PCR target sequence: The experimental result of the conventional PCR specific examination was not satisfying. Templates of Clonorchis sinensis genomic DNA and DNA extract from negative fecal samples showed positive results in PCR reaction, while no significant similarity found in the BLAST aligning. Using QIAamp DNA Stool Kit, the DNA extracted from all artificial positive fecal samples all showed positive results. In current study, all serum samples were negative by PCR.In this study, swirling purification method was used to purify schistosomal eggs from liver tissue. It enriched the research of PCR technique to detect schistosoma infection. The SYBR Green Real-time copro-PCR detection system established in present study is a method of high sensitivity and specificity, can be used to estimate the Schistosoma japonicum infectiosity, and its sensitivity was evaluated precisely. This method was used to evaluate several different fecal sample extraction method. With Schistosoma japonicum specific highly repeated DNA sequence as conventional PCR template, our study also explored the possibility of setting up a copro-PCR method suitable for field study.