Identification Of Cell Free Circulating DNA From Schistosoma Japonicum For Diagnosis Of Schistosomiasis

Pathogenic helminth infections are responsible for severe health problems and economic losses worldwide.Timely and accurate diagnosis of helminth infections is critical for adopting suitable strategies for pathogen control.The nucleic acid-based diagnostic methods,including polymerase chain reaction,quantitative qPCR,loop-mediated isothermal amplification and recombinase polymerase amplification methods have been available for the detection of helminth infections.These methods offer considerable advantages such as precision,timeliness and sensitivity of parasite detection.Schistosomiasis is a major public health problem that is included in the neglected tropical diseases.The early diagnosis and detection of the pathogen is of critical for the control of the disease.Traditional diagnosis methods include the detection of worm's eggs in fecal examination,or detection of circulating antigens in immunological based assays.These traditional strategies lack sensitivity in an earlier detection of the schistosomiasis.Cell-free DNA(cfDNA)that includes the fragments of parasitic DNA circulating in the body fluids of host offers an alternative mean for the rapid detection of pathogen and thus is a useful diagnosis tool.In this study,we explored the useful mitochondrial cfDNA markers for the diagnosis of schistosomiasis from the experimentally infected hosts(rabbit and mice).We first analyzed the efficacy of three different cfDNA isolation kits and found the QIAamp Circulating Nucleic Acid Kit the most productive one.Then tested several genes of the mitochondrial DNA of Schistosoma japonicum,and identified mitochondrial ribosomal RNA,rrnL(large subunit rRNA 16S),and cytochrome b gene as persistent and useful cfDNA markers for the early detection of schistosomiasis.Furthermore,we also tested an established barcoding marker of helminths the internal transcribed spacer 2(ITS2)of nuclear genome,and found it too a useful cfDNA marker.We evaluated the sensitivity of these cfDNA markers with varying numbers of cercaria.Altogether,our results suggest that these three cfDNA markers can be useful for developing diagnostic tool for the detection of S.japonicum infections.