Development of a Novel Quantitative PCR Assay for Detecting Schistosoma japonicum in Field-Captured Rodents

In the context of the current low endemicity of schistosomiasis in China, Schistosoma japonicum (S. japonicum) infections increasingly have been reported in field-captured rodents, highlighting the need for rapid and scalable tools to support surveillance during the elimination stage. This study developed and evaluated a quantitative polymerase chain reaction (qPCR) assay targeting a highly repetitive S. japonicum sequence for detecting infection in liver tissues. Analytical performance was assessed using adult-worm genomic DNA, DNA from non-target pathogens, egg-spiked mouse liver samples, experimentally infected BALB/c mice, and liver samples from field-captured rodents collected in Dongzhi and Duchang counties. The optimized assay generated a single melting peak at 79 ± 0.5 °C, used Ct < 33 as the positivity cutoff, and showed no apparent cross-reactivity with the non-target pathogens tested. Using serially diluted adult-worm genomic DNA, the assay consistently detected S. japonicum DNA at concentrations as low as 1 pg/μL. In the egg-spiked liver model, as few as two eggs were detected in 200 mg of liver tissue. In the experimentally infected model, all infected mice tested positive, whereas all non-infected controls tested negative, indicating complete agreement between the qPCR results and the experimentally defined infection status. In the field evaluation, qPCR yielded a higher positivity rate than parasitological examination in Dongzhi County (66.81% vs. 51.72%) and identified six positive samples in Duchang County, where all samples were negative by parasitological examination. These findings indicate that the qPCR assay can facilitate sensitive and high-throughput detection of S. japonicum infection in field-captured rodents and may provide a useful complementary tool for surveillance during schistosomiasis elimination.