Abstract:

Objective To develop a method for rapid detection of specific gene fragments of Schistosoma japonicum using a multienzyme isothermal rapid amplification (MIRA) combined with fluorescent probing. Methods The S. japonicum non-long terminal repeat retrotransposons (SjR2) fragment was selected as the target sequence, and three pairs of primers and fluorescent probes were designed and synthesized to establish a fluorescent MIRA reaction system, with which, fluorescence quantitative PCR was performed, and amplification curves were plotted to compare and screen out primer pairs and probe concentrations with better amplification effects. To evaluate the sensitivity of this method, adult S. japonicum genomic DNA at different concentrations of 1 fg/μl, 5 fg/μl, 10 fg/μl, 100 fg/μl, 1 pg/μl, and 10 pg/μl, were detected, respectively. To evaluate the methd specificity, genomic DNA extracted from Paragonimus westermani, Clonorchis sinensis, C. orientalis, Gnathostoma, and Toxoplasma gondii were examined using the fluorescence MIRA method. To assess the detactable limit of serum gene DNA of the method, rabbit blood was collected from the ear vein for separating serum to prepare simulated positive serum samples containing 1 fg, 5 fg, 10 fg, 100 fg, 1 pg, and 10 pg DNA of adult S. japonicum, which were detected using the fluorescence MIRA method. Results The amplification efficiency of primer pair 1 (SjR2-1) was high, and the fluorescent product was seen at the 22nd cycle (11 min) with a maximum fluorescence value of 170 000; the probe amount at 0.6 μl/reaction displayed better fluorescence intensity with low fluorescence background. The fluorescent products were amplified at the 26th cycle (13 min) at 39 ℃. The minimum detection limit of the established fluorescence MIRA method for detecting Schistosoma adult worm DNA was 1 fg/reaction. The electrophoresis of amplification products showed that electrophoresis bands appeared when the template of Schistosoma genomic DNA was 10 pg, 1 pg, 100 fg, and 10 fg, respectively, and the detection limit was 10 fg/reaction and the product size was 186 bp. The reaction tube containing only the DNA of S. japonicum adult worm showed fluorescence amplification curves, while no significant fluorescence amplification curves were observed in the detection of genomic DNA of P. westermani, C. sinensis, C. orientalis, Gnathostomum and T. gondii. When the DNA content in the simulated positive serum of different concentrations of Schistosoma was 1 pg and 10 pg, positive amplification curves appeared, and positive fluorescence amplification signals appeared as early as the 16th cycle (within 8 min). The minimum detection limit for detecting Schistosoma adult worm DNA in simulated positive serum by this method was 1 pg/reaction. Conclusion A fluorescent MIRA method detecting specific gene fragments of S. japonicum was successfully developed. The method is rapid, sensitive and specific in use, showing potential diagnostic application value for schistosomiasis japonica.

Key words: Schistosoma japonicum, Multienzyme isothermal rapid amplification, Fluorescent probe, Gene fragment