| Objective: To develop the LAMP for rapid detecting Schistosoma japonicum, Pneumocystis carinii and Plasmodium falciparum respectively.Methods: Four primers were designed to target gene by Primer Explorer V3. The DNA of Schistosoma japonicum, Pneumocystis carinii and Plasmodium falciparum was extracted respectively by phenol-chloroform extraction. The appropriate amount of target pathogen DNA was added and amplified under isothermal conditions (65°C) for 60 min by the LAMP, then, LAMP results were judged by naked eye, SYBR Green I stain and electrophoretic analysis. The specificity of LAMP products were also confirmed by digestion of LAMP products using restriction enzymes. To evaluate the specificity of the LAMP assay, target pathogen and negative controls were detected by LAMP and PCR. To evaluate the sensitivity of the LAMP, target pathogen were 10-fold serially diluted and was amplified by LAMP and PCR.Results: After LAMP reaction, ladder patterns unique to the LAMP assay were observed with three LAMP or the target pathogens. Both naked eye , SYBR Green I stain and electrophoresis analysis were able to detect the products in the LAMP assay. Amplification was not observed when negative control was tested. The specificity of LAMP products for the target pathogen was also confirmed by restriction enzymes. The specificity of LAMP was similar to that of correspondence conventional PCR assay. Three LAMP with equal sensitivity to conventional PCR or higher than that of conventional PCR assay, the detection sensitiveness of LAMP assay for Schistosoma japonicum, Pneumocystis carinii and Plasmodium falciparum was 1 pgDNA/μL,1pgDNA/μl,1.5 Plasmodium /107RBC, respectively. Conclusion: As a consequence, we established specific, sensitive and rapid LAMP detection method for schistosoma japonicum; pneumocystis carinii and plasmodium falciparum respectively. |
