Rapid Detection Of Seven Pathogens With Loop-Mediated Isothermal Amplification

Objective: To develop the LAMP or RT-LAMP for detecting T. gondii, EHEC O157:H7, S.dysenteriae, V.cholera,V. parahaemolyticus, Rotavirus G3 and Norovirus GII ,respectively.Methods: Four or six primers were designed to target gene by PrimerExplorer V3 . DNA was extracted by TIAamp Genomic DNA Kit or phenol-chloroform extraction,and the viral RNA was extracted with a QIAamp viral RNA mini kit. Through optimizing the conditions needed for the specific amplification of the target gene, we have established seven LAMP or RT-LAMP for the correpondens pathogens. Target pathogen DNA amplified under isothermal conditions(65°C)for 60 min, 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 seven LAMP or RT-LAMP for the target pathogens. Both naked eye ,SYBR Green I stain and electrophoretic analysis were able to detect the products in the LAMP assay. Amplification was not observed when negative control were tested. The specificity of LAMP products for the target pathogen were also confirmed by restriction enzymes. The specificity of LAMP was similar to that of correpondens conventional PCR assay. Seven LAMP or RT-LAMP with equal sensitivity to conventional PCR or higher than that of conventional PCR assay,the detection limit of LAMP assay for T. gondii, EHEC O157:H7, S.dysenteriae, V.cholera,V. parahaemolyticus, Rotavirus G3 and Norovirus GII was 1 pg/μL, 10 cfu/ mL, 12 cfu/mL, 20 cfu/mL, 15 cfu/mL, 15.6 pg/tube and 25 pg/tube,respectively.Conclusion: As a consequence,we established seven specific,sensitive and rapid LAMP or RT-LAMP detection method for T. gondii, EHEC O157:H7, S.dysenteriae, V.cholera,V. parahaemolyticus, RV G3 and Norovirus GII , respectively.