No abstract

According to Chinese Pharmacopoeia, Escherichia coli should not be detected in oral drugs. In this study, a visual LAMP method for rapid detection of Escherichia coli was developed to replace the traditional microbial limit test method. Methods Four specific primers (FIP, BiP, F3, B3) for loop mediated isothermal amplification were designed by using uidA gene of Escherichia coli, and calcein-Mn2+ dye was added to the system to establish a visual loop mediated isothermal amplification detection method for Escherichia coli Finally, the visual LAMP system was used to detect the presence of E.coli and the sensitivity of the system in drugs. Results The specificity of the optimized system was good, using this system to detect 10 different strains, the results showed that only Escherichia coli genome appeared, the system turned green, the other systems were brown yellow; the detection limit of gradient dilution Escherichia coli was 102 CFU/mL by LAMP. Visual LAMP was used to detect the oral drugs sold in the market and not passed the test of the drug administration, and no E.coli was detected, but the oral drugs that were artificially contaminated with E.coli were visualized by LAMP. The color detection limit of the system in oral drugs was 1.8×102 CFU/mL. Conclusion The visual LAMP detection system can be used for the preliminary detection of oral drugs containing Escherichia coli.

Objective According to the Chinese Pharmacopoeia, Salmonella should not be detected in oral drugs. In this experiment, LAMP technology was used to detect Salmonella quickly, and the detection results were visible to the naked eye with calcein-Mn2+ dye, so as to provide pharmaceutical companies with the initial screening of drugs containing Salmonella. Methods Four primers (FIP, BiP, F3, B3) were designed to detect Salmonella by loop mediated isothermal amplification using inv A gene of Salmonella, and then the loop mediated isothermal amplification detection method was established. To verify the specificity of the method, LAMP detection was carried out on 10 kinds of laboratory preserved strains. The sensitivity of the method was obtained by detecting 10 times gradient dilution of Salmonella bacteria solution. Finally, the LAMP system of Salmonella was used to detect the oral drugs sold on the market and not tested by the Drug Administration, and the sensitivity of the system in the drug was determined. Based on these methods, Calcein-Mn2+ dye was added to test the availability of the dye. Results After optimization, the specificity of the system was good. For 10 different strains of LAMP detection, only Salmonella appeared trapezoidal band, and other strains did not detect the band; Through the experimental verification, the sensitivity of the system was 3.2 × 101 CFU/mL; The system was used for the oral drugs sold in the market and not passed the inspection of the drug administration The results of LAMP detection showed that no Salmonella was detected. However, the results of LAMP detection showed that there were trapezoidal bands in the test results. The sensitivity of the system in the detection of Salmonella in oral drugs could reach 3.8 × 101 CFU/mL. Adding calcein-Mn2+ dye into the system, the presence or absence of Salmonella can be distinguished by naked eye observation, and the detection limit is about 102 CFU/mL. Conclusion The visual LAMP detection system for Salmonella can be used for the preliminary screening of oral drugs containing Salmonella.