The Application Of LAMP And Realamp In The Detection Of Some Bacterial Biological Warfare Agents

Objective:Francisella tularensis,Burkholderia pseudomallei,Brucella and Yersinia pestis are important biological warfare agents with highly pathogenic and infectious,and can pose a great threat to human health and even survival.This study aims to establish a detectable technology for the bacteria,which is rapid,accurate,reliable and easily promoted,in order to prevent and deal with the possible bioterrorism threat.Method:This study was based on Loop-mediated isothermal amplification(LAMP)and real-time fluorescence loop-mediated isothermal amplification(RealAmp),and in order to establish and optimized best detection system of the involved bacteria.The LAMP primers were designed separately for FopA gene to detect Francisella tularensis,for FliC gene to detect Burkholderia pseudomallei,for Omp25gene to detect Brucella;and for F1 gene to detect Yersinia pestis.The specificity of the detection system was evaluated by using genomic DNA of strains with high genomic homology as template,and the sensitivity of the detection system was evaluated by using genomic DNA and plasmid of target strains as template.Simulated soil samples containing plasmid bacteria were used to evaluate the detection limit and the application value of the detection system in the actual environment,and parallel comparison with real-time fluorescent quantitative PCR reagents to evaluate the accuracy and the detection rate.Results:(1)Screening of the FopA gene of Francisella tularensis,the FliC gene of Burkholderia pseudomallei,the Omp25 gene of Brucella and the F1 gene of Yersinia pestis,respectively,established a loop-mediated isothermal amplification technique and real-time Fluorescent loop mediated isothermal amplification techniques and optimize the reaction conditions and systems;(2)Using non-target bacterial strain genomic DNA and aphid genome total DNA as a template,no non-specific amplification occurred;(3)Using the genomic DNA of Francisella tularensis as a template,the sensitivity of LAMP and RealAmp techniques can reach 4.9×10~2 cfu/mL and 4.9×10~1 cfu/mL,respectively.With plasmid as template,the sensitivity can reach 8copies/?L and 5 copies/?L,respectively;Using the genomic DNA of Burkholderia pseudomallei as a template,the sensitivity of LAMP and RealAmp techniques can reach 4.9×10~3 cfu/mL and 4.9×10~2 cfu/mL,respectively.Using plasmid as template,the sensitivity can reach 1×10~2copies/?L and 1×10~1 copies/?L,respectively;Using the genomic DNA of Brucella as a template,the sensitivity of LAMP and RealAmp techniques can reach 4.9×10~2 cfu/mL and 4.9×10~1 cfu/mL,respectively.With plasmid as template,the sensitivity can reach 1×10~1 copies/?L and 8copies/?L,respectively;Using the genomic DNA of Yersinia pestis as a template,the sensitivity of LAMP and RealAmp techniques can reach 4.9×10~4 cfu/mL and 4.9×10~3 cfu/mL,respectively.With plasmid as template,the sensitivity can reach 1×10~1 copies/?L and 8 copies/?L,respectively;(4)Based on Francisella tularensis,Burkholderia pseudomallei,Brucella and Yersinia pestis,a simulated sample was established in soil-contaminated soil,except for LAMP detection of Yersinia pestis The detection limit was 44 cfu/g,and the detection limits of LAMP and RealAmp for other plasmids were 4.4 cfu/g;A total of 24soil simulation samples with high,medium and low concentrations(4.4×10~1 cfu/g to 4.4×108 cfu/g)were detected in parallel with real-time PCR reagents.The results were consistent with expectations.Conclusion:The loop-mediated isothermal amplification technology used for detection of Francisella tularensis,Burkholderia pseudomallei,Brucella and Yersinia pestis has strong specificity,high sensitivity,simple instrument,low cost and fast,which can be completed in 1 hour,and it's easy to be popularized at the establishment unit.And the real-time fluorescence loop-mediated isothermal amplification is more sensitive,simpler and faster,the amplification results of which can be seen in20–40 minutes.Compared with the domestic real-time fluorescence quantitative PCR reagents in parallel to detect the samples,the two detection technologies shared similar accuracy and detection rate.This project is expected to provide effective,reliable and rapid detection methods for clinical diagnosis,environmental pollution detection,bioterrorism threats caused by the dangerous bacteria and ensure the public safety.