Study On New Methods For The Detection Of Biotoxins And Pathogenic Bacteria Based On Isothermal Amplification Technology

Isothermal amplification technology has the characteristics of nucleic acid amplification under simple constant temperature conditions without thermal cycler,high reaction efficiency and sensitivity.Thence,it’s diffusely used in the biosensors,food safety,clinical diagnosis and so on.In this article,three new strategies were developed by isothermal amplification technology for the detection of abrin,ricin,and Escherichia coli O157:H7.First,a method based on the combination of exponential amplification and bio–barcode was proposed and used for the detection of abrin:（1）the magnetic microparticle probe was prepared by modifying abrin on the magnetic microparticle;the gold nanoparticle probe was prepared by modifying the antibody of abrin and bio–barcode on the gold nanoparticle;（2）the abrin to be measured could bind to the antibody on the surface of gold nanoparticle probe,leading to the suppression of the generation of magnetic microparticle probe–gold nanoparticle probe complex;（3）the bio–barcode DNA would be released from the complex through DTT washing,triggering the exponential amplification reaction;（4）the highly sensitive detection of abrin could be achieved by detecting amplification products with real-time fluorescence system.The results show that:the detection range for abrin detection was measured to be 10 ng L^–1–1mg L^–1（R²=0.9939）,and the limit of detection was 5.64 ng L^–1（3σ）;this method showed good specificity for other toxins and proteins,and good anti-interference ability to ions and proteases.The result of toxin analysis in the actual sample indicates that this method has good application prospects.Second,a method based on the combination of exponential amplification and 3D–DNA walker was proposed and used for the detection of ricin:（1）freezing method was used to construct the walker probe and track probe,respectively;（2）ricin could compete with walker probes to bind aptamer,promoting the relative motion DNA walker reaction between the walker probe and track probe;（3）the ss DNA produced by the"recognition–cleavage–relative motion"process could trigger the exponential amplification reaction;（4）the highly sensitive detection of ricin could be achieved by detecting amplification products by molecular beacon method.The results show that:the detection range for ricin was measured to be 10 pmol L^–1–50 nmol L^–1（R²=0.9963）,and the limit of detection was2.50 pmol L^–1（3σ）.This method has good specificity,repeatability and stability,and shows good performance in the analysis of toxins in actual samples.Third,a method of CRISPR-Cas9 triggered two-step isothermal amplification based on the Ui O66 platform was established and used for the detection of E.coli O157:H7:（1）two Cas9（H840A）:sg RNA complexes could recognize and cleave the virulence gene loci of E.coli O157:H7 to generate the nick;（2）the strand displacement reaction that occur at the nick could continuously produce short ss DNA;（3）the circular probe could hybridize with the ss DNA,synthesizing long ss DNA with repeat sequences through rolling circle amplification;（4）the fluorescence probe departed from Ui O66 and hybridized with the long ss DNA resulting in the fluorescence recovery,enabling a highly sensitive,quantitative detection of E.coli O157:H7.The detection range was 1.3×10²–6.5×10⁴ CFU m L^–1;and the limit of detection was 40.00 CFU m L^–1（3σ）.This method exhibited excellent selectivity for different pathogenic bacteria.In summary,we have successfully developed some new methods of isothermal amplification reaction combined with other techniques for the detection of biological toxins and pathogenic bacteria,and have great results and practical application potential.We believe that with the further development of isothermal amplification method,it will have broader prospects in the field of applied research.