Detection Of Yersinia Pestis By Loop-mediated Isothermal Amplification Combined With Immunomagnetic Beads Techniquces

Plague is a kind of infectious disease caused by Yersinia pestis,which has many characteristics such as strong infectivity,high mortality,rapid transmission speed and natural foci.In human history,there have been three plague pandemics,causing hundreds of millions of people to death,brought hideous disaster to humans.In our country,plague is classified as class A infectious diseases.With the continuous development of modern medical technology,plague epidemic has been effectively controlled,but in recent years,the prevalence of inter-animal plague epidemic continues to expand,the situation of human plague was on the rise.At the same time,Yersinia as a typical biological warfare agents and bioterrorism agents,always threaten the safety of mankind.Thus,the monitoring of plague on human health and safety,economic development and social stability is of great significance.It is well known that rapid and effective detection of Yersinia pestis is an important means of controlling its infection.At present,the conventional detection of Yersinia pestis mainly includes bacteriological testing,immunological detection,molecular biology detection and so on.Bacteriological examination is generally carried out in four steps-microscopic microscopy,bacterial culture,plague phage lysis experiments,animal experiments.The method is time-consuming and requires a lot of manpower and material resources,and does not apply to a large number of monocytogenes screening and field testing.Molecular biology detection,that is,through the expansion of specific nucleic acid fragments in vitro to achieve the detection of Yersinia pestis,although the method has a fast,sensitive,specific advantages,but it often requires a specific instrument can be carried out,and expensive,inconvenient operation.Therefore,does not apply to remote areas of laboratory and field testing.Immunological method is a detection method based on antigen-specific antibody response,mainly colloidal gold,enzyme-linked immunosorbent assay and other methods as the representative.These methods have the advantages of simple operation,high safety,good specificity and high sensitivity.However,they need to be cultured in a large amount of time before testing,and the anti-interference ability of the method is poor.Therefore,the establishment of a fast,efficient,suitable for remote areas,and anti-interference ability detection method is very important.Loop-mediated isothermal amplification of DNA(LAMP)is a new molecular biology detection technique reported by Notomi et al.In 2000.This technique designed specific primers for 4-6 specific regions of the target gene.In the presence of a DNA polymerase(Bst DNA polymerase)with chain displacement activity,the target gene was rapidly grown at constant temperature,Efficient,specific testing.LAMP amplification reaction will form a large number of white magnesium pyrophosphate(Mg2P2O7)precipitation,therefore,by the naked eye or the use of real-time turbidimeter to determine the amount of precipitation generated to determine the extent of the amplification reaction.If the reaction before the reaction system to add calcium cyanogens this complex fluorescein,you can directly observe the naked eye by the reaction system color from yellow to green to determine whether the reaction occurred.In the actual test,the sample to be tested is usually a complex mixture,a variety of impurities tend to interfere with the test results,it is difficult to directly detect.Immunomagnetic beads techniquces can separate the target from impurities andachieve enrichment of the target.Immune magnetic bead technology is a kind of separation technology based on the principle of immunology in the 1970 s.This technique is a biological technique that utilizes magnetic microspheres with superparamagnetism as a carrier to capture and enrich the target.This microspheres with superparamagnetism can be combined with biological groups or biological ligands after a series of activations to form immunomagnetic beads to achieve specific separation of the analyte.In this study,immunomagnetic separation technology(IMS)was combined with loop-mediated isothermal amplification of DNA(LAMP).Based on LAMP specific assay,DNA was captured with magnetic beads or Immunomagnetic beads were used to capture the cells as the conditions,respectively,the establishment of a magnetic bead capture DNA combined with ring-mediated constant temperature amplification technology rapid detection of Yersinia and immunomagnetic beads capture bacteria combined with ring-mediated thermostat amplification technology for rapid detection of Yersinia pestis.The LAMP detection system,magnetic bead capture system and immune magnetic bead preparation system of the two detection methods were optimized,and the sensitivity,specificity,anti-interference ability and actual sample application of each method were carried out.Comprehensive evaluation.In this study,LAMP primers were designed based on the conserved sequence of Yersinia 3a,and the best primers were selected from the set of primers designed with amplification efficiency and specificity.Based on the best primers selected,the reaction conditions of Bst DNA polymerase,d NTPs addition and magnesium ion concentration were optimized.Finally,25?l volume of LAMP reaction system was established : 10 ?l Thermopol buffer 2.5 ?l,1.5 ?l of Mg SO4(100 m M),4 ?l of d NTP MIX(2.5 m M),0.4 ?l of FIP(100 m M),0.4 ?l of BIP(100 m M),0.5 ?l of F3(10 m M),0.5?l of B3(10 m M),0.2? l of LF(10 m M),1? l of Bst DNA Polymerase,Large Fragment(8,000 U / ml),3? l of water,and 1? l of template.65 ° C for 60 min.And the sensitivity,specificity and anti-interference ability of the established LAMP reaction system in the detection of Yersinia were evaluated.The experimental results show that the established LAMP reaction system has good specificity and anti-interference ability in the detection of Yersiniae,and the sensitivity is up to 100 copy / ml.The LAMP technique was combined with the magnetic bead capture DNA technique in the study of the rapid detection of the lepidoptera by the magnetic particle capture DNA method combined with LAMP technique.The magnetic beads capture DNA system was optimized according to the amount of magnetic beads and DNA enrichment system.And the sensitivity,specificity and anti-interference ability of the detection method were evaluated,and the method was compared with the conventional PCR detection technique.The results showed that the specificity of the method was good,and the sensitivity of detection of Yersiniae was 10 copy / ml,which was 100 times higher than that of conventional PCR technique.In the spleen,lung interference in the case of its sensitivity can still reach 10 copy / ml,1000 times higher than conventional PCR technology.In the case of liver suspension interference,the sensitivity of the assay was slightly reduced to 103 copy / ml,which was still 10 times higher than that of conventional PCR.In the study of the rapid detection of Yersinia pestis by immuno-magnetic bead-capturing bacterial method combined with LAMP technique,the monocytogenes of monocytogenes of the leprosy flora F1 were used,and the mice were treated with EDC / NHSS chemical coupling.Preparation of immunomagnetic beads.The results showed that the sensitivity,specificity and anti-interference ability of the method were optimized by the method of actual capture efficiency,antibody coupling rate,antibodyaddition amount and amount of immunomagnetic beads in the detection method,and the method Compared with conventional PCR detection techniques.The experimental results show that the method can specifically capture the monocytogenes in the mixture to be tested,the sensitivity of 1copy / ml,100 times higher than the sensitivity of conventional PCR technology.In the spleen,lung interference in the case of its sensitivity of 10 copy / ml,its sensitivity than conventional PCR technology 1000 times higher.In the case of liver interference sensitivity 100 copy / ml,100 times higher than conventional PCR technology.Subsequently,monoclonal antibodies against Plasmodium monocytogenes different from F1 antibody were prepared by immunization with mice immunized with attenuated Yersinia pestis,immunized with mice,cell fusion,hybridoma cell screening,cell cloning,ascites preparation and antibody purification,And the prepared monoclonal antibody was applied to immunomagnetic beads capture cell method combined with ring-mediated constant temperature amplification technique to detect Yersinia pestis.The results showed that the prepared antibody had higher titer and had better sensitivity in the detection method,and could be applied to the rapid detection of Yersinia pestis.In this study,two more mature methods of detection of Yersinia were established.The two methods were simple and easy to operate.They needed no large instruments,low cost,high sensitivity,strong specificity,strong anti-interference ability,Therefore,these two detection techniques are suitable for field screening,field operations and remote areas of the application of detection work.In the existing immunological detection method,the F1 antigen expressed on the surface of the Yersinia pestis has become the only target,and the Yersinia pestis can only form F1 antigen on the surface under the condition of 37 ? artificial culture or mammalian body,The optimum growth temperature is 26 ?,and the temperature inthe natural environment is much lower than 37 ?.Therefore,the surface antigen of Plague is not expressed in the natural environment.The existence of this phenomenon,the existing Yersinia immunological methods to detect a lot of inconvenience.The monoclonal antibodies prepared in this study have good specificity for the Yersinia pestis which does not express F1 antigen on the surface,which makes up the loopholes in immunological detection at present.It provides a broader prospect for the detection of Yersinia pestis.