Research On The Main New Technology Of Nucleic Acid Isothermal Amplification

Loop mediated isothermal amplification(LAMP)is a technique of in vitro amplification of nucleic acid based on the design of four specific primers for six different regions of the target gene and the constant temperature environment with strand replacement DNA polymerase as the reaction condition.African swine fever is caused by African swine fever virus.It is an acute,severe and highly contagious disease with a mortality rate of 100%.In this study,based on lamp method,a fast visual detection method for ASFV was established.The gene of European strain(i.e.Georgia2007 / 1 strain)matches the reported sequence of African swine fever virus(acfv)in a farm in Shenbei New District,Shenyang City,Liaoning Province,China.We constructed ASFV plasmid based on the target sequence and designed lamp primers F3,B3,LF,LB,FIP and BIP.Through the optimization of primers,temperature,time and system,a loop mediated isothermal amplification method for detection of African swine fever virus was established.The sensitivity of the method was tested with five different template concentrations.The results show that the minimum detection limit is 2 copies in the sensitivity experiment,the detection time is only 20 minutes,and the detection results can be judged according to the color,and the field visualization detection can be carried out.In addition,based on LAMP technology,a rapid detection method of bovine sex was established.The male and female common primers and male specific primers of LAMP were designed to study the subsequent lamp methods.By optimizing the reaction time,reaction temperature,primer ratio and reaction system,a loop mediated isothermal amplification method was established to detect bovine sex.Nine different concentrations of templates were used to detect the sensitivity of the method.The results showed that the lowest detectable concentration of common primers for male and female was 2pg/?L,and the lowest detectable concentration of specific primers for male was 5pg/?L.The specificity test showed that there was no cross reaction between cattle samples and sheep and pig samples,and the detection accuracy of male and female common primers and male specific primers was 100%.The results showed that the method had high sensitivity and specificity.Recombinase polymerase amplification(RPA)is a new thermostatic in vitro nucleic acid amplification technology,which uses the combination of recombinase and single strand binding protein to achieve the specific combination of primer and template at room temperature,to replace the denaturation and renaturation process of traditional PCR thermal cycle.In this study,a method based on recombinase polymerase amplification was established to detect bovine sex.According to the conserved region gene sequence of cattle,the male and female common primers and male specific primers of RPA were designed respectively.The specificity of the method was verified by the samples of cattle,sheep and pigs with different genders,and the sensitivity of the method was detected by seven different concentrations of templates.The results of specificity showed that the common primers of male and female could detect female and bull samples,while the male specific primers could only detect bull samples,indicating that there was no cross reaction between cattle samples and sheep and pig samples.In the sensitivity test,the lowest detectable concentration of common primers for male and female was 1pg/?L,and the lowest detectable concentration of specific primers for male was 2pg/?L.The sensitivity of RPA was higher than LAMP.The method based on RPA technology has strong specificity and high sensitivity.The detection time is only 5 minutes.It does not need special equipment and is easy to operate.In conclusion,three detection methods were established in this study,including LAMP based detection of African classical swine fever virus and bovine sex identification method,and RPA based bovine sex identification method.The three methods have strong specificity,high sensitivity and short reaction time,which are suitable for rapid detection in laboratory or field.