Study On A Loop Mediated Isothermal Amplification For The Detection Of MTBC And NDM-1

Abstract: We established a rapid detection method of Mycobaeteriumtuberculosls（MTB）and New Delhi Metallo-β-Lactamase Gene (NDM-1)based on Loop-mediated Isothermal Amplification (LAMP). To meet theclinical laboratory testing requirementsMethods:With the application of LAMP, we designed many sets ofLAMP primers, using IS6110gene and NDM-1gene as the target sequence,and selected the set of optimal primers. Meanwhile, we established optimalreaction systems and conditions to carry out the sensitivity and specificityexperiments.Results:(1)In the experiment of sensitivity, IS6110gene had a detectionlimit of4copies in each reaction. And NDM-1gene had a detection limit of6copies in each reaction.(2)In the experiment of specificity, we detectedIS6110gene and NDM-1gene in4pathogen strains (Klebsiella pneumoniae,Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae), andthe total DNA from intestinal microbes and the total DNA from soilmicrobes. We had not detected the amplification reactions.(3)In the197clinical experiments of Mycobacterium tuberculosis, the level of sensitivity、specificity、true positives and true negatives were increased, the coefficientof kappa had high consistency.The detection method established could rapidly detect NDM-1gene andvisualize the experiment result. The method is easy to operate and has highsensitivity and specificity and thus has great application value in basicresearch laboratories, emergent detection and spot detection. Conclusion:(1)Established the optimization of IS6110gene andNDM-1gene reaction system,The experiment results showed character-istics of high sensitivity and specificity.(2)The experiment results showedthat the whole detection process took only one hour and could be observedvisually. The study demonstrated that LAMP methods could perform the testin a simple laboratory space without specialized equipment. It could meet thedemands of the clinical emergency detection and monitoring, and otheraspects of the use.(3)In the clinical detection of197patients, the coefficientof kappa had high consistency. To illustrate that the LAMP reaction systemenhanced the efficiency of clinical detection samples and had a statisticalsignificance obviously.