Improvement Of Immunochromatographic Strip And Application Of High Resolution Melt Curve Preliminarily For Flora Species Analysis

Methods for rapid detection and classification of microorganisms are hot anddifficult areas of applied microbiology. In the field of rapid detection ofmicroorganisms, more than50%food poisoning attributes to foodborne pathogensinfection. In the field of classification of microorganisms, denaturing gradient gelelectrophoresis and temperature gradient gel electrophoresis are widely applied, andhigh resolution meting curve technique is rarely reported.Immunochromatographic strip (ICT) technique is widely used in the field offood safety rapid screening, and gradually expanded to foodborne pathogens siterapid detection and diagnosis because of its simple, rapid and low-cost advantages.However, conventional immunochromatographic strip needs screening high specificand affinity antibodies against objective pathogens, which is laborious andtime-consuming. Furthermore, the sensitivity of the strip needs to improve. Thus, inour study, we adopt Cronobacter and Vibrio parahaemolyticus as detection objectsand improve the conventional immunochromatographic strip from two parts. Firstly,we utilize nucleic acid hybridization instead of antigen-antibody reaction anddevelop a nucleic acid immunochromatographic strip for rapid detection ofCronobacter. Secondly, we also develop immunochromatographic strip for detectionof Vibrio parahaemolyticus. Gold nanoparticles conjugated goat anti-mouse IgG isused for enhancing strip color intensity to improve sensitivity. Moreover, in order toenrich the classification of microbial flora, high-resolution melting curve technologyis applied for the first time in this study.Results show the sensitivity of ICT for detection of Cronobacter is105CFU/mLin pure culture and106CFU/mL in powdered infant formula along with108CFU/mLSalmonella Typhimurium. On the other hand, when good nanoparticles conjugatedgoat anti-mouse IgG is added after sample migrating5min, Signal intensity of thedeveloped ICT for detection of V. parahaemolyticus improves5folds. The sensitivity is106CFU/mL in pure culture and107CFU/g in meat samples along with108CFU/mL Cronobacter and Shigella.High resolution melting curve (HRM) can detect single base difference of theDNA fragments and show difference in melting temperature (Tm). Our studyamplifies the V3region of No.3Bifidobacterium bifidum. Urea, formamidedeionized and guanidine hydrochloride are tested. Results show urea and formamidecan decrease Tm and guanidine hydrochloride increase Tm. Then the Lactobacillusis amplified from intestinal microorganisms, and different concentrations of DNAdenaturing agents are added. The flora species are successfully distinguished byHRM.Our study develops the methods for rapid detection of Cronobacter and V.parahaemolyticus, and provides a research model for other pathogens detection.Furthermore, to our knowledge, HRM used for flora species analysis has not beenpublished up to now, and provides a new idea for flora species analysis.