The Establishment Of A Rapid Detection Method For The Isothermal Amplification Of Babesiosis Nucleic Acids

ObjectiveBabesia is a parasitic protozoan parasitizing in mammalian red blood cells and can be transmitted through blood transfusion to cause babesiasis.Babesia subtypes transmitted by blood transfusion mainly include B.microti,B.divergens,and B.duncani.Currently,there is no routine method for the detection of Babesia infestation in China.We combine the isothermal amplification technology mediated by molecular beacons that we have the independent intellectual property rights with general-purpose nucleic acid chromatographic detection methods.We use the high specificity of molecular beacons and the visualization of results read to develop a method that can be applied to point-of-care technology（POCT）.And the nucleic acid detection method is time-saving,efficient,and easy-to-use in the detection of Babesia.MethodsFirstly,the human pathogenic gene sequence of this parasite was searched in Genebank,and the appropriate molecular beacon and corresponding primers were designed and screened.The reaction system was optimized and the template sequence synthesized was first isothermally amplified.We observe the template sequences’sensitivity and specificity and then verify with a plasmid.The amplified sample is added to the test strip and the results are read within the schedule time.Last,we observe the specificity and sensitivity of the established rapid isothermal amplification method.ResultsFirst,we determined that Beacon’s optimal reaction concentration was 50 pM,and then optimized the reaction system,determined the optimal concentration of the primer reaction was 5 nM,the optimal reaction temperature was 37℃,and the optimal incubation time was 30 min.The optimal Mg²⁺concentration was 15 mM,and the optimal DMSO reaction dose was 1.2μL.Then we used the optimized reaction system and the selected Beacon ring with the highest amplification efficiency to detect the template,it showed a lower sensitivity of about 50 pM and good specificity and had no cross reaction with several similar species transmitted by blood transfusion.Finally,the Babesia plasmid constructed by us can also be successfully detected.ConclusionsWe designed universal molecular beacons for the sequences of various Babesia subgenus genomes transmitted by blood transfusion,and optimized the reaction conditions.We establish a molecular beacon-mediated isothermal amplification method to detect Babesia.This method shows good specificity on the basis of high amplification efficiency.Compared with conventional detection methods such as microscopy,the method shortens the analysis time,and gets over the need of precision instrumentation equipment and the dependence of high-quality professionals.And the method can be applied to point-of-care technology（POCT）.SignificanceAiming at the technical shortages in the emergency detection and detection of emerging blood-infectious diseases and throughing technology integration and innovation,we develop an isothermal amplification method with immediate test potential and molecular beacon-mediated isothermal amplification,which may fill up regional blood safety assurance technologies and the absence of devices.The safety of emergency blood collection is further enhanced.