Establishment,Evaluation And Application Of Multiplex Detection Of Rodent-borne Pathogens Based On QIAxcel Electrophoresis System

BackgroundRodent-borne diseases are an important infectious diseases caused by rodents and rodent parasites that endanger the health of humans and animals.Rodents can transmit a variety of pathogens such as bacteria,viruses,pathogens and rickettsia to humans and animals,causing huge economic losses and potential safety hazards.Detection and identification of pathogens in time are the key to prevent and control rodent-borne diseases.Traditional detection methods such as bacteria culture and serological test had low flux and sensitivity.Some nucleic acid molecule technologies represented by Real-time fluorescence quantitative PCR(qPCR)and gene chip can simultaneously detect multiple pathogens,but those are expensive and complicated,which is not conducive to popularization and use.Therefore,it is urgent to establish a sensitive,simple,and high-throughput method for detection of multiplex rodent-borne pathogens.ObjectivesThe aim of this study was to establish a multiplex PCR method for detection of rodent-borne pathogens based on QIAxcel capillary electrophoresis system,and to evaluate the sensitivity,specificity and repeatability of this method.In this approach,seven rodent-borne pathogens were detected simultaneously in a single tube,including Orientia tsutsugamushi(Ot),Anaplasma phagocytophilum(Ap),Rickettsia typhi(Rt),Francisella tularensis(Ft),Coxiella burnetii(Cb),Leptospira interrogans(Lep)and Bartonella(Bar).MethodsBased on the Microbial data analysis cloud platform(https://analysis.mypathogen.org),specific genes of seven pathogens were screened and specific primers were designed.Specific chimeric primers were constructed according to TSP(Temperature Switch PCR).The reaction system had seven pairs of specific chimeric primers and one pair of general primers,and the general primers were amplified as the dominant primers through increasing concentration of general primers and reaction cycles in this stage.The specificity of the reaction system was tested by the nucleic acid of the target pathogens.The sensitivity and repeatability of the reaction system were tested by plasmid standards with different dilutions.PCR products were detected by QIAxcel capillary electrophoresis and agarose gel electrophoresis.2.The simulated samples were prepared by mixing the target pathogen nucleic acid of different concentrations with the specific pathogen free mouse spleen nucleic acid.The specificity and sensitivity of the multiplex PCR assay were preliminarily verified.A total of 86 mouse spleen samples were tested,and the results were compared with those of single routine PCR assay and single qPCR assay.PCR products were detected by QIAxcel capillary electrophoresis.Results1.The multiplex PCR method for detection of seven kinds of rodent-borne pathogens showed good specificity,and no cross reaction.The capillary electrophoresis sensitivity of multi-primers single-template was within the range of 11—16 copies/μl;.It’s 10 times more sensitive than agarose gel electrophoresis.The capillary electrophoresis sensitivity of multi-primers multi-templates was within the range of 20—200 copies/μl.Rt,Ft,Cb,Lep and Bar were detected at 2×101 copies/μl,which was 10 times more sensitive than agarose gel electrophoresis.Seven pathogens were detected at 2×102 copies/μl.2.Using this method to detect simulated samples,the sensitivity of single-pathogen infected samples was within the range of 10—10-4 ng/μl,and the sensitivity of Cb,Ft and Bh was 10-4 ng/μl1.The sensitivity of multi-pathogens infected samples was within the range of 10—10-4 ng/μl,and the sensitivity of Ft and Bh was 10-4 ng/μl.The sensitivity of Cb decreased by 10 times when we put multi-pathogens infected samples into test.Eighty-six field mouse spleen samples from Beijing were tested by multiplex PCR assay.The results showed that the detection ability of the multiplex PCR was comparable to the qPCR and was higher than the routine PCR.Multiplex PCR and qPCR methods have different effect for different pathogens.The infection rate of the eighty-six samples was 25%,and all positive samples were infected by single pathogen,with Bar,Lep and Ap infection rates of 21%,3%and 1%,respectively.ConclusionsIn this study,an efficient,high throughput and rapid multiplex PCR assay was established,which can detect seven kinds of rodent-borne pathogens.This method has good specificity and high sensitivity,providing an effective mean for the diagnosis and prevention of a series of rodent-borne pathogens.