The Establishment Of GeXP Multiple Detection Methods For Pathogens Such As CSFV, HP-PRRSV, PEDV And ASFV And The Preliminary Assembly Of Kits

Objective:To establish a GeXP based multiplex PCR assay to detect CSF V,HP-PRRSV,PEDV,ASFV.Then a test kit was assembied and the applicatio n value was explored by comparing the detection results of 64 clinical sample s using the test kits to the results using the standard single pathogenic quaranti ne methods.The final goal of our research is to establish a high-throught diagn ostic technology of the major hazard swine viral pathogens based on GeXP m ultiplex detection technology by integrating the achievements of this research and other similar research in our laboratory.Methods:1.Nucleic acd was selected as pathogenic diagnosis indicator.The pre-r esearchs showed that the sequences of CSFV 5UTR,PRRSV NSP2 gene,PED V M gene and ASFV B646L gene were specific among viruses but conservati ve among strains.Thus,the four sequences were chosed and downloaded from NCBI website,and then,these sequences was aligned to find out the conservati ve sections which were used to pick up the primers.In particular,the primers f or diagnosing HP-PRRSV were specially designed to distinguish HP-PRRSV strains from PRRSV strains.2.Four single pathogenic diagnosis PCR methods were established respe ctively.The specificity of the PCR method was analyzed with pathogenic vacc ine strains and laboratory reference strains isolated from clinical samples.CSF V,HP-PRRSV,PEDV RNA standards were built with T7 in vitro transcription kit.ASFV B646L gene was artificially synthesized and then the gene was shift ed to vector pUC57 to construct a new plasmid pUC57-ASFV.The RNA stand ards and plasmids were used to identify the sensitivity of the four PCR metho ds.Later,experimental results of specificity check and sensitivity check were a nalyzed,the primers could be used to establish a GeXP multiplex PCR assay o nly when the experimental results were in line with the expected results.3.The identified primers were renewedly synthesized with universal sequ ences labelled at 5' end and a GeXP multiplex PCR assay was established with the instructions from<GeXP user's guide>to detect the four pathogens in thi s research.The specificity of the GeXP method was analyzed with pathogenic vaccine strains and laboratory reference strains isolated from clinical samples.The RNA standards and plasmids were used to identify the sensitivity of the GeXP methods.The feasibility of four pathogenic GeXP multiplex detection method was evaluated according to the specificity check and sensitivity check results.4.A multiplex detection kit was assembled according to experimental res ults.64 clinical samples collected from all the regions of Sichuan province wer e detected with standard single pathogenic quarantine methods and the multipl ex detection kit at the same time.The relative specificity,relative sensitivity an d coincidence rate were analyzed by comparing the detection results of clinica 1 samples using the multiplex detection kits to the detection results of clinical samples using the standard single pathogenic quarantine methods when the sta ndard single pathogenic quarantine methods were used as reference.The clinic al application value of the kit was analyzed accoding to the experimental resul ts.Results:1.The experimental results of single pathogenic PCR method showed tha t the primers could specially detect CSFV RNA,HP-PRRSV RNA,PEDV RN A and ASFV plasmid,and couldnt detect PRRSV RNA,JEV RNA,FMDV RN A,PPV DNA,PCV DNA,PRV DNA,Mh DNA,Er DNA,Pm DNA.The sensitiv ity of single pathogenic PCR method was 102 copies/?l.Primers designed acco rding to the HP-PRRSV NSP2 gene deletion region could specially identify H P-PRRSV strains from PRRSV strains.Primers designed in this experiment w ere in line with the expects of experimental designs and the primers could be used in the establishment of the multiple detection assay.2.The four single pathogenic diagnosis GeXP method was established wi th the instructions from<GeXP user's guide>.The GeXP method could special ly check out CSFV,HP-PRRSV,PEDV reference strains and ASFV plasmids,but couldn't detect PRRSV,JEV,FMDV,PPV,PCV,PRV,Mh,Er,Pm reference s trains.The primers for diagnosing HP-PRRSV could distinguish HP-PRRSV s trains from PRRSV strains.The single pathogenic sensitivity of GeXP method was 102 copies/?l to 103 copies/?l and the multiplex pathogenic sensitivity was 103copies/?l.This established GeXP multiplex detection method had good sp ecificity and sensitivity,and could be used to assemble GeXP multiplex detect ion kit and to explore the clinical application value.3.The multiplex detection kit was assembled based on the achievements of GeXP experiments.64 clinical samples collected from all the regions of Sic huan province were detected with standard single pathogenic quarantine meth ods and the multiplex detection kit at the same time.The relative specificity an d relative sensitivity of the multiplex detection kit assembled in this study was 100%,100%respectively,and the coincidence rate was 97%to 100%.Conclusion:A multiplex detection method based on GeXP technology was successfully established for detecting the pathogens of CSFV,HP-PRRSV,P EDV,ASFV in this study,and a detection kit was assembled.The kit showed a great value in clinical application by analyzing the detection results of clinica 1 samples between the multiplex detection kit and the standard single pathoge nic quarantine methods.