Multiplex Real-time Quantitative QPCR Detection Of 11 Bovine Pathogens

Objective: With the development of intensive and large-scale breeding,the complexity of bovine disease is increasing,with increased number of new pathogens and more cases of secondary infection and mixed infection,bringing great pressure to the prevention and control of the animal disease.Rapid identification of pathogens is the key critical for the disease treatment and prevention.However,conventional laboratory detection methods based on single target detection require multiple tests to determine the pathogens,which is time-consuming.In addition,some conventional detection methods require complicated operation,or possess poor specificity and low sensitivity.This study intends to establish multiplex real-time fluorescent Quantitative PCR(q PCR)technology for the rapid,sensitive,specific and high-throughput detection of 11 bovine pathogens.Methods:(1)Preparation of positive reference materials for 11 bovine pathogens: According to the literature investigation,the conservative genes of bovine herpes virus(BHV),bovine parainfluenza(BPIV),filamentous Mycoplasma filamentous subspecies SC(Mmm SC),bovine respiratory syncytial virus(BRSV),bluetongue virus(BTV),bovine viral diarrhea virus(BVDV),bovine rota virus(BRV),peste des petits ruminants virus(PPRV),Rinderpest virus(RPV),foot-and-mouth disease virus(FMDV)and Vesicular stomatitis virus(VSV)conserved genes were identified as the target genes.The specific primers were designed according to the principle of primer design,and the target genes were cloned and in vitro transcribed to prepare the positive reference materials for these pathogens.(2)Establishment of single-fold q PCR detection method for 11 bovine pathogens: Specific primers and probes were designed using the reference materials as templates.After carefully optimization of the annealing temperature,primer concentration and probe concentration for PCR amplification,single-fold Taq Man q PCR detection methods was established for the rapid detection of 11 bovine pathogens.The specificity,sensitivity and repeatability of these methods were investigated using positive reference materials and clinic samples.(3)Establishment of multiplex q PCR detection method for 11 bovine pathogens: Based on the 11 single-fold q PCR method,the number of detection targets was gradually increased by optimizing the annealing temperature,primer concentration and probe concentration of each pathogen.Finally,several multiplex Taq Man q PCR systems based on 4 different fluorescent tags were established for the rapid detection of 11 bovine pathogens.The specificity,sensitivity and stability of each system were investigated.Finally high-throughput parallel detection of 11 different bovine pathogens were realized using these multiplex Taq Man q PCR systems under the help of a multi-channel q PCR instrument.Results:(1)The positive reference materials for the partial nucleic acid fragments of 11 bovine pathogens were succefully prepared.These references materials is stable for a long time under the condition of-80 in a specific preservation solution.(2)Single-fold q PCR detection method were established for the rapid,specific and sensitive detection of BHV,BPIV,Mmm SC,BRSV,BTV,BVDV,BRV,PPRV,RPV,FMDV and VSV.These methods did not shown non-specific amplification in the system containing different pathogens reference materials or positive samples,exhibiting high specificity.The minimum detection limit of BHV,BPIV,BRSV,FMDV and VSV was 10copies/μL and that of Mmm SC,BRV,BVDV,BTV,PPRV and RPV was 100copies/μL,showing high sensitivity.The variable coefficient(CV)of repeated tests for in and between batches in using 3 different concentrations of each pathogen was less than 2%,exhibiting good stability.(3)A high-throughput detection method based on multi-channel q PCR instrument and several multiplex q PCR systems was successfully established for the simultaneously detection of 11 bovine pathogens.No non-specific amplification was observed in the multiplex q PCR system.The minimum detection limit of BHV,BPIV,BRSV and VSV was 10 copies/μL,those of BRV,BVDV and FMDV was 50copies/μL,and those of Mmm SC,BTV,PPRV and RPV was 100 copies/μL.The CV of both in and between batches was within 2%.Conclusion: In this study,11 positive reference materials were successfully prepared,including BHV,BPIV,Mmm SC,BRSV,BVDV,BRV,PPRV,RPV and FMDV.11 single-fold Taq Man q PCR assays were established for the rapid,specific and sensitive detection of these 11 pathogens.A high-throughput detection method based on multi-channel q PCR instrument and several multiplex Taq Man q PCR systems was finally established for the simultaneously detection of 11 bovine pathogens.The established single-fold and multiplex q PCR detection methods exhibit high specificity,sensitivity and repeatability.The high-throughput and rapid detection of bovine pathogens using this method will benefit the rapid diagnosis,epidemic monitoring and epidemiological investigation of associated diseases in intensive and large-scale culture,providing guidance for the formulation of prevention and control measures of these diseases.