Development And Application Of PagN PCR Method And Monoclonal Antibodies Against PagN Protein For Salmonella Detection

Salmonella is a facultative intracellular parasitic zoonotic pathogen,which can widely infects many animals.It can also be transmitted to humans through meat,eggs,milk and even fruits and vegetables,causing gastroenteritis and other diseases and even death.Efficient and rapid detection methods are important for effective Salmonella prevention and control.The traditional detection method of Salmonella is microbial culture,which is complicated and required about 1 week to complete the detection.It is difficult to meet the demand of rapid detection of Salmonella.PCR detection methods based on molecular biology,which show advantages in simple operation and experiment period,is an important research content of rapid detection method of Salmonella.The outer membrane proteins of Salmonella are generally located on the surface of the bacteria.It has important potential in diagnostic technology development and vaccine development.The PagN protein is an outer membrane protein of Salmonella with four extracellular regions which means structure of 8 times transmembrane.It is widely distributed and highly conserved in different Salmonella serotypes,but not in non-Salmonella strains such as E.coli.It is expected to be a new target for specific detection of Salmonella.Monoclonal Antibodies(mAbs)can recognize specific antigens and play an important role in the prevention and control of Salmonella with the advantages of high sensitivity and little or no cross reaction.The immunomagnetic beads prepared based on monoclonal antibody can rapidly enrich and separate samples under the action of magnetic field.It plays an important role in sample pretreatment.Monoclonal antibody against PagN protein was developed and immune magnetic beads were prepared in this study to realize specific enrichment and isolation of Salmonella in samples.The combination of immune magnetic beads and PCR can be used to detect Salmonella rapidly.In this study,a PCR method was established to detect the outer membrane protein gene of pagN of Salmonella with high specificity and sensitivity.It can be applied to the detection of Salmonella in laboratory simulated contaminated samples,chicken embryos samples and pork samples collected in the market.Meanwhile,the recombinant expressed PagN(rPagN)proteins of Salmonella was prepared by E.coli prokaryotic expression system.Monoclonal antibodies specific to PagN proteins of Salmonella were obtained by using B cell hybridoma technique.Furthermore,the immune magnetic beads based on monoclonal antibody against PagN protein of Salmonella was prepared and used to combine with the established pagN PCR method for specific enrichment and identification of Salmonella in pork samples rapidly.Totally,this study established a method of immunomagnetic beads combination with PCR for rapid detection of Salmonella which targeted the outer membrane protein PagN of Salmonella.1 Establishment and application of pagN PCR method for rapid detection of SalmonellaA PCR method was developed to detect the pagN gene of Salmonella.The distribution and similarity of pagN in 10318 Salmonella strains and some non-Salmonella strains were analyzed based on bacterial genome sequences from NCBI.The results showed that pagN gene existed in 99.73%of Salmonella strains and the similarity reached 86.2%,while the gene is not present in non-Salmonella strains.The PCR amplification system and amplification conditions of pagN gene of Salmonella were established and optimized with high specificity and sensitivity.The results of PCR amplification of 18 different Salmonella stains of serum group A-F as well as 14 strains of non-Salmonella strains showed that the specific target band of 556 bp could be amplified with Salmonella strain as template,while no specific target band appeared with non-Salmonella strains,indicating the specificity of pagN PCR assay.Sensitivity analysis showed that the detection limit of pagN PCR could reach 10 CFU/mL of Salmonella or 32.2 ng/mL of Salmonella genomic DNA which is consistent with the widely used stn PCR method previously established in the laboratory.The PCR method was used to detect Salmonella in simulated contaminated pork samples.The samples contaminated with at least Salmonella of 104 CFU/10 g could be detected after enrichment culture for 6 h.There was positive result for samples contaminated with Salmonella of 1 CFU/10 g when the enrichment cultured increased to 10 h.The method was further applied to the detection of Salmonella in pork samples and chicken embryo samples collected.40 pork samples and 21 chicken embryo samples were inoculated by BPW.PCR and traditional microbial culture methods were used to detect Salmonella from the enrichment cultured medium of 40 pork samples and 21 chicken embryo samples simultaneously.The results showed that 8 positive samples were detected by pagN PCR,5 of which were consistent with the results of traditional microbial culture method.However,the PCR method takes only 26 h to complete the detection,which is about 30%time of traditional microbial culture method.The PCR method established in this study can be used as a new method for rapid detection of Salmonella with high sensitivity and specificity.2 Preparation and identification of monoclonal antibodies against PagN protein of SalmonellaThe pagN gene was amplified using the genome of Salmonella Enteritidis C50041 as template.The recombinant plasmid was constructed by ligating the amplified pagN gene fragment with plasmid vectors of pCold I and pGEX-6P-1,respectively.After PCR and sequencing verified the correctness,the recombinant plasmids were transferred to BL21(DE3)for induced expression and purification of PagN proteins with His(His-PagN)and GST(GST-PagN)tags,respectively.SDS-PAGE analysis showed that His-PagN protein was mainly expressed as inclusion body.After purification,27 kDa recombinant protein with high purity was obtained by renaturation.GST-PagN is mainly expressed in soluble form.After direct purification,46 kDa recombinant protein with high purity was obtained.The molecular weight of the proteins was consistent with their theoretical value.Western Blot analysis showed that the two recombinant proteins showed a single band in the expected molecular weight with corresponding labeled antibody as the primary antibodies,indicating their good immune reactivity.Hybridoma cells were obtained from prepared single splenocytes by cell fusion with SP2/0 after BALB/c mice were immunized with His-PagN.Indirect ELISA method used GST-PagN as detection antigen was used to detect hybridoma cell culture supernatant and screen positive hybridoma.Eight hybridoma cells with stable PagN monoclonal antibodies production capacity were obtained after three subclones.Among of them,the ascites titers of 1D9,3B3,3F10,4C4 and 5B1 were all above 2.0×105.The affinity,reactivity,purity and reaction spectrum of 1D9 mAb were further analyzed by SDS-PAGE,Western Blot,ELISA and pagN gene deletion Salmonella.The affinity of 1D9mAb was 4.62×108 M-1 and showed good reactivity with His-PagN recombinant protein.There was no cross-reaction with other Salmonella proteins except PagN based on the Western Blot result of 1D9 with pagN gene deletion and wide type Salmonella strains.The results show that 1D9 has good specificity.The results of monoclonal antibody reaction spectrum analysis showed that 1D9 monoclonal antibody reacted with different serotypes of Salmonella and had a single band,but does not react with non-Salmonella bacteria.These results indicated that the monoclonal antibody could be used as a candidate monoclonal antibody for specific detection of Salmonella.It has good application potential in the prevention and control of Salmonella.3 Preparation and application of immunomagnetic beads with PagN mAb of SalmonellaImmunomagnetic beads were prepared by coupling 1D9 monoclonal antibody with nano magnetic beads,which were used for the enrichment of Salmonella.And a rapid detection method for Salmonella was set up by immunomagnetic beads enrichment combined with pagN PCR detection.In this study,the time of magnetic beads coupling antibody,the amount of used magnetic bead,and the time of Salmonella capture by magnetic bead antibody complex were optimized.The preparation of PagN immunomagnetic beads of Salmonella was prepared with 2.5 mg carboxyl magnetic beads coupled with antibodies for 1 h and incubated with the samples for 1.5 h.The enrichment rates of the immunomagnetic beads for Salmonella with different amount of 102 CFU,103 CFU and 104 CFU were all above 80%,and showed best capture efficiency of 88.66%when respond to 103 CFU Salmonella.Different strains of Salmonella serotypes and non-Salmonella were used to evaluate the specificity of the prepared immunomagnetic beads.The enrichment rate of the immune beads against different Salmonella serotypes strains was all above 75%,which showed a best enrichment efficiency for Salmonella Enteritidis strain with rate of 86.99%.The non-specific capture rate of the immune beads against non-Salmonella strains was all less than 6%.Finally,the immunomagnetic bead was combined with pagN PCR to develop an IMBs-PCR method to detect Salmonella in pork samples.Twenty pork samples randomly collected from the market were cultured by BPW.The bacterial enrichment solution was taken for simultaneous detection of Salmonella by IMBs-PCR and traditional microbial culture method.The results showed that one same positive sample was detected by both methods,indicating a 100%coincidence rate of IMBs-PCR method to the traditional microbial culture method.More importantly,the detection time of IMBs-PCR method is only 30%of the traditional method,which greatly improving the efficiency of Salmonella detection.In this study,an IMBs-PCR detection method based on immunomagnetic bead combined PCR which target to PagN protein and pagN gene was established with advantages of simple operation,convenience and high specificity in rapid detection of Salmonella.