Abstract:[Background] Brucella, infectious bovine rhinotracheitis virus (IBRV), and Neospora caninum are major pathogens that cause abortions in dairy cows. Current serological methods for detecting the above pathogens are characterized by low specificity and complex operation, while PCR and RT-PCR methods are only capable of detecting single pathogens. Multiplex PCR enabling the detection of multiple pathogens at the same time greatly increases the detection efficiency. [Objective] To establish a multiplex PCR assay for rapidly detecting Brucella, IBRV, and N. caninum in clinical samples. [Methods] Specific primers were designed for Brucella omp25, IBRV gB, and N. caninum SRS2, and the annealing temperature, primer concentration, extension time, and number of cycles of the multiplex PCR assay were optimized. The specificity of the multiplex PCR assay was examined with the standard nucleic acids of Salmonella, Escherichia coli, Cryptosporidium, Toxoplasma gondii, and circovirus. The sensitivity of the multiplex PCR assay was tested with the recombinant plasmids carrying the target genes. Finally, the established assay was employed to test 55 vaginal swabs from cows suffering from abortions. [Results] The optimal conditions of the multiplex PCR assay were annealing temperature of 59 ℃, the extension time of 40 s, 30 cycles, and the primer concentration of 1 μmol/L. The lower limit of detection was 4×101 copies for omp25 and SRS2 and 4×102 copies for gB. The established assay showed no cross-reactivity with other pathogens. Nine out of 55 samples were detected positive for Brucella, 3 positive for IBRV, and 7 positive for N. caninum. Particularly, one sample was subjected to mixed infection by Brucella and N. caninum. [Conclusion] A multiplex PCR assay for three pathogens was successfully constructed, enabling rapid and accurate detection of clinical samples.

Abstract:[Background] Bovine pasteurellosis, caused by serotypes A, B, and E of Pasteurella multocida (Pm), is an acute infectious disease of cattle. Polymerase chain reaction (PCR) is an effective means to diagnose and control this disease. [Objective] To establish a multiplex PCR assay for rapid detection of serotypes A, B, and E of Pm and provide technical support for the rapid and accurate clinical diagnosis of bovine pasteurellosis. [Methods] According to the conserved regions of hyaD-hyaC, bcbD, and ecbJ genes of Pm, we designed three pairs of specific primers for PCR and determined the appropriate annealing temperature (Tm) by temperature-gradient PCR assay. Chessboard test was employed to optimize the primer concentration and then a multiplex PCR assay was established. The recombinant plasmid standard and positive strain were used to determine the sensitivity (limit of detection) of the established assay. The nucleic acid samples of 8 common bovine pathogens (Mannheimia hemolytica C1655, Escherichia coli C237, Listeria monocytogenes C1597, Staphylococcus aureus C3053, Salmonella dublin C79351, Mycobacterium paratuberculosis C1625, bovine infectious rhinotracheitis virus CAV1546, and Mycoplasma bovis C65-1) were used to determine the specificity of the multiplex PCR assay. Three batches of diagnostic reagents were prepared to perform inter-batch and intra-batch tests on sensitive and specific samples to determine the repeatability of the assay. Three different models of PCR instruments were used to detect sensitive and specific samples with the established method to determine the applicability of the assay. The performance of the assay in clinical application was evaluated by detection of clinical samples and simulated infection samples. [Results] The optimal multiplex PCR assay was established under the following conditions: Tm of 55 ℃ and the three pairs of primers at the concentrations of 0.25, 0.30, and 0.20 μmol/L, respectively. The established method could simultaneously detect Pm serotypes A (821 bp), B (203 bp), and E (363 bp). The multiplex PCR assay had high sensitivity. It showed the limits of detection of 43.080, 3.710, and 4.350 copies/μL for recombinant plasmid standards pMD-A, pMD-B, and pMD-E, respectively, as well as the limit of detection of 102 CFU for the positive bacterial liquid. Moreover, the established assay had strong specificity as it only produced bands for the serotypes A, B, and E of Pm and no bands for other pathogens. The consistent results of the inter-batch and intra-batch tests indicated good repeatability of the assay. The detection results of clinical samples and simulated infection samples showed a 100% coincidence rate with the pathogen isolation and identification. [Conclusion] The multiplex PCR assay for the detection of serotypes A, B, and E of Pm was successfully established, which provided technical support for the identification and epidemiological investigation of Pm.

Abstract:This text involved with a pre-enrichment medium for the simultaneous recovery of Salmonella spp.，Escherichia coli and Staphylococcus aureus.This medium is named buffered saline broth(BSB)，which contains peptone 10g，beef exact 3g，disodium hydrogen phosphate 9g，potassium dihydrogen phosphate 1.5g，additive 50g，deionized water 1 000mL，pH7.2.1cfu per one milliliter of Salmonella spp.，Escherichia coli and Staphylococcus aureus in saline were stimutaneously added to 97 mL of BSB，buffered peptone water，lactose broth，nutrient broth，Escherichia coli broth，rappaport-vassiliadis enrichment broth，7.5% sodium chloride enrichment medium，respectively，and incubated for 18h at 37℃.The results showed BSB was the best enrichment medium，in which Salmonella spp.，Escherichia coli and Staphylococcus aureus multiplied at nearly same speed，and reached at 106、106、107cfu/mL，respectively.Multiplex PCR produced specific amplicons of expected sizes，284bp for Salmonella spp.invA gene，622bp for Escherichia coli phoA gene，484bp for Staphylococcus aureus nuc gene.In contrast，the three bacteria couldnt multiply harmoniously in the other six media.So BSB might be considered as the medium，which could enrich above mentioned three bacteria.

Abstract:A rapid multiplex PCR (m-PCR) method that allows the simultaneous detection,in a single tube, of two commonly encountered food-borne pathogens in meat and other animal products was developed. The invasion protein gene (invA) of Salmonella spp. and rfbE gene of Escherichia coli O157 were used as the gene targets. The multiplex PCR assay was specific and rapid,with a turnround time of 9 h~10 h.While the detection limit is 2.4×102 cfu/mL of Salmonella spp. and 2.2×102 cfu/mL of E.coli O157 respectively when detecting the artificially contaminated pork meat with incubation at 37℃ for 4 h. The m-PCR assay developed in this study could provide a cost-effective and informative supplement to conventional microbiological methods for routine monitoring of food.

Abstract:Four primers against the genes encoding (cpa, cpb, etx, and iA) four major toxins(α, β, ε, ι) of Cl. perfringens were designed and the colony multiplex PCR of identification and genotyping of Cl. perfringens strains were developed. Cl. perfringens reference strains stored in china institute of veterinary drug control including A, B, C, D and E genotyping were genotyped using the colony muitiplex PCR assay. The expected sequences were obtained successfully by the colony multiplex PCR assay. But the sequences were not obtained from Cl. novyi, Cl. septicum and Cl. tetani. The expected sequences were obtained from Cl. perfringens individual colony diluted to 100 times with 0.85% saline solution.13 Cl. perfringens strains isolated from diferent animals were genotyped using the colony multiplex PCR assay, and the results were comparaed with the results of toxins neutranization test in mice. The two assays showed good accordance. These results showed that the development of the colony multiplex PCR is very important for early and fast identification and genotyping of Cl. perfringens in china.

Abstract:[Background] The bovine diarrheal syndrome, caused by single or multiple pathogen infections, significantly restricts the development of the cattle industry. Rapid and accurate pathogen diagnosis is crucial for disease control. [Objective] To establish a one-step multiplex PCR/RT-PCR method for detecting 11 bacterial and viral pathogens causing bovine diarrhea, facilitating rapid pathogen diagnosis. [Methods] We identified the main pathogens causing bovine diarrhea and appropriate high-coverage primers by literature searching. According to the searching results, we selected primers for the following target genes: Clostridium perfringens α-toxin, Salmonella enterica invA, Escherichia coli K99, bovine enterovirus (BEV) 5-UTR, bovine astrovirus (BAstV) ORF1a, bovine rotavirus (BRoV) VP6, bovine kobuvirus (BKoV) D4, bovine norovirus (BNoV) ORF1, bovine coronavirus (BCoV) N, bovine torovirus (BToV) N, and bovine viral diarrhea virus (BVDV) 5-UTR. After optimization of the annealing temperature with the temperature gradient PCR method as well as the primer concentration and cycle number with the single factor test method, we established a one-step multiplex PCR/RT-PCR method for detecting the 11 pathogens. The specificity, sensitivity, and repeatability of the method were evaluated, and then the method was employed to detect clinical samples. [Results] The optimal annealing temperature was 54.4 ℃. The optimal concentrations of primers for the target genes in C. perfringens, S. enterica, E. coli, BEV, BAstV, BRoV, BKoV, BNoV, BCoV, BToV, and BVDV were 0.20, 0.25, 0.25, 0.20, 0.25, 0.25, 0.35, 0.50, 0.25, 0.25, and 0.30 μmol/L, respectively. The optimal number of cycles was 35. The established method demonstrated high specificity, yielding positive results only for the target pathogens and negative results for Mannheimia haemolytica, Streptococcus pyogenes, and Mycoplasma bovine. This method exhibited high sensitivity, with the lowest limits of detection of the recombinant plasmid standards being 7.5×103, 7.5×104, 7.5×103, 7.5×101, 7.5×104, 7.5×102, 7.5×103, 7.5×102, 7.5×102, 7.5×104, and 7.5×103 copies/μL, respectively. The method showed good reproducibility, with consistent inter-batch and intra-batch test results. We then used the established method to detect 490 clinical samples from Jiangsu. The positive rates for BEV, BAstV, BRoV, BKoV, BNoV, BCoV, BToV and BVDV were 0.61%, 0.41%, 0.61%, 0.21%, 27.14%, 3.27%, 0.21%, and 1.02%, respectively. The results of the one-step multiplex PCR/RT-PCR method in clinical samples were consistent with those of single PCR. Sequencing verification of 50 randomly selected positive PCR products confirmed the presence of genes corresponding to the identified pathogens. [Conclusion] We successfully established a one-step multiplex PCR/RT-PCR method for the simultaneous detection of 11 major pathogens causing bovine diarrhea.

Abstract:[Background] Aeromonas hydrophila is pathogenic to aquatic animals, livestock, poultry, and human. Hemolysin, aerolysin, and enterotoxin are major virulence factors particularly important in the early detection and prevention of A. hydrophila. There are few reports using colony PCR to extract templates for multiplex PCR. [Objective] Based on colony PCR, a multiplex PCR assay was established for rapid detection of five genes including hemolysin gene, enterotoxin gene, and 16s rRNA gene of A. hydrophila. [Methods] We used the selective Rimler-Shotts (RS) medium to enrich, isolate, and identify A. hydrophila in the sample. Subsequently, we established and optimized the multiplex PCR conditions for the 16S rRNA, ast, alt, aerA, and act of A. hydrophila and compared the results of multiplex PCR with the DNA templates extracted by different methods in colony PCR. Finally, we evaluated the specificity of the established method by using A. veronii, A. sobria and A. salmonicida. [Results] After 16S rRNA identification of single colonies on RS plates, the colony morphology of A. hydrophila and other cultivable bacteria was preliminarily characterized, and the enrichment degree of A. hydrophila was visually identified. The optimization results of the multiplex PCR system showed that the optimal primer concentration ratio was 16s rRNA:ast:alt:aerA:act=1:2:2:3:4. PCR for fresh bacterial liquids treated with both boiling-refrigerated centrifugation method and boiling-centrifugation method could produce clear bands, while the PCR of single colony required the former method. The established multiplex PCR method was specific. [Conclusion] Colony multiplex PCR can be used to easily and intuitively detect A. hydrophila and its virulence genes without the need of kit extraction of DNA templates, and it was specific.

Abstract:A multiplex PCR assay for detection of Escherichia coli O157:H7 was developed by using 3 sets of primers that specifically amplify segments of the rfbE、fliC and eaeA genes. The target genes fragment of the PCR assay were 291 bp, 625 bp and 368 bp, respectively. Analysis of 30 strains demonstrated that this PCR system was specific. The detection limit of the PCR was 91.35 pg with genomic DNA. This multiplex PCR assay did not cross-react with the background Salmonella?typhimurium and could detect 1.4 CFU/mL in artificial inoculated meat sample after enrichment at 37℃ for 6 h. Three of 30 meat samples were detected positive. These results indicated that the multiplex PCR assay can be used for specific and sensitive detection of E. coli O157:H7.

Abstract:A rapid, sensitive and specific method of DNA microarray combined with multiplex PCR that allow the simultaneous detection and identification of Shigella dysenteriae, Salmonella spp. and E. coli O157. The invasion-associated plasmid antigen H of S. dysenteriae(ipaH), Salmonella spp. enterotoxin gene(stn) and Escherichia coli O157 Shiga-Toxin gene(slt) were used as target genes, and then three pairs of primers and captured oligonucleotide probes were designed and synthesized. The multiplex PCR products were hybridized with DNA microarray, which contained specific probes of three foodborne pathogenic microorganisms. Genomic DNAs from 26 bacterial strains were detected, and only 3 index bacteria were positive. The detection limit of this assay was around 8 pg genomic DNA. In addition, this method was applied to detect artificially contaminated food samples, and the detection limit was 50 CFU/mL for non-cultured samples. These results suggested that detection of pathogenic microorganisms by DNA microarray is an effective procedure with high specificity and sensitivity. The DNA microarray assay developed in this study could provide an informative supplement to conventional microbiological methods for routine monitoring of food.

Abstract:[Background] Colibacillosis and salmonellosis are the most common bacterial diseases in poultry, causing economically devastating to poultry industries. Salmonella and avian pathogenic E. coli (APEC) are important zoonotic pathogens, also with potential threat to human health. Thus, it is necessary to strengthen the rapid detection and differential diagnosis of these bacterial diseases. [Objective] We developed multiplex PCR to efficiently detect APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum. [Methods] The special genes of APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum were screened and selected for primers designing. A multiplex PCR method was developed by optimization of conditions. Then, the sensitivity and usage for detection of clinical isolates were determined. [Results] A multiplex PCR was established for accurately and effectively detection of APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum. The sensitivity of the multiplex PCR was 103 bacterial colony forming units (CFUs) and 100 pg genomic DNA. The results of multiplex PCR for detection of clinical isolates agreed well with those of traditional serological assays. [Conclusion] Multiplex PCR developed in this study could efficiently detect APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum.