Establishment and Application of Multiplex PCR for Simultaneously Detecting Escherichia coli, Salmonella, Klebsiella pneumoniae, and Staphylococcus aureus in Minks

Abstract

To establish a multiplex PCR for simultaneous detection of Escherichia coli ( E. coli ), Salmonella, Klebsiella pneumoniae ( K. pneumoniae ), and Staphylococcus aureus ( S. aureus ), four pairs of specific primers were designed according to the conservative regions of phoA gene for E. coli, invA gene for Salmonella, khe gene for K. pneumoniae, nuc gene for S. aureus . The quadruple PCR system was established through optimization of multiplex PCR and detection of specificity, sensitivity, and stability. The results showed that target gene bands of E. coli (622 bp), Salmonella (801 bp), K. pneumoniae (303 bp), and S. aureus (464 bp) could be amplified by this method specifically and simultaneously from the same sample containing the four pathogens, with a detection sensitivity of 100 pg/μL. Meanwhile, no bands of common clinical bacteria, including Clostridium perfringens, Pseudomonas aeruginosa, Pasteurella multocida, Streptococcus pneumoniae, Streptococcus pneumoniae, Proteus mirabilis, Staphylococcus sciuri, Staphylococcus pseudintermedius, Acinetobacter baumannii, Enterococcus faecalis , and Bacillus subtilis were amplified. In addition, 380 tissue samples were detected by multiplex and single PCR established in current study, respectively. Among the 368 carcass samples, positive detection rates of E. coli, K. pneumoniae, Salmonella , and S. aureus were 33.7, 12.0, 10.6, and 13.9%. Among the 12 visceral tissue samples, positive detection rates of E. coli, K. pneumoniae, Salmonella , and S. aureus were 41.7, 25.0, 16.7, and 8.3%, respectively. Positive detection rates of multiplex PCR were consistent with that of single PCR. Compared with single PCR, the multiplex PCR method had the advantages of time-saving, high specificity and high sensitivity. The results showed that the minks in these farms had mixed infection of these four pathogens, and the method established in this study could be applied to the rapid and accurate detection and identification of these four bacteria. In conclusion, the multiplex PCR method has stable detection results, good repeatability, and short detection time. It is suitable for the rapid and accurate detection of four kinds of bacteria above the carcass of fur animals, which could be suitable in microbial epidemiology investigation. It can provide a reliable technical reference for rapid clinical diagnosis and detection.

The Detection of Foodborne Pathogenic Bacteria in Seafood Using a Multiplex Polymerase Chain Reaction System

“…From current sensitivity results, the LOD of E. coli in our study was similar to the results previously reported [ 12 ], with a detection limit of 10 pg. Nogva et al performed PCR amplification using the HlyA gene of L. monocytogenes , and the results showed a detection limit of 1 pg of L. monocytogenes [ 31 ], similar to the results of the current experiment.…”

“…Hernandez et al successfully detected 1~10 CFU of Shigella and Salmonella from food samples within 24 h at low detection limits [ 11 ]. Li et al also used multiplex PCR to detect the genomic DNA of four pathogenic bacteria with a detection limit of 100 pg/μL [ 12 ]. Ashraf et al successfully detected pathogenic bacteria from milk samples at a concentration of 10 4 CFU/mL [ 13 ].…”

Development of two multiplex PCR assays for rapid detection of eleven Gram-negative bacteria in children with septicemia

“…Additional strength of the mPCR assays was its capability to detect DNA concentrations as low as 100 pg which was similar to the previous reports [ 29 , 30 ], although these targeted different pathogens. This outcome is attributable to the methodology of PCR that enables it to amplify small concentrations to readily detectable quantities.…”

Development and application of an indirect ELISA and nested PCR for the epidemiological analysis of Klebsiella pneumoniae among pigs in China

“…A gene designated khe , which encodes a hemolysin of K. pneumoniae , has been proposed as a potential species-specific gene probe ( Yin-Ching et al, 2002 ) and was utilized to detect the signature for clinical K. pneumoniae isolates from hospitals ( Escobar Pérez et al, 2013 ; Chen et al, 2014 ; He et al, 2016 ). Li et al (2020) designed a multiplex PCR method to detect K. pneumoniae in minks with the khe gene. These studies suggested that khe is a specific gene of K. pneumoniae .…”