Search

Rapid, sensitive, point-of-care detection of pathogenic bacteria is important for food safety. In this study, we developed a novel quantum dot nanobeads-labelled lateral flow immunoassay strip (QBs-labelled LFIAS) combined with strand displacement loop-mediated isothermal amplification (SD-LAMP) for quantitative Salmonella Typhimurium (ST) detection. Quantum dot nanobeads (QBs) served as fluorescence reporters, providing good detection efficiency. The customizable strand displacement (SD) probe was used in LAMP to improve the specificity of the method and prevent by-product capture. Detection was based on a sandwich immunoassay. A fluorescence strip reader measured the fluorescence intensity (FI) of the test (T) line and control (C) line. The linear detection range of the strip was 102–108 CFU·mL−1. The visual limit of detection was 103 CFU·mL−1, indicating that the system was 10-fold more sensitive than AuNPs-labelled test strips. ST specificity was analyzed in accordance with agarose gel outputs of PCR and SD-LAMP. We detected ST in foods with an acceptable recovery of 85%–110%. The method is rapid, simple, almost equipment-free, and suitable for bacterial detection in foods and for clinical diagnosis.

Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. are common food-borne pathogens in milk that may cause serious diseases. In the present study, we established a simple, rapid, and specific method to simultaneously detect viable E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk. Three specific genes, fliC from E. coli O157:H7, cgcA from Cronobacter spp., and invA from Salmonella spp., were selected and used to design primers and probes. False-positive results were eliminated with the use of a combined sodium deoxycholate (SD) and propidium monoazide (PMA) treatment. Using the optimized parameters, this SD-PMA treatment combined with multiplex real-time PCR (SD-PMA-mRT-PCR) detected E. coli O157:H7, Cronobacter spp. and Salmonella spp. respectively, at 102 cfu/mL in pure culture or artificially spiked skim milk samples. A reasonable recovery rate (from 100 to 107%) for detection of viable bacteria using the SD-PMA-mRT-PCR assay was obtained in the presence of dead bacteria at 107 cfu/mL. The SD-PMA-mRT-PCR method developed in this study can accurately detect and monitor combined contamination with E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk and milk products.

Salmonella serotyping is a critical step for the identification of pathological isolates in humans and animals. Whole-genome analysis is a proven method for Salmonella serovars identification. In this study, we analysed 1668 genomic sequences of 23 serogroup B serovars and 129 genomic sequences of 80 non-target serovars to detect serovar-specific molecular targets for serogroup B (O:4 epitopes). Serogroup B is the most common serogroup found in China, whose presence is increasing in recent years. We identified five, eight, one, and seven new serovar-specific genes for Indiana, Derby, Typhimurium, and Agona serovars, respectively. We then designed specific PCR primer sets for these genes to validate our results. All target Salmonella serovars could be detected successfully by PCR in different types of food samples. The analytical sensitivity of PCR for 19 out of 21 pairs of primers was 101–103 CFU/mL and 102–105 CFU/g in pure cultures of the bacteria and artificially contaminated fresh pork samples, respectively. Furthermore, we performed a functional analysis of the newly-identified serovar-specific genes. We found that the function was unknown for most of the genes, although for some of them we were able to identify their generic functional categories (membrane protein genes and secreted proteins genes). Our findings revealed that whole-genome analysis is a valuable tool to identify new serovar-specific targets, which could be used to develop rapid and accurate molecular detection methods and provide additional information about different Salmonella serogroup B serovars.

Accurate serotyping of Salmonella is essential for epidemiological investigations and surveillance of Salmonella infections. Compared to traditional methods, polymerase chain reaction (PCR) is more sensitive and identifies Salmonella to the serotype level. Few genes have been used as a target for PCR serotyping; thus, research to identify specific targets of common serotypes is needed. The Salmonella E serogroup contains multiple pathogenic serotypes that have been associated with recent cases of human salmonellosis. Thus, this study mined new molecular targets for rapid detection of the E serogroup of Salmonella serovars. Using pan-genome analysis and the online BLAST program, specific genes of Salmonella serovars Weltevreden, London, Meleagridis, and Senftenberg were obtained. Primer sets were evaluated by PCR using target strains, 41 nontarget Salmonella serotypes and 22 non-Salmonella strains. There were 11, 5, 5, and 1 genes specific to S. Weltevreden, S. London, S. Meleagridis, and S. Senftenberg, respectively, with detection limits as low as 102 or 103 CFU/mL per assay for pure culture. Moreover, the primer sets satisfactorily identified Salmonella serotypes in artificially contaminated foods. These results demonstrated that pan-genome analysis identified new specific targets, and PCR serotyping could be a primary screening method or a supplement to the classical detection method.

Salmonella is one of the most common pathogens that cause foodborne diseases in humans, resulting in high medical and economical costs worldwide. The aim of this study was to develop a rapid and accurate approach for simultaneous detection of viable Salmonella spp. in raw meat, including Salmonella enterica serovars Paratyphi B, S. Typhimurium, and S. Enteritidis. To reduce detection time and improve sensitivity, immunomagnetic separation (IMS) was used as a pre-concentration and separation method. In addition, false positive and false negative results were removed by combining propidium monoazide (PMA) treatment with internal amplification control. Results showed that the detection limit of IMS-PMA combined with sextuplex polymerase chain reaction (PCR) assay reached as low as 101 CFU/mL in pure culture and 102 CFU/g in spiked raw meat (ground pork and ground beef), and the total assay time took less than 6 h. Thus, the novel IMS-PMA-mPCR assay developed in this study holds promise for routine screening of viable Salmonella serovars in meat and other samples.

Salmonella is a food-borne pathogen that causes worldwide infections in humans and animals. Over 2600 Salmonella serovars have been identified, and serogroup C1 (O:6, 7 epitopes) contains highly diverse serovars associated with high pathogenicity. Here, we aimed to develop a reliable polymerase chain reaction (PCR) method for detecting serovar-specific markers of Salmonella serogroup C1, with greater reliability and lower complexity than current methods. We identified three, two, four, and one novel genes unique to the Infantis, Rissen, Braenderup, and Virchow serovars. The PCR primers targeting these genes were specific for the target serovars. The detection limits of the PCR assays using 9 pairs of primers were 102–103 colony-forming units (CFUs)/mL and 104–105 CFU/g in pure bacterial cultures and artificially contaminated raw pork samples, respectively. No false-positive or false-negative results were obtained with 45 food samples, as determined by comparison with biochemical tests and traditional serotyping methods. The potential functions of all serovar-specific genes identified are unknown, except for one Rissen-specific gene that encodes a secreted protein. Our results imply that all 10 novel, specific genes can be considered as useful targets for detecting the Salmonella Infantis, Rissen, Braenderup, and Virchow serovars in food.

Escherichia coli O157:H7 and Salmonella spp. in milk are 2 common pathogens that cause foodborne diseases. An accurate, rapid, specific method has been developed for the simultaneous detection of viable E. coli O157:H7 and Salmonella spp. in milk. Two specific genes, namely, fliC from E. coli O157:H7 and invA from Salmonella spp., were selected to design primers and probes. A combined treatment containing sodium deoxycholate (SDO) and propidium monoazide (PMA) was applied to detect viable E. coli O157:H7 and Salmonella spp. only. Traditional culture methods and SDO-PMA-multiplex real-time (mRT) PCR assay were applied to determine the number of viable E. coli O157:H7 and Salmonella spp. in cell suspensions with different proportions of dead cells. These methods revealed consistent findings regarding the detected viable cells. The detection limit of the SDO-PMA-mRT-PCR assay reached 102 cfu/mL for Salmonella spp. and 102 cfu/mL for E. coli O157:H7 in milk. The detection limit of SDO-PMA-mRT-PCR for E. coli O157:H7 and Salmonella spp. in milk was significantly similar even in the presence of 106 cfu/mL of 2 nontarget bacteria. The proposed SDO-PMA-mRT-PCR assay is a potential approach for the accurate and sensitive detection of viable E. coli O157:H7 and Salmonella spp. in milk.