Simultaneous detection of viable Salmonella spp., Escherichia coli, and Staphylococcus aureus in bird's nest, donkey-hide gelatin, and wolfberry using PMA with multiplex real-time quantitative PCR.

Salmonella spp., Escherichia coli, and Staphylococcus aureus are common microbial contaminants within the homology of medicine and food that can cause serious food poisoning. This study describes a highly efficient, sensitive, specific, and simple multiplex real-time quantitative PCR (mRT-qPCR) method for the simultaneous detection of viable Salmonella spp., E. coli, and S. aureus. Primers and probes were designed for the amplification of the target genes invA, uidA, and nuc. Dead bacterial genetic material was excluded by propidium monoazide (PMA) treatment, facilitating the detection of only viable bacteria. This method was capable of detecting Salmonella spp., E. coli, and S. aureus at 102, 102, and 101 CFU/ml, respectively, in pure culture. PMA combined with mRT-qPCR can reliably distinguish between dead and viable bacteria with recovery rates from 95.7% to 105.6%. This PMA-mRT-qPCR technique is a highly sensitive and specific method for the simultaneous detection of three pathogens within the homology of medicine and food.

Dual-signal amplification strategy: Universal asymmetric tailing-PCR triggered rolling circle amplification assay for fluorescent detection of Cronobacter spp. in milk.

Cronobacter spp. are important opportunistic foodborne pathogens in powdered infant formula that cause many serious diseases in neonates and infants. In this study, a novel assay based on dual signal amplification strategy was developed by coupling asymmetric tailing PCR (AT-PCR) with rolling circle amplification (RCA) for the detection of Cronobacter spp. in milk. The tailing single-stranded DNA was generated through AT-PCR and used to initiate RCA, generating tandem repetitive G-quadruplex sequences. In the presence of the fluorescence dye thioflavin T that could intercalate into the G-quadruplex structures, the fluorescence signal was detected with a microplate reader. The AT-PCR coupled with RCA assay was specific for Cronobacter spp. detection because of the highly specific primers chosen for the AT-PCR. The limits of detection were 4.3 × 101 cfu/mL in pure culture and 4.5 × 102 cfu/mL in spiked milk, respectively. The fixed sequences designed in the hairpin DNA allowed this AT-PCR coupled with RCA assay to serve as a universal platform for the detection of other pathogens by modifying the specificity of the PCR primers.

Rapid and quantitative detection of viable emetic Bacillus cereus by PMA-qPCR assay in milk.

Emetic Bacillus cereus is one of the causative agents of foodborne diseases which can cause vomiting-type food poisoning after ingestion of contaminated food. To minimize B. cereus food poisoning, propidium monoazide (PMA) combined with quantitative polymerase chain reaction (qPCR) called PMA-qPCR was applied for detecting viable emetic B. cereus in milk. The cereulide synthetase gene of emetic B. cereus (cesB) was chosen for the primer, and PMA treatment was optimized at 3 µg/mL to inhibit the PCR amplification of DNA from dead cells. Under optimized assay parameters, the limit of detection (LOD) using this method were 102 CFU/mL in both pure culture and in spiked milk matrix. The cycle threshold (Ct) values obtained for this assay was not significantly affected by the presence of non-target bacteria such as E. coli O157:H7 which indicated the high selectivity of the assay for emetic B. cereus. The PMA-qPCR assay used in this study has the potential for sensitive detection of viable emetic B. cereus in milk.

Simultaneous quantitative detection of viable Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. using sodium deoxycholate-propidium monoazide with multiplex real-time PCR.

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.

Rapid and simultaneous quantification of viable Escherichia coli O157:H7 and Salmonella spp. in milk through multiplex real-time PCR.

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.

Sensitive Detection of Staphylococcus aureus with Vancomycin-Conjugated Magnetic Beads as Enrichment Carriers Combined with Flow Cytometry.

A novel sandwich strategy was designed to detect Staphylococcus aureus. The strategy is based on an antibacterial agent that captures bacterial cells and a fluorescein-labeled antibody that acts as the signal-output probe. Vancomycin (Van), which exerts a strong antibacterial effect on Gram-positive bacteria, was utilized as a molecular recognition agent to detect pathogenic bacteria. To effectively concentrate S. aureus, we used bovine serum albumin (BSA) as the amplification carrier to modify magnetic beads (MBs), which were then functionalized with Van. To improve the specificity of the method for S. aureus detection, we adopted fluorescein isothiocyanate (FITC)-tagged pig immunoglobulin G (FITC-pig IgG) as the signal probe and the second recognition agent that bound between the Fc fragment of pig IgG and protein A in the surface of S. aureus. To quantify S. aureus, we measured the fluorescence signal by flow cytometry (FCM). The use of multivalent magnetic nanoprobes (Van-BSA-MBs) showed a high concentration efficiency (>98%) at bacterial concentrations of only 33 colony-forming units (CFU)/mL. Furthermore, the sandwich mode (FITC-pig IgG/SA/Van-BSA-MBs) also showed ideal specificity because Van and IgG bound with S. aureus at two distinct sites. The detection limit for S. aureus was 3.3 × 101 CFU/mL and the total detection process could be completed within 120 min. Other Gram-positive bacteria and Gram-negative bacteria, including Listeria monocytogenes, Bacillus cereus, Cronobacter sakazakii, Escherichia coli O157:H7, and Salmonella Enteritidis, negligibly interfered with S. aureus detection. The proposed detection strategy for S. aureus possesses attractive characteristics, such as high sensitivity, simple operation, short testing time, and low cost.