Acid-tolerant Lactiplantibacillus plantarum ZDY2013 shows a colonization niche preference and interacts with enterotoxigenic Bacillus cereus in specific-pathogen-free mice.

Probiotics have long been utilized as functional food and modulate gut microbial homeostasis, but their colonization niche is mostly unclear and transient, which restrains the development of microbiome-targeted strategies. Lactiplantibacillus (L.) plantarum ZDY2013 is an allochthonous species of the human gastrointestinal tract with acid-tolerant properties. It serves as an antagonistic agent against the food-borne pathogen Bacillus (B.) cereus and a potent regulator of the gut microbiota. However, there is a knowledge gap regarding the colonization dynamics of L. plantarum ZDY2013 in the host intestine and the colonization niche of its interaction with pathogens. Here, we designed a pair of specific primers targeting L. plantarum ZDY2013 based on its whole genome sequence. We evaluated their accuracy and sensitivity with other host-derived strains and confirmed their availability with artificially spiked fecal samples from different mouse models. Additionally, the content of L. plantarum ZDY2013 was quantified by qPCR in fecal samples from BALB/c mice, followed by the analysis of its colonization niche preference. Moreover, the interactions between L. plantarum ZDY2013 and enterotoxigenic B. cereus HN001 were also elucidated. The results revealed that the newly designed primers could identify L. plantarum ZDY2013 with high specificity and were resistant to the influence of the complex fecal matrix and gut microbes from different hosts. Interestingly, the content of mixed L. plantarum ZDY2013 and B. cereus HN001 when orally administered remained higher when compared with the single strain group in BALB/c mice upon discontinuation of intragastric administration. In addition, L. plantarum ZDY2013 was mainly enriched in the large intestine during the ingestion period and maintained the highest content in the stomach after discontinuing supplementation on day 7. Moreover, L. plantarum ZDY2013 colonization neither damaged the intestine nor ameliorated the damage triggered by B. cereus in BALB/c mice. Overall, our study constructed two efficient specific primers targeting L. plantarum ZDY2013 and provided the potential to explore the underlying mechanism of competition between L. plantarum ZDY2013 and pathogens in host species.

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.

Sensitive Detection of Staphylococcus aureus by a Colorimetric Biosensor Based on Magnetic Separation and Rolling Circle Amplification.

Staphylococcus aureus (S. aureus) is a common foodborne pathogen that causes fever, vomiting, and other intestinal symptoms, and seriously affects human health and social safety. As a result, a reliable and sensitive detection technique for S. aureus must be developed. In this work, we proposed a sandwich assay on vancomycin functionalized magnetic beads (Van-MNPs) for S. aureus detection based on the specific binding between IgG and targets. The Van-MNPs were used as a tool for the separation of target bacteria. The biotin-modified IgG mediates binding between DNA nanoflowers (DNFs) and the target bacteria via interacting with streptavidin. The DNFs prepared by rolling circle amplification (RCA) were employed as a nano-container to enhance the capacity of biotins, and the streptavidin-horseradish peroxidase (SA-HRP) was loaded onto DNFs to catalyze the color change of TMB. Therefore, a colorimetric biosensor based on magnetic separation and rolling circle amplification was developed. The proposed methods for S. aureus detection showed a limit of detection (LOD) of 3.3 × 103 CFU/mL and excellent specificity. The biosensor has a certain reference value for the detection of S. aureus in juice.

Hybrid RCA-DLS assay combined with aPCR for sensitive Salmonella enteritidis detection.

Salmonella infection could come from eating contaminated meat or raw eggs, and drinking milk or water contaminated by Salmonella enteritidis. Therefore, it is necessary to explore a fast and easy method for the detection of S. enteritidis in these diverse samples. For this purpose, a novel particle size sensing tool was designed for ultrasensitive and accurate S. enteritidis detection. This assay consisted of rolling circle amplification (RCA) with dynamic light scattering (DLS) using gold nanoparticles (AuNPs) modified with DNA probe as DNA-AuNPs as the capture surface into a hybrid RCA-DLS assay combined with asymmetric polymerase chain reaction (aPCR) and subsequent detection. Under optimal experimental conditions, the novel hybrid RCA-DLS assay combined with aPCR for S. enteritidis reached a limit of detection (LOD) as low as 3 × 100 CFU/mL in pure culture. In spiked milk samples, the LOD was 2.0 × 100 CFU/mL without pre-enriched bacteria. The total time of RCA-DLS assay was about 6 h which including genomic DNA extraction, aPCR, RCA and DLS determination. The hybrid RCA-DLS assay combined with aPCR holds promise in the specific and sensitive S. enteritidis detection.

Fluorescence detection of Staphylococcus aureus using vancomycin functionalized magnetic beads combined with rolling circle amplification in fruit juice.

Staphylococcus aureus is a common foodborne pathogen that can cause a suppurative infection after eating contaminated food. Detection of S. aureus plays an important role in the food industry. In this study, a strategy for the detection of S. aureus using magnetic separation (MS) technology combined with rolling circle amplification (MS-RCA) was proposed. The strategy used antibiotics to capture bacteria and employed RCA products as signal output probes. Vancomycin (Van), as a commonly used antibiotic, can recognize peptidoglycan on the cell wall of Gram-positive bacteria and can effectively identify target bacteria. Therefore, we prepared BSAylated-Van functionalized magnetic beads (Van-MBs) for the pre-enrichment of S. aureus. To ensure the selectivity of this method, we used biotin-pig IgG to bind S. aureus. In addition, to amplify the output signal of the MS-RCA strategy, we introduced streptavidin (SA) and successfully obtained the Van-MBs@S. aureus@biotin-pig IgG@SA@biotin-RCA probe complex and used the biotin-avidin-system (BAS) by combining magnetic separation technology and RCA technology to realize the enrichment and specific detection of S. aureus. Furthermore, by optimizing the experimental conditions such as the magnetic separation time and the amount of Van-MBs, the detection performance of this method was improved. Under the optimal conditions, the detection limit of this method for S. aureus was 3.3 × 102 CFU/mL in fruit juice, and it was less affected by other bacteria.

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.

[Research progress of biosensors in the detection of foodborne pathogens].

As the main factor leading to foodborne illnesses, foodborne pathogens have been attached great importance by people. The development of simple, rapid, high-sensitivity and low-cost food-borne pathogen detection methods is of great significance in reducing the incidence of foodborne diseases. Biosensor technology is a new micro-analysis technology developed by multi-disciplinary cross-infiltration. It has the characteristics of high sensitivity and fast analysis speed, and is widely used in the detection of food-borne pathogens. This paper introduces the basic principles of biosensors, summarizes the application of common biosensors in the detection of foodborne pathogens, and prospects for future development.

[Progress in rolling circle amplification in biological detection].

Rolling circle amplification is a rapid, sensitive and isothermal single-stranded DNA amplification technique that can be used with staining or probes to amplify the detection signal. This technology has been widely used in biological detection and other aspects. The present paper introduces how to design rolling circle amplification, summarize its application in the detection of pathogens, nucleic acid tumor markers, proteins, biological small biomolecules, and viruses in recent years and prospects for future development.

Sensitive fluorescent detection of Listeria monocytogenes by combining a universal asymmetric polymerase chain reaction with rolling circle amplification.

A new, facile, low-cost, and highly sensitive method for detection of Listeria monocytogenes involving a combination of asymmetric polymerase chain reaction (aPCR) and rolling circle amplification (RCA) had been developed. The aPCR-RCA processes were not new but components of the processes made the assay useful. Twenty-one thymine (21-T) tagged forward primer generated universal twenty-one adenine (21-A) aPCR amplicons after aPCR amplification. A poly-T sequence dumbbell-like RCA template was produced through the blunt-end ligation activity of T4 DNA ligase. After the mixture of aPCR amplicons and dumbbell-like RCA template, the RCA reaction would initiate when the addition of phi29 DNA polymerase, then a large number of G-quadruplex sequences were produced which allowed the intercalation of Thioflavin T (3,6-dimethyl-2-(4-dimethylaminophenyl) benzo-thiazolium cation, THT) for easy fluorescence detection. Under the optimal conditions, the assay showed a limit of detection (LOD) of 4.8 × 101 CFU/mL in pure culture and 4.0 × 102 CFU/g in spiked lettuce homogenates. By changing the aPCR primer, the aPCR-RCA method developed in this study had a potential to detect other bacteria without the design an RCA template for each bacterium.

Catalytic hairpin assembly combined with graphene oxide for the detection of emetic Bacillus cereus in milk.

A fluorescence assay combined with PCR, catalytic hairpin assembly (CHA), and graphene oxide (GO) was established to detect emetic Bacillus cereus in milk samples. The processes of the assay are not new, but components of the processes make the assay useful. Two partially complementary hairpin probes (H1 and FAM-H2) were designed according to the target single-strand DNA (ssDNA). The CHA reaction could be initiated only by the target ssDNA, which was generated by the denaturation of PCR amplicons. In the absence of the target ssDNA, CHA reaction could not be triggered, which caused the H1 and FAM-H2 adsorbing on the surface of GO and exhibiting a low fluorescence intensity. Addition of the target ssDNA resulted in opening of the hairpin H1 that subsequently hybridized with H2. Then, target ssDNA would be replaced from the H1 and recycled to promote another CHA reaction. Through the CHA reaction, multiple H1-H2 duplexes were generated that could not adsorb on the surface of GO. Thus, a strong fluorescence signal would be obtained. The assay showed a limit of detection for emetic B. cereus of 6.2 × 101 cfu/mL in pure culture and 5.9 × 102 cfu/mL in spiked milk without enrichment. By changing the PCR primer, the assay developed in this study had potential to detect other bacteria.

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.