Development of a Simple,Rapid Multiplex PCR Method Using the 16S rRNA Gene to Determine the Country of Origin of Brackish Water Bivalve <Emphasis Type="Italic">Corbicula japonica</Emphasis>

In this study, a multiplex PCR detection method was developed to identify the country of origin of Corbicula japonica (clams), a commercially important bivalve in Asia. Specific primer sets that have a single nucleotide mismatch at the 3′ terminus were designed after sequencing the mitochondrial 16S rRNA gene of clams identified as C. japonica originating from Korea, China, and Japan. Using this method, each origin was clearly identified based on the PCR products: three bands for Korean C. japonica (100, 283, and 384 bp), one band for Chinese C. japonica (384 bp), and two bands for Japanese C. japonica (384 and 100 bp). These results indicate that the 16S rRNA gene, which is usually used to identify species, can distinguish the country of origin within C. japonica. Our multiplex PCR assay should be a useful tool for the fair trade of the species.     

Saltatory Rolling Circle Amplification (SRCA): a Novel Nucleic Acid Isothermal Amplification Technique Applied for Rapid Detection of <Emphasis Type="Italic">Shigella</Emphasis> Spp. in Vegetable Salad

Shigella spp. are enteric pathogens that pose a serious threat to public health worldwide. A novel saltatory rolling circle amplification (SRCA) assay was developed to detect Shigella spp. in food targeting the ipaH gene. SRCA as an isothermal amplification method requires no expensive thermocycle instrument and could avoid electrophoresis as visualization results was successfully applied for SRCA. In order to confirm the specificity of this assay, 34 strains including 11 strains belonging to different Shigella species and 23 non-Shigella bacteria were detected with pure cultures. The sensitivity of Shigella flexneri by SRCA was evaluated using agarose gel electrophoresis, which was 7.3 × 10¹ fg/μL. In addition, the amplification results were also determined by adding the fluorochrome, SYBR Green I (1 μL of 1000×), allowing naked eye visualization of results, and the sensitivity was 7.3 × 10⁰ fg/μL. Moreover, the sensitivity of PCR was 7.3 × 10² fg/μL, showing that the sensitivity of SRCA by electrophoresis and SYBR Green I fluorescence were 10- and 100-fold higher than that of PCR, respectively. The detection limit of SRCA was also evaluated with artificially inoculated vegetable salad without enrichment, and it was 4.7 × 10² and 4.7 × 10¹ CFU/g by electrophoresis and fluorescence, respectively. The detection limit by PCR was 4.7 × 10³ CFU/g, which was 10- and 100-fold higher than that of SRCA. Therefore, SRCA is a potentially reliable tool for rapid and specific detection of Shigella in food and could be useful in underdeveloped countries with limited resources.     

Rapid Detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Food Using a Recombinase Polymerase Amplification-Based Assay

Staphylococcus aureus (S. aureus) is of great importance and is a leading cause of food poisoning, which is a public health concern in terms of the frequency and seriousness of the disease. In the present study, RPA and real-time RPA assays were developed and validated to detect S. aureus with high sensitivity and specificity by targeting the nuc gene for the first time. The analytical sensitivity of real-time RPA was 10² copies/reaction, which was higher than the sensitivity of the real-time PCR method. The analysis time was reduced to 10 min, but this method was as reliable as real-time PCR. Furthermore, the potential use of RPA to detect S. aureus was validated with five different artificially contaminated foods. In conclusion, the RPA and real-time RPA assays developed here, similar to real-time PCR, are rapid and simple and exhibit with high sensitivity and specificity. These assays serve as efficient tools for the detection of S. aureus in less advanced laboratories and are suitable for point-of-care detection.     

Real-Time Recombinase Polymerase Amplification Assay for the Detection of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> in Seafood

Vibrio cholerae, the most hazardous Vibrio species, is threatening aquacultured animals and even human beings. In recent years, a few genetic markers that target V. cholerae have been reported, but their application was limited by techniques or themselves. The outer membrane lipoprotein gene lolB has been identified as a specific marker by using PCR and quantitative PCR methods. In this study, we developed a real-time recombinase polymerase amplification (RPA) assay targeting lolB gene in an attempt to provide a sensitive, rapid, and reliable detection method of V. cholerae. The sensitivity of RPA assay was determined by amplifying the standard plasmid dilutions. The detection limit down to five copies was achieved within 10 min, which was lower than that of qPCR (ten copies after 25 cycles within 1.5 h). The reproducibility of RPA assay was verified by eight independent experiments, presenting a high R ² value of the standard regression line (0.97). In addition, the specificity was confirmed with DNA extracted from other bacteria, shrimps, clams, and fishes using both methods. Finally, V. cholerae in shrimp samples were quantified using real-time RPA and qPCR with consistent outcomes, while no amplification was obtained from clam and fish samples using both methods. The applicability in the food industry was confirmed by quantitative detection of natural seafood.     

Polymerase chain reaction for the detection of<Emphasis Type="Italic"> Phaseolus vulgaris</Emphasis> beans in chestnut purée

A method based upon polymerase chain reaction (PCR) for the detection of Phaseolus vulgaris beans in chestnut purée was developed. The method involves DNA isolation by the GeneSpin kit and PCR with primers oriented to a sequence of a nuclear gene encoding PvLEA-18, a late embryogenesis-abundant protein of beans. The PCR method was shown to be specific for Ph. vulgaris, producing a 100 bp fragment with six Ph. vulgaris cultivars, and negative results with four non-Ph. vulgaris Phaseolus samples and five non-Phaseolus legumes. When evaluated with model mixtures of Ph. vulgaris beans and the chestnut purée, a detection limit of 1% was determined.     

A Rapid and Visual Single Primer Isothermal Amplification-Based Method for the Detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Raw Pork Products

Staphylococcus aureus (S. aureus) is an important food-borne pathogen which poses a severe threat to public health worldwide. Rapid detection of S. aureus with high sensitivity is of particular importance for food safety. In this study, a novel single primer isothermal amplification (SPIA) method was established to detect S. aureus in food, targeting the accessory gene regulator (agr) gene with a DNA/RNA primer. The developed SPIA method has the advantages of visualization and avoiding tedious electrophoresis. In order to confirm the specificity of this method, 7 S. aureus strains and 26 non-S. aureus strains were detected with their pure cultures. The sensitivity and detection limit of S. aureus with artificially inoculated raw pork products by SPIA were evaluated through fluorescence and turbidity by naked eye and the amplification curve, which were 4.3?×?10⁰ CFU/mL and 5.6?×?10⁰ CFU/g, respectively. Compared with the conventional PCR method, the SPIA has 100-fold higher sensitivity and 100-fold lower detection limit. Therefore, the developed SPIA method is a potentially reliable tool for rapid and visual detection of S. aureus in food.     

Enterohemorrhagic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EHEC) in water from karst springs: detection with real-time PCR and isolation of strains

Within 2 months, two water sources in a karst area in Switzerland were sampled 9 times each, and analyzed by real-time PCR for 6 EHEC O-types, Shiga-like-toxin (stx1 and stx2) and intimin (eae) genes. With the exception of O111, 5 O-types were recorded regularly and at high frequencies (O26: 33.3 %; O157: 33.3 %; O104: 66.6 %; O103: 72.2 %; O145: 94.4 %). Genes for Shiga-like-toxins and intimin were almost omnipresent (stx1: 77.8 %; stx2: 83.3 %; eae: 77.8 %). Strain isolation was undertaken for O-groups 26, 103, 104, 145 and 157. Sample selection for strain isolation was based on Cq-values for the O-groups and stx1, stx2 and eae. From selected samples, frozen enrichment cultures were cultivated on EHLY-agar and 50 typical colonies screened for the O-type and genes encoding for stx1, stx2 and eae. With this approach, only one virulent EHEC-strain could be isolated (Escherichia coli O103, stx1 +; stx2 ?; eae +). We carried out one extensive testing with 800 colonies of O-group O145, and no virulent strain was isolated. Our findings showed that PCR-results are not sufficient to formulate epidemiological conclusions and that the isolation of strains is necessary. However, as the detection procedure of EHEC in foods is cumbersome and expensive, the appropriateness of such an approach in official food control is a matter of debate.     

Detection of Viable <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Cells in Seafood Using a Real-Time Visual Loop-Mediated Isothermal Amplification Combined with Propidium Monoazide

Vibrio cholerae is an important foodborne pathogen causing severe intestinal infectious diseases that have high incidence and mortality. Almost all of rapid testing methods including immunological and molecular assays for V. cholerae are incapable of distinguishing live cells from dead ones, which may overestimate the number of bacteria and result in many false positive results. To address the problems, live cell-specific dye such as propidium monoazide (PMA) is employed. The loop-mediated isothermal amplification (LAMP) assay is a nucleic acid amplification method that is fast, specific, and sensitive. In this study, we developed a real-time visual LAMP assay using PMA dye to detect thyA gene, thereby identifying viable V. cholerae cells. The results showed that only V. cholarae strains could be detected, and there was no cross-reaction with non-V. cholarae strains. Besides, the sensitivity of the PMA-LAMP assay was 1.1 × 10² CFU/mL and the entire reaction could be accomplished within 1 h. The sensitivity was on par with that of the PMA-qPCR assay. The detection limit in different artificially inoculated samples was 5 CFU/25 g materials for the tested pathogens. In the practical test, the PMA-LAMP assay performed well in comparison with PMA-qPCR and the culture method. Hence, PMA-LAMP assay can provide a highly effective and rapid approach for detecting viable V. cholerae.     

Dynamic changes of bacterial communities and nitrite character during northeastern Chinese sauerkraut fermentation

Northeastern Chinese sauerkraut is a well-known traditional fermented vegetable in China. Incomplete identification of the microorganisms’ (bacteria in spontaneous fermentation) diversity and accumulation of nitrite make it difficult to normalize the fermentation process and product qualities of northeastern Chinese sauerkraut. Conventional culturing and polymerase chain reaction-denaturing gradient gel electrophoresis methods were combined to describe microbial structure and diversity. Lactobacillus, Leuconostoc, Enterobacter, Accumulibacter, Thermotoga, Pseudomonas, Clostridium, Rahnella and Citrobacter were predominant microorganisms in different fermentation periods. The pH value and nitrite concentration presented a certain relevance to the amount of lactic acid bacteria. Lactobacillus and Leuconostoc had the ability to decrease nitrite by inhibiting nitrate-reducing bacteria such as Enterobacter. Therefore, Northeastern Chinese sauerkraut should not be eaten until 4 weeks of fermentation for the safety and quality of fermented foods. Northeastern Chinese sauerkraut is rich in lactic acid bacteria, which demonstrate its ability as an excellent probiotic for applications in functional foods.     

Quantification of <Emphasis Type="Italic">Penicillium nalgiovense</Emphasis> on Dry-Cured Sausage ‘Salchichón’ Using a SYBR Green-Based Real-Time PCR

To evaluate the effective implantation of a specific protective culture of Penicillium nalgiovense, a real-time quantitative PCR (qPCR) using SYBR Green methodology was developed. Two specific primers were designed on the basis of the published partial sequences of the Internal Transcribe Spacer (ITS)1–5.8S-ITS2 region of various strains of P. nalgiovense. Using the developed method, a PCR product of 51 bp with a T _m value 81.34 °C was detected. T _m values of the amplified product allowed specific differentiation between P. nalgiovense and the remaining mould species tested. The developed qPCR method was tested on inoculated slices of dry-cured sausage (‘salchichón’) showing an efficiency of 97.24 %, a R ² value of 0.99 and a detection limit of P. nalgiovense of 1 log colony-forming units (cfu)/cm². The qPCR method demonstrated that the protective strain of P. nalgiovense grew and competed against an ochratoxin A (OTA)-producing Penicillium verrucosum strain on commercial dry-cured sausage. This qPCR method provides a specific, accurate and sensitive detection and quantification of P. nalgiovense on dry-cured sausage salchichón in order to estimate its colonization during their processing. This assay will improve strategies to prevent and control unwanted mould colonization and OTA risk in dry-cured meat commodities.     

Detection of histamine-producing bacteria using polymerase chain reaction techniques and DNA probes

The polymerase chain reaction technique was used to enable the detection of DNA specific amplicons, corresponding to gene fragments coding for histidine decarboxylase present in the histidine decarboxylating bacteria frequently found in canned fish. The level of histamine in several fish product samples was quantified by HPLC and the DNA of the samples containing histamine was isolated and amplified with hdcA histidine-decarboxylase gene-specific primers. The primer sets used, CL1/CL2, JV16HC/JV17HC and CL1/JV17HC, amplified 150 bp, 370 bp and 500 bp products respectively. Non-expected fragments (100 bp, 150 bp and 250 bp) were also amplified by JV16HC/JV17HC. Some amplified fragments were sequenced automatically, presenting high homologies with the Clostridium perfringens hdcA gene. A DNA probe from a C. perfringens pure strain was hybridized with the DNA fragments amplified from the contaminated samples. The hybridization blots were then detected by colorimetry, revealing that the samples were contaminated by C. perfringens.     

Rapid Detection of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> in Shellfish by Real-Time Recombinase Polymerase Amplification

Vibrio parahaemolyticus (V. parahaemolyticus) is a zoonotic pathogen generally found in seafood. To detect the foodborne pathogen rapidly and accurately for food safety measures, we developed a real-time recombinase polymerase amplification (RPA) method. An evaluation of the specificity and sensitivity of the method is discussed here. A set of primers and probe was specially designed to target the tlh gene, which is usually regarded as a marker of total V. parahaemolyticus strains. During the reaction, target DNA was amplified and tagged with specific fluorophore within 10 min and at an incubation temperature of 40 °C. In addition to fast amplification and low temperature, the fluorescence signal was synchronized with the amplification of products for the generation of real-time data. The detection limit of this assay was 0.4 pg/μL of DNA, which is comparable to assays that use the bacterial culture as template, 4?×?10³ cfu/mL. The real-time RPA method had a stable performance when testing the spiking shellfish samples at the same level of contamination by the pathogen in different kinds of shellfish. Thus, the real-time RPA method shows great potential for on-site detection of V. parahaemolyticus, especially in low-resource settings.     

Evaluation of <Emphasis Type="Italic">eryC</Emphasis> as a Molecular Marker for the Quantitative Detection of <Emphasis Type="Italic">Brucella</Emphasis> Spp. by Real-Time PCR in Food Samples

We evaluated the capacity of the Brucella sp. eryC gene as a diagnostic marker for brucellosis by quantitative real-time PCR. eryC gene encodes the enzyme d-erythrulose-1-phosphate dehydrogenase that plays an important role in the erythritol metabolism and is related with the Brucella survival in the intracellular environment of the macrophage. The assay includes an internal amplification control (IAC) in order to avoid false negative results. It was 100% specific, with an analytical sensitivity of 1 genome equivalent (GE) in 43% of the reactions, being the quantification highly linear (R ² > 0.9953) and efficient (PCR efficiency >0.8820) over a 6-log dynamic range, down to 10 GE. Finally, the applicability of this assay was evaluated with artificially contaminated biological matrices implicated in the transmission of this bacterium such as sheep raw milk and pig blood. The eryC-IAC real-time PCR assay allowed detection of as few as ten Brucella cells per 25 ml of sheep raw milk or per 1 ml of pig blood. In conclusion, we present an alternative for the detection of Brucella genus and therefore facilitate the establishment of preventive and prophylactic measures in food and farm environments.     

Development of Real-time PCR Assays for the Detection of the <Emphasis Type="Italic">pin</Emphasis> II Terminator (t<Emphasis Type="Italic">pin</Emphasis>II) Used in GM Constructs and Its Donor Organism,Potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>)

Two singleplex TaqMan methods were developed for the detection of potato targets: one for the detection of the tpinII terminator, which is an emerging terminator used in GM constructs, and one for the detection of the endogenous StLS gene of potato. Performance criteria such as specificity and sensitivity were successfully tested for the two methods, taking into account the recommendations of international guidelines. The presence of the StLS target was checked in 16 potato cultivars. The StLS target is present at low copy number and can be used for quantitation purposes, as demonstrated on transgenic potatoes in this paper. The StLS target is an excellent candidate to replace the presently recommended endogenous target based on the UGP gene, which shows several disadvantages due to its high copy number and lack of specificity. The research also indicates that DNA can easily be extracted from different parts of potato tubers with a classical cetyltrimethylammonium bromide method.     

Quantitative Detection of <Emphasis Type="Italic">Clostridium perfringens</Emphasis> by Real-Time PCR in Raw Milk

Clostridium perfringens causes a broad spectrum of diseases in both humans and animals and is an important cause of foodborne illness. We developed and tested a real-time (Q-)PCR assay for the species-specific detection of C. perfringens that targets the phospholipase C (plc) gene and includes an internal amplification control (IAC), making it possible to identify false-negative results, which are common due to the high level of PCR inhibition by food compounds. The CPplc-IAC real-time PCR (RTi-PCR) assay was 100% selective, as shows with 36 Clostridium strains and 85 non-Clostridium strains, with an analytical sensitivity of 1 genome equivalent (GE) in 23% of the reactions and 10 GE in 100% of the reactions. The quantification was linear (R ² = 0.9990) over a 7-log dynamic range, down to 10 GE, with a PCR efficiency E = 0.841. The applicability of this RTi-PCR assay was assessed in milk samples. The assay detected as few as 300 spores in 25 mL of artificially contaminated raw sheep milk with 78% probability and 30 spores in 25 mL with 50% probability. It also has accuracy of 83.03 to 151.18%, as shown by an evaluation of the correspondence between RTi-PCR assay results and the number of spores per milliliter determinated by standard plating. This RTi-PCR method was effective for the detection and quantification of C. perfringens in milk having an important applicability in the control of this pathogen in the dairy food industry.     

Simultaneous Analysis of Free/Bound Phytosterols and Diosgenin in Taiwanese Yams

Gas chromatography (GC) and high performance liquid chromatography (HPLC) methods were developed for the simultaneous analysis of phytosterols and diosgenin in two representative Taiwanese yams, Dioscorea pseudojaponica Yamamoto and Dioscorea alata L. The major phytosterols found were β-sitosterol, stigmasterol, and campesterol. GC afforded good separation of phytosterols and diosgenin, with the diosgenin peak appearing between stigmasterol and β-sitosterol. For HPLC, UV detection had higher sensitivity (detection limits 0.03–0.09 μg); however, evaporative light scattering detection (ELSD) achieved higher noise tolerance and did not show baseline interference from solvent gradients. In addition, mixed solvents for extraction before acid hydrolysis, which is necessary to avoid fibers and acid hydrolysates binding to the target compounds, were evaluated. Phytosterol contents of 30.1 and 9.9 mg/100 g were found in D. pseudojaponica Yamamoto and D. alata L., respectively. Diosgenin was only found in D. pseudojaponica Yamamoto at a content of 15.2 mg/100 g.     

Antibacterial activities of a cinnamon essential oil with cetylpyridinium chloride emulsion against <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium in basil leaves

This study examined the antibacterial activities of two different cinnamon essential oil emulsions against Escherichia coli O157:H7 and Salmonella Typhimurium on basil leaves. Cinnamon oil (0.25%) treatments containing CPC (0.05%) exhibited greater effects on the pathogenic bacteria than cinnamon oil treatment without this emulsifier (p < 0.05). Treatment with cinnamon bark and leaf oil emulsions (CBE and CLE, respectively) reduced the populations of E. coli O157:H7 by 4.10 and 5.10 log CFU/g, and S. Typhimurium by 2.71 and 2.82 log CFU/g, respectively. Scanning electron micrographs showed morphological changes in the two pathogenic bacteria following emulsion treatment. In addition, there was no difference in the color or ascorbic acid content of the basil leaves by the emulsion treatment. These results suggest that CBE or CLE treatment can be an effective way to ensure the microbial safety of minimally processed vegetables and a good alternative to chlorination treatment in the fresh produce industry.     

Comparative Study on the Inactivation and Photoreactivation Response of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> Seafood Isolates and a <Emphasis Type="Italic">Listeria innocua</Emphasis> Surrogate after Pulsed Light Treatment

The inactivation and photoreactivation response of six seafood-isolated Listeria monocytogenes and one Listeria innocua strain after pulsed light (PL) treatment was evaluated. The lower inactivation levels found after exposure of treated samples to daylight during the first 90 min of storage confirmed that both L. innocua and L. monocytogenes have the capability to photorepair PL-induced DNA damage upon appropriate conditions. Photoreactivation levels from 0.2 to 2.1 log CFU cm^?2 were observed depending on treatment intensity (fluence) and Listeria strain. Complete photorepair of PL-caused damage was not found even after treatments inducing low inactivation levels. Photoreactivation increased up to 2.1 log with the applied fluence up to a threshold able to cause between 2.4 and 5.4 log reductions under dark storage. Photorepair was not avoided but lower photoreactivation was observed after higher fluence inducing more than 6 log reductions under dark storage. Both L. innocua and L. monocytogenes serotype 1/2b exhibited the highest photoreactivation levels whereas serotypes 1/2a showed the lowest ones. The overall inactivation and photoreactivation responses of tested Listeria strains were comparable indicating that L. innocua may be a good surrogate for the safe evaluation, optimization and validation of PL technology to control L. monocytogenes in food products and food processing facilities.     

Viability of Probiotics in Goat Cheese During Storage and Under Simulated Gastrointestinal Conditions

The objective of this study was to evaluate the suitability of two strains of probiotic bacteria (Bifidobacterium animalis subsp. lactis and Lactobacillus rhamnosus) incorporated individually into Boursin-type goat cheese and their in vitro resistance through the passage of the gastrointestinal tract in the cheese. The viability of B. lactis and Lb. rhamnosus cultures was unaffected throughout 35 days of storage at 4 °C, with a final count of >?7 log CFU/g. No significant difference (p?>?0.05) was observed between probiotic treatments and control in relation to pH and titratable acidity. B. animalis presented greater resistance to the artificial gastric and enteric juices than Lb. rhamnosus, with mean decreases in the initial populations of 0.2 and 4.0 log CFU/g within 35 days of storage, respectively. The addition of probiotic cultures did not affect the consumer acceptance of the goat cheeses. Three segments of consumers with different liking were identified. The results demonstrated that “Boursin” goat cheese is a promising matrix for the incorporation and protection of B. animalis subsp. lactis.     

Effect of Tannin Extracts on Biofilms and Attachment of <Emphasis Type="Italic">Escherichia coli</Emphasis> on Lettuce Leaves

Lettuce is often involved in foodborne outbreaks caused by pathogenic Escherichia coli. Current control strategies have often proved ineffective to ensure safe food production. For that reason, the present study compared the efficacy of tannin extracts and chlorine treatments on the reduction of E. coli ATCC 25922 adhered to lettuce leaves. E. coli was inoculated artificially on leaf surfaces of fresh crisp lettuce. Effectiveness of water, chlorine (200 mg/L), and three commercial available tannin extracts from Acacia mearnsii De Wild. (tannin AQ (2 %, w/v), tannin SG (1 %, v/v) and tannin SM (1 %, v/v)) treatments was evaluated using the viable plate count method and scanning electron microscopy (SEM). SEM results revealed that bacterial cells are attached as individual cells and in clusters to the leaf surface after 2 h of incubation. Biofilm formation was observed after 24 h of incubation. The tannin SM treatment was able to reduce counts in approximately 2 log CFU/cm² on leaf segments. However, treatment was less effective in the reduction of E. coli counts after 24 h of incubation when compared to 2 h incubation of the same extract. The results suggest that the tannin SM extract diminishes E. coli counts adhered to and under biofilm formation on lettuce leaves and its effect is similar to the use of chlorine solutions.