基于不同靶基因的荧光定量PCR快速检测蜡样芽孢杆菌的研究

为了建立一种基于不同靶基因的快速灵敏检测食品中产与不产呕吐毒素蜡样芽孢杆菌的荧光定量PCR方法,本研究采用煮沸法提取基因组DNA,用普通PCR方法验证引物的特异性,通过在米饭中添加不同浓度的产呕吐毒素蜡样芽孢杆菌模拟受污染的食品,采用本研究构建的荧光定量PCR方法对米饭进行检测。结果表明基于不同靶基因设计的两对引物具有特异性强和扩增效率高等优点,荧光定量PCR方法能对污染米饭中蜡样芽孢杆菌准确定量,检测限能达到101CFU/g。建立的荧光定量PCR方法特异、灵敏和准确,适用于食品中蜡样芽孢杆菌的快速定量检测。      

Establishment of a novel multiplex PCR assay and detection of toxigenic strains of the species in the Bacillus cereus group

Five different enterotoxins and one emetic toxin of Bacillus cereus have been characterized. To amplify all of the enterotoxin and emetic-specific sequences of the species in the B. cereus group, a multiplex PCR with 12 primer pairs was established. In developing the assay method, a common terminal sequence at the 3' ends of all primers was chosen and a hot start Taq polymerase was used to overcome primer dimer formation. The assay was successfully applied to analyze the toxigenic potential of 162 food-poisoning and food-related strains. Results showed that there were 10 toxigenic patterns for all the test strains. All of the B. cereus strains carried at least one toxin gene. More than 70% of Bacillus mycoides strains carried no known toxin genes. The toxin profiles and toxin genes of B. mycoides strains were significantly different from B. cereus strains (P < 0.05), although the two species were closely related. The results suggest that many B. mycoides strains might be less prone to cause food poisoning. They also indicate the importance of detecting the toxin genes together with the detection of the species in the B. cereus group.     

Emetic toxin-producing strains of Bacillus cereus show distinct characteristics within the Bacillus cereus group

One hundred representative strains of Bacillus cereus were selected from a total collection of 372 B. cereus strains using two typing methods (RAPD and FT-IR) to investigate if emetic toxin-producing hazardous B. cereus strains possess characteristic growth and heat resistance profiles. The strains were classified into three groups: emetic toxin (cereulide)-producing strains (n=17), strains connected to diarrheal foodborne outbreaks (n=40) and food-environment strains (n=43), these latter not producing the emetic toxin. Our study revealed a shift in growth limits towards higher temperatures for the emetic strains, regardless of their origin. None of the emetic toxin-producing strains were able to grow below 10 degrees Celsius. In contrast, 11% (9 food-environment strains) out of the 83 non-emetic toxin-producing strains were able to grow at 4 degrees Celsius and 49% at 7 degrees Celsius (28 diarrheal and 13 food-environment strains). non-emetic toxin-producing strains. All emetic toxin-producing strains were able to grow at 48 degrees Celsius, but only 39% (16 diarrheal and 16 food-environment strains) of the non-emetic toxin-producing strains grew at this temperature. Spores from the emetic toxin-producing strains showed, on average, a higher heat resistance at 90 degrees Celsius and a lower germination, particularly at 7 degrees Celsius, than spores from the other strains. No difference between the three groups in their growth kinetics at 24 degrees Celsius, 37 degrees Celsius, and pH 5.0, 7.0, and 8.0 was observed. Our survey shows that emetic toxin-producing strains of B. cereus have distinct characteristics, which could have important implication for the risk assessment of the emetic type of B. cereus caused food poisoning. For instance, emetic strains still represent a special risk in heat-processed foods or preheated foods that are kept warm (in restaurants and cafeterias), but should not pose a risk in refrigerated foods.     

Development of real-time PCR methods to quantify patulin-producing molds in food products

Patulin is a mycotoxin produced by different Penicillium and Aspergillus strains isolated from food products. To improve food safety, the presence of patulin-producing molds in foods should be quantified. In the present work, two real-time (RTi) PCR protocols based on SYBR Green and TaqMan were developed. Thirty four patulin producers and 28 non-producers strains belonging to different species usually reported in food products were used. The patulin production was tested by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair F-idhtrb/R-idhtrb and the probe IDHprobe were designed from the isoepoxydon dehydrogenase (idh) gene, involved in patulin biosynthesis. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the idh gene copy number and Ct values for the different patulin producers tested. The ability to quantify patulin producers of the developed SYBR Green and TaqMan assays in artificially inoculated food samples was successful, with a minimum threshold of 10 conidia g⁻¹ per reaction. The developed methods quantified with high efficiency fungal load in foods. These RTi-PCR protocols, are proposed to be used to quantify patulin-producing molds in food products and to prevent patulin from entering the food chain.     

Detection of meat species using TaqMan real-time PCR assays

Species-specific real-time PCR (TaqMan) assays were developed for detection of beef, pork, lamb, chicken and turkey. Assays were developed around small (amplicons <150 base pairs) regions of the mitochondrial cytochrome b (cytb) gene. Speciation was achieved using species-specific primers. For detection purposes, two TaqMan probes were developed; the first was specific to the mammalian species (beef, lamb and pork), the second to the poultry species (chicken and turkey). Normal end-point TaqMan PCR conditions were applied; however, PCR was limited to 30 cycles. Applying the assays to DNA extracts from raw meat admixtures, it was possible to detect each species when spiked in any other species at a 0.5% level. The absolute level of detection, for each species, was not determined; however, experimentally determined limits for beef, lamb and turkey were below 0.1%.     

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R² values of 0.9969 and 0.9958 respectively. Linear correlations between the log₁₀ input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10¹ CFU/mL to 1.65 × 10⁶ CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.     

Occurence of potentially enterotoxigenic Bacillus cereus in infant milk powder

Considering that powdered infant milk formula effectively supports the growth of numerous pathogens, this study investigates the prevalence of potentially pathogenic Bacillus cereus in dried milk products by evaluating the occurrence of B. cereus and the presence of virulence-associated genes. The approach consisted of enriching, isolating and biochemical identifying isolates, followed by PCR assays aimed at the hbl (C, D, A, B), nhe (A, B, C) and cytK enterotoxin genes coding HBL complex, NHE complex and cytotoxin K, respectively. Among cytK-positive strains, the discrimination of two different forms for cytotoxin K, cytK-1 and cytK-2 was performed. Bacillus cereus was detected in powdered infant milk formula samples. All the strains harbored at least one gene of the cytK, HBL and NHE enterotoxins. Because of an increasing trend in invasive infections by B. cereus in infants and immunocompromised children, our PCR findings highlight the need to implement an adequate control plan in order to guarantee the health of potentially fragile consumers. From a hygiene point of view, intensive and continuous monitoring of potentially pathogenic B. cereus may be crucial for powdered infant milk formula safety and even recommended in order to assess the infant health risk, as proposed by Commission Regulation (EC) no. 1441/2007 on microbiological criteria for foodstuffs. Furthermore, the detection in this study of B. licheniformis, B. subtilis and B. mycoides strains raises significant health issues regarding Bacillus spp. in powdered infant milk formula.     

Improved multiplex PCR assay for simultaneous detection of Bacillus cereus emetic and enterotoxic strains

Toxin producing Bacillus cereus can cause enterotoxic and/or emetic food poisoning. In the present study, a multiplex PCR assay was developed to detect all toxin genes known to be involved in food poisoning of B. cereus in a single reaction. Specific primers for the detection of enterotoxic (entFM, hblC, nheA, and cytK) genes and emetic toxin production (2 primer pairs: ces, CER) were designed based on the GeneBank sequences. The developed multiplex PCR assay was evaluated in pure culture and artificially inoculated milk, using 43 B. cereus strains and non-target strains. In brief, sensitivity in pure culture was 10-fold or more higher than artificially inoculated milk in multiplex PCR detection limit assay. The presented PCR assay is a developed molecular tool for the rapid simultaneous detection of emetic and enterotoxin producing B. cereus strains.     

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148^T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.     

Development of a multiplex real-time PCR to quantify aflatoxin, ochratoxin A and patulin producing molds in foods

The antimicrobial activity of 8 Bacillus spp. and 2 Lysinibacillus spp. representing the predominant aerobic sporeformers during traditional maari fermentations, a traditional fermented baobab seeds product from Burkina Faso, was investigated. The antimicrobial activity was assessed against a total of 31 indicator organisms representing various Gram-negative and positive pathogens. The screening showed that 3 Bacillus subtilis strains (B3, B122 and B222) in particular had antimicrobial activity against some Gram-positive organisms and were selected for further studies. It was found that the antimicrobial substances produced were heat stable, in-sensitive to catalase, sensitive to protease and trypsin but resistant to the proteolytic action of papain and proteinase K and equally active at pH values ranging from 3 to 11. Bacteriocin secretion started in late exponential growth phase and maximum activity was detected during the stationary growth phase. The production of bacteriocin by B. subtilis B3, B122 and B222 was dependent on the aeration conditions. Maximum production of bacteriocin was observed under reduced aeration. Specific primers were used to screen isolates B3, B122 and B222 for genes involved in the synthesis of the bacteriocins subtilosin A, subtilin, sublancin and ericin. Amplicons of the expected sizes were detected for iywB, sboA, sboX, albA and spaS involved in the biosynthesis of subtilosin and subtilin, respectively. The translated nucleotide sequences had 100% identity to the YiwB, SboX and SboA amino acid sequences of the subtilosin A producing B. subtilis subsp. subtilis strain 168. Interestingly there was a 3 amino acid deletion at the N-terminal part of AlbA in B3, B122 and B222 that probably alters the activity of this enzyme. Analysis of the spaS gene sequences of B3, B122 and B222, encoding a subtilin precursor peptide, showed that the translated nucleotide sequence had 98% identity with the corresponding SpaS amino acid sequence of subtilin producing B. subtilis subsp. spizizenii strain ATCC6633.     

Development of three real-time PCR assays to detect peanut allergen residue in processed food products

Hypersensitivity to peanut is a public health problem, since the ingestion of even low amounts of peanut can trigger severe allergic reactions. Allergic consumers rely on the information provided on the label of foodstuffs to identify products that might endanger their health. In order to protect the allergic consumer methods are required for the detection of allergenic ingredients. For this purpose we have developed three real-time polymerase chain reaction (PCR) assays, based on TaqMan chemistry, that are capable of detecting peanut specific DNA sequences in food products. The peanut specific sequence targeted for detection is located within the gene family of the allergen Ara h 3. The occurrence of multiple Ara h 3 sequences in the peanut genome increases the chance to achieve a good sensitivity. DNA extraction is also known to affect detection by PCR, therefore the efficiency of several different DNA extraction methods was compared. The methods reported here are capable of detecting 2.5 pg peanut DNA (less than one copy of peanut genome equivalent) and all three assays were successfully applied to detect peanut traces in a model food product where they could detect 10 mg kg⁻¹ peanut.     

Development of real-time PCR assays for the detection of lupin residues in food products

Lupin is a legume from the Leguminosae family that is used, amongst other, for human nutrition. In Europe, lupin is used as a substitute for soy in bakery and dietary products and recently its consumption has increased significantly. Unfortunately lupin is known to trigger allergic reactions in sensitised individuals and therefore its use in food products requires a mandatory declaration on the label in accordance with Directive 2007/68/EC. To protect the allergic consumer the availability of detection methods for the identification of lupin in food products is required. Here we present the development of two real-time polymerase chain reaction (PCR) methods that allow the detection of lupin-specific DNA as a marker for the presence of this allergenic ingredient in food products. Genomic DNA sequences coding for conglutin genes were chosen as targets for the detection of lupin. One primer set and probe was designed for the amplification of a 153 bp fragment of α-conglutin; another primer set and probe was designed for the detection of a 150 bp δ-conglutin amplicon. Lupin at a level of 10 mg/kg food matrix could be detected in cookies baked from a lupin containing dough using the α-conglutin method. Since lupin is used in bakery products the effects exerted by heat treatments on lupin detection by real-time PCR have been investigated. Enzyme-linked immunosorbent assay (ELISA) analyses were performed in parallel to compare the detection of lupin DNA with that of lupin protein in market products. Qualitative ELISA results confirm results obtained by the real-time PCR methods targeting α- and δ-conglutin.     

Diversity of Bacillus cereus strains in extended shelf life

Characterisation of 49 Bacillus cereus strains obtained from extended shelf life (ESL) milk and filler nozzles was done using (GTG)₅ rep PCR fingerprinting, determining the presence of the virulence genes cytK, nheA, cer and hblA, and discrimination of psychrotrophic and mesophilic strains with 16S rDNA. Fourteen isolates were selected for 16S partial sequencing. Fingerprinting and sequencing showed evidence of filler nozzles contaminating ESL milk despite high heterogeneity existing between the isolates. While there is high prevalence of cer, hblA and nheA; cytK was not widely distributed. There was 100% and 8% prevalence of mesophilic and psychrotrophic signatures, respectively. Despite the large diversity of the B. cereus strains in this study, there is evidence that filler nozzles and raw milk are a source of contamination of B. cereus in ESL milk.     

Development and verification of a quantitative real-time PCR method to identify and quantify gelatin derived from animal hide

The species origin of hide gelatin is a crucial issue with respect to health concerns and religious restrictions. Analysis of the animal-derived ingredients of gelatin by reliable methods is necessary to ensure its authenticity. However, due to the highly processed nature of gelatin, it remains a challenge to identify gelatin end products accurately and robustly. Our study established and verified a quantitative real-time PCR (qPCR) method based on careful selection of target genes and a DNA extraction method. The middle products of the gelatin production streamline were investigated to explore the influence of each critical processing step on the method. Gelatin reference samples were used to quantify the levels of target species. Commercial gelatin commodities were surveyed to highlight the mislabeling situation. In summary, the qPCR method was demonstrated to be highly specific and sensitive, with limits of detection (LOD) of 0.1 to 1 pg/µL and gelatin LODs of 0.1% to 5% (w/w). The transition from decoction to concentrated gel was found to have the most severe effect on the qPCR. Intensification of pressure or temperature or employment of enzyme hydrolysis aggravated the DNA damage, resulting in elevated Cq values. Quantitation of gelatin products was feasible; gelatin products produced from 5% target hide and 95% matrix hide mixtures showed 2.9% to 5.2% target species. The 26% relative error for low gelatin content is acceptable for semiquantitation purposes. A market survey showed that 52.6% of the gelatin products were mislabeled as being of animal origin.     

A rapid and high-throughput real-time PCR assay for species identification: application to stockfish sold in Italy

In some parts of Italy, there is a tradition of eating special, highly prized species of cod, fished and dried in Norway. In order to safeguard the value of this food product, in 2005, the Italian government legislated that the commercial term “stockfish” can only be attributed to Gadus morhua (Gm). In this study, we present an improved real-time PCR assay for efficient identification of Gm with respect to other gadiforms of commercial interest. The method is applied to 437 stockfish samples, collected in various Italian regions, in order to verify whether labelling regulations had been respected and to report instances of fraud in the Italian stockfish market. The PCR method employed here allowed rapid and economical identification of the species, with a very high percentage of correct identifications.     

Diversity and toxigenicity among members of the Bacillus cereus group

Members of the Bacillus cereus group were isolated from rice products by centrifugation-plating and conventional spread-plating methods. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) results showed broad diversity among the strains and revealed some associations among isolates from raw and cooked rice samples, at the genotypic level. A comparatively greater diversity among strains was observed in isolates from raw rice than those from cooked rice and, generally, the RAPD profiles of isolates from raw and cooked rice were different, with only a few of them common to both types of rice. The toxigenic potential of the isolates was also determined by molecular and immunoassay analyses. The results revealed that most isolates from the B. cereus group were potentially or actually toxigenic, and some isolates could produce both diarrhoeal and emetic toxins. Generally, isolates belonging to the B. cereus group with the same RAPD pattern were shown to have a similar profile of enterotoxigenicity.     

银白色葡萄球菌实时荧光PCR快速检测方法的建立

本文旨在建立一种银白色葡萄球菌实时荧光PCR检测方法。根据对NCBI数据库中银白色葡萄球菌非核糖体多肽合成酶(NRPS)基因序列的比对分析,设计引物及探针,并基于所设计的引物及探针建立Taq-man 实时荧光PCR的检测方法。结果表明,本实验所建立的方法特异性较强,只能扩增出银白色葡萄球菌,而其他常见菌株的检测结果呈阴性;该方法的灵敏度为 10 pg/uL;在对实验室通过传统方法分离的金黄色葡萄球菌的检测中,发现有两个分离菌株呈阳性,其结果与普通PCR方法完全一致。     

溶藻弧菌实时荧光定量PCR快速检测方法的建立

为建立溶藻弧菌（VA）的快速检测方法,本研究以VA Collagenase基因为靶基因设计合成引物及Taq Man探针,建立了实时荧光定量PCR快速检测VA的方法。结果显示,对15株实验菌株进行荧光定量PCR检测,只有VA菌株检测为阳性,表明该检测方法特异性强;该方法的灵敏度为18 cfu/m L;稳定性和重复性实验结果表明,同一样品重复检测4次Ct值的变异系数均小于2%;利用该检测方法对采集的165份样品进行检测,共计检出2份VA阳性样品,与SN/T2564-2010行标法检测结果一致,显示了良好的实用性。该检测方法灵敏度高、特异性强,具有良好的实用性。