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.     

基于qPCR快速检测米饭中产呕吐毒素蜡样芽孢杆菌的研究

为了建立基于QPCR的快速方法检测米饭中产呕吐毒素蜡样芽孢杆菌,首先采用煮沸法提取蜡样芽孢杆菌基因组DNA,并利用普通PCR方法验证引物特异性,然后通过在米饭样品中添加目标菌,模拟受污染的实际样本,用QPCR技术定量检测米饭中产呕吐毒素蜡样芽孢杆菌。结果表明该方法具有快速、特异性强、灵敏度高和稳定性好的优点,能对产呕吐毒素蜡样芽孢杆菌定量。不经过增菌培养,实际样品的检测限为9.8×101CFU/g;经过2 h的增菌培养,检测限能达到100CFU/g;并且米饭中添加其他杂菌后,不影响对蜡样芽孢杆菌的检测。建立的QPCR方法适用于米饭等相关淀粉类食品中产呕吐毒素蜡样芽孢杆菌的检测,从而为监测该菌所致食品污染以及早期相关食物中毒提供快速定量检测方法。     

基于PCR方法检测蜡样芽孢杆菌的研究进展

蜡样芽孢杆菌是引起食物中毒的常见食源性致病菌之一,如何快速准确检测食品中的蜡样芽孢杆菌是控制其污染食品的关键环节。本文介绍了蜡样芽孢杆菌引起食物中毒的原因,并综述了基于PCR方法检测蜡样芽孢杆菌的研究进展。      

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。      

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.     

Multiplex PCR assay and lyophilization for detection of Salmonella spp., Staphylococcus aureus and Bacillus cereus in pork products

Food Science and Biotechnology - Multiplex PCR (m-PCR) has the potential for more rapid detection of pathogens compared to simple PCR through the simultaneous amplification of multiple gene targets...     

Environment driven cereulide production by emetic strains of Bacillus cereus

The impacts of growth media and temperature on production of cereulide, the emetic toxin of Bacillus cereus, were measured for seven well characterised strains selected for diversity of biochemical and genetic properties and sources of origin. All strains carried cereulide synthase gene, ces, on a megaplasmid of ca. 200 kb and all grew up to 48-50 degrees C, but produced cereulide only up to 39 degrees C. On tryptic soy agar five strains, originating from foods, food poisonings and environment, produced highest amounts of cereulide at 23 to 28 degrees C, whereas two strains, from human faeces, produced cereulide similarly from 23 to 39 degrees C, with no clear temperature trend. These two strains differed from the others also by producing more cereulide on tryptic soy agar if supplemented with 5 vol.% of blood, whereas the other five strains produced similarly, independent on the presence of blood. On oatmeal agar only one strain produced major amounts of cereulide. On skim milk agar, raw milk agar, and MacConkey agar most strains grew well but produced only low amounts of cereulide. Three media components, the ratio [K(+)]:[Na(+)], contents of glycine and [Na(+)], appeared of significance for predicting cereulide production. Increase of [K(+)]:[Na(+)] (focal variable) predicted (P<0.001) high cereulide provided that the contents of glycine and [Na(+)] (additional variables) were kept constant. The results show that growth medium and temperature up and downregulate cereulide production by emetic B. cereus in a complex manner. The relevance of the findings to production of cereulide in the gut and to the safety of amino acids as additives in foods containing live toxinogenic organisms is discussed.     

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.     

Occurrence of emetic toxin producing Bacillus cereus in the dairy production chain

A collection of 5668 Bacillus cereus isolates, resulting from investigations on sources of contamination to milk in 10 different dairies and 10 farms, was screened for the presence of emetic toxin producing strains, based on phenotypic traits, a specific RAPD-PCR pattern, a sperm motility inhibition test, and was confirmed chemically. No emetic strains (<0.2%) were found in milk at a farm during the grazing period and none was found in environmental samples. Emetic strains were generally rare in milk (<1.0–3.8%) during the stall period but extensive clonal development occurred on one farm in cubicles with deep sawdust bedding. Among 3401 isolates obtained from samples taken along the dairy processing line and in monthly samplings of silo tanks, only 0.05% were emetic toxin producers. However, evidence for an emetic house flora was found in one silo tank. It may be important for the dairy industry to increase surveillance for emetic strains in milk powder and to further optimize the cleaning routines of silos.     

Production of Bacillus cereus emetic toxin (cereulide) in various foods

To determine the role of Bacillus cereus as a potential pathogen in food poisoning, the production of an emetic toxin (cereulide) by B. cereus was quantified in various food sources. The amount of emetic toxin in 13 of 14 food samples implicated in vomiting-type food poisoning cases ranged from 0.01 to 1.28 microg/g. A vomiting-type strain, B. cereus NC7401, was inoculated into various foods and incubated for 24 h at 20, 30, and 35 degrees C. In boiled rice, B. cereus rapidly increased to 10(7)-10(8) cfu/g and produced emetic toxin at both 30 and 35 degrees C. In farinaceous foods, the production of emetic toxin was as high as that in the food samples implicated in food poisoning. Low levels of emetic toxin were detectable in egg and meat and their products and a small quantity of toxin was detectable in liquid foods such as milk and soymilk when not aerated. Bacterial growth and toxin production was inhibited in foods cooked with vinegar, mayonnaise, and catsup, supposedly by the decreased pH of acetic acid. This is the first report that has quantified emetic toxin of B. cereus in various foods.     

Potential of selected infant food formulas for production of Bacillus cereus emetic toxin, cereulide

Cereulide producing Bacillus cereus was isolated from randomly chosen commercial infant foods. The cereulide production in infant food formulas was investigated. When the reconstituted foods were inoculated with >10(5) cfu ml(-1) of cereulide producing B. cereus, 2 to 200 microg of cereulide per 100 ml of food accumulated during 24 h of non-refrigerated storage. The amount of cereulide measured in the foods by the accurate chemical assay (LC-MS) matched with that found by sperm micro assay, proving the cereulide was the sole heat stable toxin in the foods and present in its toxic form. The infant formulas containing both cereal and dairy ingredients were the most supportive for cereulide production. Cereulide accumulation was affected by the infant food composition as well as by the handling of the food. Diluting the reconstituted food with water resulted in increased toxin production expressed as mug per volume. More cereulide was accumulated when the food was incubated stationary compared with moderate shaking. The amount of cereulide accumulated within 24 h at room temperature per 100 ml of cereal and dairy or in rice-nondairy reconstituted infant formulas, inoculated with >or=10(5) cfu ml(-1) of B. cereus strain F4810/72, was higher or similar to the amounts reported for foods implicated in emetic type of food poisonings. Thus mishandling and temperature abuse of infant foods may cause food poisoning when emetic B. cereus is present.     

Bacillus cereus, the causative agent of an emetic type of food-borne illness

Bacillus cereus is the causative agent of two distinct forms of gastroenteritic disease connected to food-poisoning. It produces one emesis-causing toxin and three enterotoxins that elicit diarrhea. Due to changing lifestyles and eating habits, B. cereus is responsible for an increasing number of food-borne diseases in the industrial world. In the past, most studies concentrated on the diarrhoeal type of food-borne disease, while less attention has been given to the emetic type of the disease. The toxins involved in the diarrhoeal syndrome are well-known and detection methods are commercially available, whereas diagnostic methods for the emetic type of disease have been limited. Only recently, progress has been made in developing identification methods for emetic B. cereus and its corresponding toxin. We will summarize the data available for the emetic type of the disease and discuss some new insights in emetic strain characteristics, diagnosis, and toxin synthesis.     

Rapid identification of Bacillus cereus based on the detection of a 28.5-kilodalton cell surface antigen

Conventional procedures for the identification of suspect Bacillus cereus isolated on mannitol-egg yolk-polymyxin (MYP) agar may need several days. To facilitate the identification of the bacterium, an enzyme-linked immunosorbent assay (ELISA) was developed. The assay was based on the detection of a 28.5-kDa cell surface antigen of B. cereus. Bacterial colonies grown on MYP agar or nutrient agar were suspended in phosphate-buffered saline (pH 7.2) containing 0.1% Teepol. The cell suspensions were heated at 100 degrees C for 5 min and added to the microtiter plates coated with antibodies against the 28.5-kDa antigen. After washing, the same antibodies labeled with horseradish peroxidase were used as secondary antibodies to reveal the signal of antigen-antibody reaction. For 38 strains of B. cereus and 127 strains of non-B. cereus bacteria (including 79 isolates of Bacillus spp.) tested, the sensitivity and specificity of the ELISA were 100 and 88.2%, respectively. Strains producing false-positive results were members of the B. cereus group (i.e., Bacillus anthracis, Bacillus mycoides, and Bacillus thuringiensis), which are genetically and biochemically similar to B. cereus. Similar ELISA results were obtained by using antibodies against another cell surface antigen with a molecular mass of 20 kDa. If members of the B. cereus group were recognized as a single species, the sensitivity and specificity of the ELISA were 100 and 99.1%, respectively. The ELISA could be used as a rapid method for presumptive identification of B. cereus grown on MYP agar.     

Influence of type of food on the kinetics and overall production of Bacillus cereus emetic toxin

Potato puree and penne pasta were inoculated with cereulide producing B. cereus 5964a and B. cereus NS117. Static incubation at 28 degrees C proved these two foods to be a better substrate for higher cereulide production (4,080 ng/g in puree and 3,200 ng/g in penne were produced by B. cereus 5964a during 48 h of incubation) compared with boiled rice (2,000 ng/g). This difference occurred despite B. cereus counts of more than 10(8) CFU/g in all three products. Aeration of cultures had a negative effect on cereulide production, causing concentrations more than 10-fold lower than in some statically incubated samples. Cereulide production remained undetectable in shaken milk, whereas it reached 1,140 ng/ml in statically incubated milk. At 12 and 22 degrees C, presence of background flora was also a determinative factor. A total B. cereus count of more than 106 CFU/ml did not necessarily lead to uniform cereulide production and was also dependent on the B. cereus strain involved. In this study, we confirm that a number of factors play a crucial role in the determination of the extent to which, if at all, cereulide will be produced. Among those, type of the food, temperature, pH, and whether additional aeration (via incubation on an orbital shaker) is induced had an important role. An important effect was also induced by the cereulide-producing strain involved.     

一蜡样芽胞杆菌食物中毒分离株的表型与基因型

目的对2003年发生在云南的一起蜡样芽胞杆菌呕吐型食物中毒案例中的分离株YN0303进行表型和基因型分析研究。方法采用16S r DNA序列分析和16S/23S r DNA ITS特征图谱分析结合形态、生理生化和基因型进行菌种鉴定;PCR检测呕吐毒素和肠毒素合成相关基因,高效液相和质谱联用(HPLC-MS)化学定量分析呕吐毒素合成情况,测试部分生理生化特性和生长温度范围,进行了pan C分型和多位点序列分型(multilocus sequence typing,MLST)。结果确认了该菌株确为蜡样芽胞杆菌Bacillus cereus,虽然该食物中毒是典型的呕吐型案例,但是发现该分离株呕吐毒素相关合成基因ces呈阴性,呕吐毒素化学定量分析结果为阴性;其生理生化特性(水杨苷发酵、淀粉水解和溶血特性)与典型呕吐型菌株也不相同。pan C分型并非呕吐型蜡样芽胞杆菌所属的Ⅲ型,而是属于细胞毒性强的IV型,而且其肠毒素基因hbl、nhe和cyt K2也均呈阳性;多位点序列分析显示该菌是一个新型别ST753,是区域性相对独立进化菌株。结论在该呕吐型食物中毒案例中可能有多个蜡样芽胞杆菌参与,该分离株YN0303并非主要致病株—呕吐型菌株,但可能作为细胞毒性强的菌株参与了共感染。     

即时食品蜡样芽孢杆菌快速检测标准曲线模型的研究

本实验研究建立了一种即时食品蜡样芽孢杆菌快速检测模型.基于实时光电检测技术,将蜡样芽孢杆菌标准菌株(CICC10041)菌液进行7次10倍递增稀释,每个稀释度进行国标法和实时光电法2种方法5次重复测定,获得35组一一对应的数据,以国标法计数结果的对数值为纵坐标,以实时光电法相应稀释度的DT值为横坐标,建立标准曲线模型,...     

原料乳中蜡样芽孢杆菌的快速检测

目前原料乳中蜡样芽孢杆菌的检测方法繁琐费时耗力,针对此缺点建立一种操作简便快速检测方法,采用过滤富集菌体后PCR技术来检测原料乳中蜡样芽孢杆菌.整个过程只需6 h左右即可完成.检测灵敏度高达101 mL-1.这种方法是对传统检测方法的有效改进,并且达到了原料乳检测快速、准确、灵敏的要求.     

Vancomycin-modified poly-l-lysine magnetic separation combined with multiplex polymerase chain reaction assay for efficient detection of Bacillus cereus in milk

In this study, a new vancomycin (Van)-modified poly-l-lysine (PLL) magnetic bead (MB) technique was developed for isolation of gram-positive bacteria. The method combines magnetic separation with a multiplex PCR (mPCR) assay and gel electrophoresis for easy and rapid detection of Bacillus cereus. Vancomycin was used as a molecular ligand between the MB and the d-alanyl-d-alanine moieties on the cell wall surface of B. cereus. The PLL served as a flexible molecular tether between the MB and Van that reduced steric hindrance maintaining the biological activity of Van. The MB-PLL-Van capture nanoprobes exhibited excellent capture and isolation efficiency for B. cereus in spiked milk matrix samples without interference from the complex food matrix. The subsequent mPCR assay showed high specificity for the 4 target genes in B. cereus, the entFM, cesB, cer, and 16S rRNA genes, that were used to achieve efficient genotyping and detection. Under optimum conditions, the limit of detection reached 10³ cfu/mL, with a dynamic range of detection at 10³ to 10⁷ cfu/mL in pure culture. Application of the MB-PLL-Van mediated mPCR assay for B. cereus in milk matrix samples achieved results similar to those of the pure culture. In addition, with a 6-h pre-enrichment of B. cereus that was spiked in milk matrix samples, the limit of detection reached 10¹ cfu/mL. The MB-PLL-Van mediated mPCR assay developed in this study could be used as a universal technology platform for the efficient enrichment and genotyping of gram-positive bacteria.     

Development of a PCR assay for detection of Enterobacteriaceae in foods

A broad-range PCR assay for the detection of bacteria belonging to the Enterobacteriaceae family was developed in this study. Primers targeting the bacterial 16S rRNA gene were newly designed and used in this PCR assay. To determine the specificity of the assay, 72 different bacterial species (of 49 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for every tested Vibrioaceae or Enterobacteriaceae strain except Proteus mirabilis. For all other bacterial strains and eukaryotes tested, results were negative. Bacterial DNA for PCR was prepared by a simple procedure with the use of Chelex 100 resin from culture after growth in brain heart infusion medium. To test this PCR assay for the monitoring of the Enterobacteriaceae family, either Escherichia coli or Salmonella Enteritidis was inoculated into various foods as an indicator. Prior to the PCR, the inoculation of 10 to 40 CFU of bacteria per g of food was followed by a 5-h enrichment culture step, and the PCR assay allowed the detection of bacterial cells. When actual examinations of the contamination of 15 noodle foods with Enterobacteriaceae by this PCR assay were conducted, 33% (5 of 15) of the samples tested positive. These results agreed with those of the Petrifilm Enterobacteriaceae Count Plate assay. Including the enrichment culture step, the entire PCR detection process can be completed within 7 h.     

基于内参的副溶血性弧菌实时荧光定量PCR快速检测方法的建立

目的建立基于内参的副溶血性弧菌实时荧光定量PCR方法,快速检测样品中的副溶血性弧菌。方法根据Gen Bank已公布的副溶血性弧菌基因组序列,筛选特异性靶基因,设计特异性引物探针,优化反应体系,并在体系中加入内参(IAC),通过标记不同荧光基团的Taq Man探针来监测IAC,进而实时监控整个PCR反应。按照5~50 cfu/25 g的细菌量人工污染样品,以评价所建立反应的体系。结果以副溶血性弧菌基因组DNA为模板,最低检测限为1 pg/μl;以10倍梯度稀释的菌液经水煮法提取的DNA为模板,最低检测限为4×102cfu/ml;以含有gyr B的质粒为模板,最低检测极限可以达到100 copies/μl;建立gyr B和gyr B-IAC标准曲线,Ct值与模板拷贝数均呈良好线性关系(r2=0.999);人工污染初始菌量为7 cfu/25 g时,样品中副溶血性弧菌增菌6 h即可检出。结论本研究所建立的gyr B-IAC实时荧光定量PCR方法,既能有效检测食品中副溶血性弧菌,又能实时监测PCR反应过程,有效防止"假阴性"的发生,结果可靠,有利于实现海产品中副溶血性弧菌实时荧光定量PCR检测方法的标准化。