Multiplex real-time PCR detection of fumonisin-producing and trichothecene-producing groups of Fusarium species

Some species of Fusarium can produce mycotoxins during food processing procedures that facilitate fungal growth, such as the malting of barley. The objectives of this study were to develop a 5' fluorogenic (Taqman) real-time PCR assay for group-specific detection of trichothecene- and fumonisin-producing Fusarium spp. and to identify Fusarium graminearum and Fusarium verticillioides in field-collected barley and corn samples. Primers and probes were designed from genes involved in mycotoxin biosynthesis (TRI6 and FUM1), and for a genus-specific internal positive control, primers and a probe were designed from Fusarium rDNA sequences. Real-time PCR conditions were optimized for amplification of the three products in a single reaction format. The specificity of the assay was confirmed by testing 9 Fusarium spp. and 33 non-Fusarium fungal species. With serial dilutions of purified genomic DNA from F. verticillioides, F. graminearum, or both as the template, the detection limit of the assay was 5 pg of genomic DNA per reaction. The three products were detectable over four orders of magnitude of template concentration (5 pg to 5 ng of genomic DNA per reaction); at 50 ng template per reaction, only the TRI6 and FUM1 PCR products were detected. Barley and corn samples were evaluated for the presence of Fusarium spp. with traditional microbiological methods and with the real-time PCR assay. The 20 barley samples and 1 corn sample that contained F. graminearum by traditional methods of analysis tested positive for the TRI6 and internal transcribed spacer (ITS) PCR products. The five corn samples that tested positive for F. verticillioides by traditional methods also were positive for the FUMI and ITS PCR products. These results indicate that the described multiplex real-time PCR assay provides sensitive and accurate differential detection of fumonisin- and trichothecene-producing groups of Fusarium spp. in complex matrices.     

串珠镰刀菌分离菌株PCR筛选鉴定方法的研究

为监测粮食中的串珠镰刀菌污染，根据rDNA18S、5．8S、28S及其间区ITS和ITS2序列，设计了1对镰刀菌属特异性引物Fu3／Fu4及2对串珠镰刀菌种特异性引物Fu5／Fu4(Ⅰ型ITS2特异性)和Fu3／Fu2(Ⅱ型ITS2特异性)，建立了串珠镰刀菌PCR及复合PCR检测方法。Fu3／Fu4、Fu3/Fu2和FuS／Fu4分剐扩增516bp、375bp和188bp的DNA片段。Fu3／Fu4 PCR的检出灵敏度为10pg，FuS／Fu4为100pg，复合PCR(Fu3、Fu5和Fu4)的检出限为100pg，分别相当于每次反应检出10^3、10^4和10^4个孢子。复合PCR可以同时鉴别镰刀菌属和串珠镰刀菌种。检测了32株从山东、浙江、安徽和河南4省区分离的串珠镰刀菌及交孢镰刀菌，26株为Ⅰ型ITS2特异性，4株为Ⅱ型ITS2特异性，两株待定。该方法可用于大量快速筛选串珠镰刀菌，有利于进一步开展串珠镰刀茵在我国的生态分布、生物地域学及系统发育学等方面的研究。该方法快速、敏感、特异，适宜粮食中串珠镰刀菌污染的监测。     

Multiplex polymerase chain reaction assay for the differential detection of trichothecene- and fumonisin-producing species of Fusarium in cornmeal

The genus Fusarium comprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In this study, a multiplex polymerase chain reaction (PCR) assay was developed for the group-specific detection of fumonisin-producing and trichothecene-producing species of Fusarium. Primers for genus-level recognition of Fusarium spp. were designed from the internal transcribed spacer regions (ITS1 and ITS2) of rDNA. Primers for group-specific detection were designed from the TRI6 gene involved in trichothecene biosynthesis and the FUM5 gene involved in fumonisin biosynthesis. Primer specificity was determined by testing for cross-reactivity against purified genomic DNA from 43 fungal species representing 14 genera, including 9 Aspergillus spp., 9 Fusarium spp., and 10 Penicillium spp. With purified genomic DNA as a template, genus-specific recognition was observed at 10 pg per reaction; group-specific recognition occurred at 100 pg of template per reaction for the trichothecene producer Fusarium graminearum and at 1 ng of template per reaction for the fumonisin producer Fusarium verticillioides. For the application of the PCR assay, a protocol was developed to isolate fungal DNA from cornmeal. The detection of F. graminearum and its differentiation from F. verticillioides were accomplished prior to visible fungal growth at <10(5) CFU/g of cornmeal. This level of detection is comparable to those of other methods such as enzyme-linked immunosorbent assay, and the assay described here can be used in the food industry's effort to monitor quality and safety.     

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.     

Fusarium verticillioides from finger millet in Uganda

Finger millet (Eleusine coracana) is a subsistence crop grown in Sub-Saharan Africa and the Indian Sub-continent. Fusarium species occurring on this crop have not been reported. Approximately 13% of the Fusarium isolates recovered from finger millet growing at three different locations in eastern Uganda belong to Fusarium verticillioides, and could produce up to 18,600?µg/g of total fumonisins when cultured under laboratory conditions. These strains are all genetically unique, based on AFLP analyses, and form fertile perithecia when crossed with the standard mating type tester strains for this species. All but one of the strains is female-fertile and mating-type segregates 13:20 Mat-1:Mat-2. Three new sequences of the gene encoding translation elongation factor 1??α were found within the population. These results indicate a potential health risk for infants who consume finger millet gruel as a weaning food, and are consistent with the hypothesis that F. verticillioides originated in Africa and not in the Americas, despite its widespread association with maize grown almost anywhere worldwide.     

Characterization of Fusarium verticillioides strains isolated from maize in Italy: fumonisin production, pathogenicity and genetic variability

Fusarium verticillioides (teleomorph Gibberella moniliformis) is the main fungal agent of ear and kernel rot of maize (Zea mays L.) worldwide, including Italy. F.verticillioides is a highly toxigenic species since it is able to produce the carcinogenic mycotoxins fumonisins. In this study, 25 F. verticillioides strains, isolated from maize in different regions of Italy were analyzed for their ability to produce fumonisins, their pathogenicity and their genetic variability. A further referenced strain of G. moniliformis isolated from maize in USA was also used as outgroup. The fumonisins B₁, B₂, and B₃ were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Pathogenicity tests were carried out by symptom observation and determination of growth parameters after inoculation of maize seeds, seedlings and wounded detached leaves. Total genomic DNA was used for Amplified Fragment Length Polymorphism (AFLP) analysis. About 20% of the analyzed strains were unable to produce fumonisins in in vitro experiments on inoculated maize flour, while, among fumonisin producers, a great variability was observed, with values ranging from 1 to 115 mg kg⁻¹. The different analyzed strains showed a wide range of pathogenicity in terms of effect on seed germination, seedling development and of symptoms produced on detached leaves, which were not correlated with the different in vitro fumonisin production. AFLP analysis indicated the presence of genetic diversity not only between the Italian strains and the American reference but also among the Italian isolates.     

Characterization of Fusarium verticillioides strains by PCR‐RFLP analysis of the intergenic spacer region of the rDNA

Thirty‐three Fusarium verticillioides strains from diverse origins and hosts have been analysed for fumonisin production and characterized in order (i) to detect the variability present in this species and (ii) to discriminate among isolates. The method used was a combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) generated by restriction endonucleases applied to the IGS region (intergenic spacer of rDNA). All the F. verticillioides strains associated with crops produced fumonisins B₁ and B₂ except those isolated from banana. Analysis of the IGS region by PCR‐RFLP proved to be useful to detect variability within F. verticillioides and allowed discrimination of two related groups of isolates belonging to distinct lineages differing in fumonisin production and host preferences: the fumonisin‐producing group associated with cereals and the fumonisin non‐producing group associated with banana. The method used facilitates early detection and characterization of F. verticillioides strains required to control both types of pathogens and to evaluate plant exposure to the toxin, quality of the raw material to be processed and the potential fumonisin contamination in order to prevent fumonisins entering the food chain. Copyright © 2005 Society of Chemical Industry     

Nested PCR Method for Early Detection of Fumonisin Producing Fusarium verticillioides in Pure Cultures,Cereal Samples and Plant Parts

Fumonisin-producing Fusarium verticillioides was detected in cereal samples by semi-nested PCR using one forward, VERTF-1, and two reverse primers, VERTR and VERTF-2. A total of 326 Fusarium species isolated from maize, paddy, sorghum, wheat and pearl-millet samples were subjected to a first round of n-PCR with the species specific VERTF-1 and VERTR set of primers which recorded 59.50% of F. verticillioides. Further, second round of n-PCR scored 53.98% of fumonisin-producing F. verticillioides with fumonisin specific VERTF-1 and VERTF-2 set of primers. Maize samples recorded the highest frequency of fumonisin-producing F. verticillioides 40.98%, followed by paddy 33.33%, and sorghum 12.50%. Sensitivity of nested PCR was conducted by whole grain experiment of the cereals, roots and leaves of the cereals by diluting the DNA 10-100 times, in which 1:50, 1:75, and 1:100 diluted samples recorded positive. This method can be used for the early detection of fumonisin producing F. verticillioides occurring on cereals.     

Impact of cycling temperatures on Fusarium verticillioides and Fusarium graminearum growth and mycotoxins production in soybean

BACKGROUND: Fusarium graminearum and F. verticillioides are two very important mycotoxigenic species as they cause diverse diseases in crops. The effects of constant and cycling temperatures on growth and mycotoxin production of these species were studied on soybean based medium and on irradiated soya beans. RESULTS: F. graminearum grew better when was incubated at 15, 20 and 15–20 °C (isothermal or cycling temperature) during 21 days of incubation. Maximum levels of zearalenone and deoxynivalenol (39.25 and 1040.4 µg g^?1, respectively) were detected on soya beans after 15 days of incubation and the optimal temperature for mycotoxin production was 15 °C for zearalenone and 20 °C for deoxynivalenol. F. verticillioides grew better at 25 °C in culture medium and at 15/20 °C and 15/25 °C on soybean seeds. Fumonisin B₁ was produced only in culture medium, and the maximum level (7.38 µg g^?1) was found at 15 °C after 7 days of incubation. CONCLUSION: When growth and mycotoxin production under cycling temperatures were predicted from the results under constant conditions, observed values were different from calculated for both species and substrate medium. Therefore, care should be taken if data at constant temperature conditions are to be extrapolated to real field conditions. Copyright © 2012 Society of Chemical Industry     

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.     

FTA滤膜用于PCR检测单核细胞增生李斯特氏菌的研究

建立单核细胞增生李斯特氏菌(Listeria moncytones,LM)快速、敏感、特异的PCR检测方法.利用FTA滤膜提取模板DNA,采用PCR特异性扩增单增李斯特菌的溶血素基因(HIyA),并评价该方法的特异性与灵敏性.引物能特异性的扩增单增李斯特的HIyA基因,而其他细菌的扩增结果均呈现阴性:利用FTA滤膜提取模板直接检测单增李斯特具有较高的灵敏度,灵敏度为l 02 cfu/mL.利用FFA滤膜提取模板,操作简便,成本低且具有较高的灵敏度,为食品中单核细胞增生李斯特氏菌的快速检测提供新的手段.     

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.     

Validated PCR assay for the routine detection of Salmonella in food

We developed a rapid and reliable PCR assay with genus-specific primers for the detection of Salmonella in food samples. With these primers, no primer-specific amplicons were detected when challenged with cultures of microorganisms other than salmonellae, and positive results, i.e., Salmonella-specific bands, were obtained with pure cultures of all 125 Salmonella isolates tested, which represented 100 serovars. The PCR assay was optimized using both pure cultures and artificially inoculated food samples. The assay results were compared with those of the Australian standard culture methods, using more than 500 "naturally" contaminated food samples, over a period of 9 years. Food samples were subjected to nonselective preenrichment in buffered peptone water followed by selective enrichment in Rappaport Vassiliadis (RV) broth and mannitol selenite cystine (MSC) broth. A simple sample preparation method was developed based on concentrating bacterial cells from 1 ml of RV or MSC broths. The PCR results were in perfect agreement with the results of the standard culture methods; no false-positive or false-negative results were obtained. However, the PCR assay was extremely rapid, and results could be obtained within 4 h of testing of enrichment broths.     

免疫磁捕获-荧光定量PCR快速检测食品中沙门氏菌

建立了免疫磁珠分离（Immunomagnetic separation,IMS）联合荧光定量PCR（Real-time PCR）技术准确、快速检测食品中沙门氏菌（Salmonella spp）的方法。采用亲和纯化的羊抗沙门氏菌抗体与Dynabeads M-280磁珠制备沙门氏菌免疫磁珠,优化反应条件,建立免疫磁珠分离方法。同时根据沙门氏菌特异性ttr基因,建立Real-time PCR体系,并检测其特异性及敏感性。结果显示,沙门氏菌可产生荧光信号,而其他细菌均未见明显荧光信号。利用所建立的免疫磁捕获-荧光定量PCR（IMS-real-time PCR）方法可以检测初始含菌量最低为6.5CFU/25g的食品样品,全过程需时约8h。150份实际食品样品中,IMS-real-time PCR方法检出27份阳性样品,免疫磁珠联合XLT4平板培养法检出18份阳性样品,传统国标法检出14份阳性样品。      

Kinetics of fumonisin B1 formation in maize ears inoculated with Fusarium verticillioides

The kinetics of fumonisin B₁ (FB₁) biosynthesis have been examined in ears of four botanical varieties Zea mays var. indentata, Zea mays var. indurata, Zea mays var. saccharata and Zea mays var. everta inoculated with F. verticillioides isolates at silking stage. The level of mycotoxin accumulated in kernels was correlated with sample harvest time (r?=?0.73) and ergosterol content (r?=?0.70). FB₁ biosynthesis was influenced by amylose, starch and moisture contents, which undergo dynamic changes during grain formation. A comparative analysis of ear infection rates and efficiency of FB₁ biosynthesis showed that these are separate characters and their expression could be independent.     

PCR法快速检测耐热霉菌雪白丝衣霉

以耐热性霉菌——雪白丝衣霉为研究对象,根据Gen-bank公布的beta-tubulin基因序列,设计两对引物,经过PCR筛选,确定引物Bn-1用于建立雪白丝衣霉的PCR检测方法。对PCR法的扩增条件退火温度和引物浓度进行优化,59℃的退火温度和0.2μM/PCR反应体系的引物浓度为最佳条件;考察该PCR方法的灵敏度和特异性,基因组DNA的灵敏度为1ng/PCR反应体系,检测特异性好。该方法为雪白丝衣霉的快速检测提供了一种重要的技术手段。     

6类致泻大肠埃希氏菌多重PCR检测体系的建

目的 构建食源性致泻大肠埃希氏菌高效、快速、特异性强的检验方法。方法 根据6类致泻大肠埃希菌11种毒力基因引物, 采用煮沸法制备细菌基因组DNA作为模板构建致泻大肠埃希氏菌单菌多重PCR检测体系; 采用试剂盒法制备细菌基因组DNA作为模板构建6类菌十重PCR检测体系, 对2种检测体系进行优化并对其进行特异性、重复性验证。结果 构建了6类致泻大肠埃希氏菌多重PCR检测体系, 包括单菌多重PCR、6类菌十重PCR检测体系。仅6类致泻大肠埃希氏菌阳性菌株有特异性条带产生; 不同类型致泻大肠埃希氏菌菌株均检出其特异性阳性条带, 该多重PCR检测方法重复性好, 稳定性强。结论 该方法可高效快速地确定待测样是否为大肠埃希氏菌及其致病型, 为食品中致泻大肠埃希氏菌的监控、预警及爆发引起食物中毒后溯源等提供参考。     

A multiplex ligation detection assay for the characterization of Salmonella enterica strains

A proof of principle of a multi-target assay for genotyping Salmonella has been developed targeting 62 genomic marker sequences of Salmonella related to pathogenicity. The assay is based on multiplex ligation detection reaction (LDR) followed by customized ArrayTube? microarray detection. The feasibility of the developed assay was verified in a method comparison study with conventional PCR using 16 Salmonella 'test' strains comprising eight serovars. Subsequently, the feasibility of the LDR microarray assay was also tested by analyzing 41 strains belonging to 23 serovars. With the exception of four serovars each serovar was characterized by a unique virulence associated gene repertoire. The LDR microarray platform proved to be a convenient, rapid and easy to use tool with potential in tracing a Salmonella contamination in the food chain, for outbreak studies, and to provide data for risk assessors that support bio-traceability models.     

Development of a multiplex real-time PCR assay for the detection of ruminant DNA

The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test.     

Combined PCR and slot blot assay for detection of Salmonella and Listeria monocytogenes

Detection of Salmonella typhimurium and Listeria monocytogenes by the polymerase chain reaction (PCR) assay coupled with slot blot detection was investigated in this study. After being extracted from diluted bacterial culture with the extraction buffer, bacterial DNA was subjected to PCR. The slot blot assay was optimized and used to detect PCR products. The lowest detection level of this method was 10(3) cfu/ml in the original culture media for both pathogens, or 5 bacterial cells in the PCR reaction. Combined with immunomagnetic separation (IMS) to separate and concentrate bacteria from samples, the detection limit could be 40 cfu/ml of bacteria from milk samples. The whole detection procedure was completed within 7 h. After multiplex PCR (amplification of DNA from two different bacteria in the same PCR tube) and slot blot, a detection level of 10(3) cfu/ml was achieved in the simultaneous detection for both pathogens, which was similar to that of individual detection for each pathogen. The combination of PCR and slot-blot seems to be highly sensitive and time-efficient, and is therefore promising for routine use in the detection of Salmonella and L. monocytogenes in food samples such as milk.