Influence of inoculum and climatic factors on the severity of Fusarium head blight in German spring and winter barley

Fusarium head blight (FHB) of small cereals is a disease of global importance with regard to economic losses and mycotoxin contamination harmful to human and animal health. In Germany, FHB is predominantly associated with wheat and F. graminearum is recognised as the major causal agent of the disease, but little is known about FHB of barley. Monitoring of the natural occurrence of FHB on Bavarian barley revealed differences for individual Fusarium spp. in incidence and severity of grain infection between years and between spring and winter barley. Parallel measurement of fungal DNA content in grain and mycotoxin content suggested the importance of F. graminearum in winter barley and of F. langsethiae in spring barley for FHB. The infection success of these two species was associated with certain weather conditions and barley flowering time. Inoculation experiments in the field revealed different effects of five Fusarium spp. on symptom formation, grain yield and mycotoxin production. A significant association between fungal infection of grain and mycotoxin content was observed following natural or artificial infection with the type B trichothecene producer F. culmorum, but not with the type A trichothecene-producing species F. langsethiae and F. sporotrichioides. Trichothecene type A toxin contamination also occurred in the absence of significant damage to grain and did not necessarily promote fungal colonisation.     

Application of a novel pathogenicity marker in a multiplex real-time PCR method to assess total and pathogenic Vibrio vulnificus in food and environmental samples

Pathogenic species of Vibrio genus, including Vibrio vulnificus, constitute a great challenge for food control agencies and a threat for consumers. V. vulnificus can appear in bivalve mollusks such as oysters, clams, and mussels. In addition, water, sediment, and plankton, have been described as reservoirs for this pathogen which constitutes the leading cause of death by consuming seafood in the United States.The aim of this study was to develop and pre-validate a rapid and reliable multiplex real-time PCR (qPCR) method for total and pathogenic V. vulnificus detection. Peptone, Sodium Chloride, Cellobiose (PNC) broth, with and without Colistin (PNCC) was evaluated according to international methods (ISO). The capacity of these broths to recover low numbers of pathogenic V. vulnificus in the presence of high numbers of interfering microorganisms was assessed. Finally, were used for food and environmental samples enrichment.In addition three different DNA extraction protocols were compared, but one of them proved to be better than the others regarding DNA concentration and purity obtained, and also regarding Ct values and final fluorescence obtained by qPCR.A qPCR efficiency above 90% was obtained, covering five orders of magnitude. The complete method achieved low limit of detection (3 cfu/25 g). All quality parameters of the method (relative sensitivity, specificity, and accuracy) returned values over 90% after analyzing 45 spiked samples. These results were obtained for all the targets analyzed (vvhA, vcgC and pilF).In this study the complete qPCR method developed was applied to 28 natural samples including a wide variety of seafood types and environmental samples (water), but no positive samples were detected for either target.     

Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus in wheat flour by SYBR® Green quantitative PCR

Aflatoxins are important mycotoxins that represent a serious risk for human and animal health. These mycotoxins are mainly produced by Aspergillus flavus and Aspergillus parasiticus, two closely related species with different array of aflatoxins. In this work, two specific quantitative PCR (qPCR) assays were developed to detect and quantify both species in wheat flour using primers based on the multicopy ITS2 rDNA target sequence. The species specificity of the assays was tested in a wide range of strains of these species and others colonizing the same commodities. The sensitivity of the assay was estimated in 2.5 pg/reaction in both species. Discrimination capacity for detection and relative quantification of A. flavus and A. parasiticus DNA were analyzed using samples with DNA mixtures containing also other fungal species at different ratios. Both qPCR assays could detect spore concentrations equal or higher than 10⁶ spores/g in flour samples without prior incubation. These assays are valuable tools to improve diagnosis at an early stage and in all critical control points of food chain integrated in HACCP strategies.     

Comparison of bacterial community in matured and degenerated pit mud from Chinese Luzhou‐flavour liquor distillery in different regions

The bacterial community in the pit mud of a Luzhou‐flavour liquor distillery in different regions was analysed by combined polymerase chain reaction–denaturing gradient gel electrophoresis (PCR‐DGGE) and quantitative PCR (qPCR) in order to distinguish a matured and a degenerated pit mud, judged according to sensory and physicochemical characteristics. The phyla Firmicutes, Cloacimonetes, Actinobacteria, Proteobacteria, Synergistetes and Unclassified Bacteria were detected. Firmicutes predominated in the pit mud. The diversity and homogeneity of the bacterial community in the matured pit mud were superior to those in the degenerated pit mud in the same distillery. There were significant differences in the bacterial community structure between the matured and degenerated pit mud. Moreover, the bacterial community in the degenerated pit mud samples was similar, which indicated that the bacterial community in the degenerated pit mud did not change within the two different regions. However, the bacterial community in matured pit mud samples was different, demonstrating that there were visible differences in the bacterial community between the samples of matured pit mud collected from the Luzhou‐flavour liquor distilleries in the two different regions. Notably, the quantity of Actinobacteria in the matured and the degenerated pit mud was found to be different by quantitative analysis. Potentially, the Actinobacteria could serve as an indicator bacteria to distinguish between matured and degenerated pit muds. Copyright © 2016 The Institute of Brewing & Distilling     

定量PCR技术检测杀菌型产品中乳酸菌

目的利用定量PCR(quantitativePCR,qPCR)技术检测杀菌型产品中乳酸菌,并与标签标示相比较,查探产品中各类菌种的异同性及数量。方法以市售的发酵乳及含乳饮料作为研究对象,设计可区分产品中常见的6种乳酸菌特异性引物及探针,利用qPCR技术对方法的特异性、灵敏度进行验证,并建立标准扩增曲线;利用模拟样品对方法进行检验,最后对市场上购买的实际样品进行检测。结果除去干酪乳杆菌,其他5种引物都能特异性扩增出其目标菌,并且对其他的乳酸菌均无扩增现象;DNA检测的灵敏度可达到2～4pg;单一标准菌种扩增曲线线性较好,相关系数r2值在0.954～0.992之间;对模拟样品的检测,与平板法差异性比较,P值均大于0.05,无显著性差异。对市售样品进行检测,产品中标记的干酪乳杆菌、双歧杆菌和嗜热链球菌与检测结果存在不匹配现象。结论本研究建立的qPCR方法可快速、准确地对产品中德氏乳杆菌保加利亚亚种、嗜热链球菌、乳酸乳球菌、嗜酸乳杆菌、双歧杆菌的鉴定和定量的分析。     

Characterization by culture-dependent and culture-independent methods of the bacterial population of suckling-lamb packaged in different atmospheres

This study offers insight into the dynamics of bacterial populations in fresh cuts of suckling lamb under four different atmospheric conditions: air (A), and three Modified Atmosphere Packaging (MAP) environments, 15%O₂/30%CO₂/55%N₂ (C, commercial), 70%O₂/30%CO₂ (O), and 15%O₂/85%CO₂ (H) for 18 days. Microbial analyses by both conventional methods and PCR-DGGE were performed. Controversial and surprising results emerged from comparing both methods in relation to the genus Pseudomonas. Thus, conventional methods detected the presence of high numbers of Pseudomonas colonies, although PCR-DGGE only detected this genus in air-packaged samples. PCR-DGGE detected higher microbial diversity in the control samples (A) than in the modified atmospheres (C, O, H), having atmosphere H the fewest number of species. Brochothrix thermosphacta, LAB (Carnobacterium divergens and Lactobacillus sakei), and Escherichia spp. were detected in all the atmospheres throughout storage. Moreover, previously undescribed bacteria from lamb meat such as Enterobacter hormaechei, Staphylococcus equorum and Jeotgalicoccus spp. were also isolated in this study by DGGE. Additionally, qPCR analysis was used to detect and characterize strains of Escherichia coli. Virulence genes (stx₁, stx₂ and eae) were detected throughout storage in 97% of the samples. A high CO₂ atmosphere was the most effective packaging combination doubling storage time in comparison with commercial atmosphere.