Fermentation in 24-well plates is an efficient screening platform for filamentous fungi.

UNLABELLED: Fermentation by filamentous fungi in Erlenmeyer flasks is a favoured method for comparing different fermentation conditions. However, significant inter-flask variation often occurs when using Erlenmeyer flasks, which makes the comparison of fermentation product levels less reliable. We have investigated the use of a 24-well plate method for citric acid, ethanol and glycerol batch fermentation using the filamentous fungi Aspergillus carbonarius and compared the relative standard deviation (RSD) from sextuplicates obtained using Erlenmeyer flasks and 24-well plates. The production levels using the Erlenmeyer flasks showed a combined average RSD of 29%, which is two and a half-fold higher than what was measured using the 24-well plates showing an average RSD of 12%. We conclude that fermentation in 24-well plates is a more reliable screening method for metabolite production by filamentous fungi and possibly for screening metabolites in general. SIGNIFICANCE AND IMPACT OF THE STUDY: Fermentation studies with filamentous fungi and especially screening experiments often struggle with high inter-vessel variations in metabolite production. This study compares two different types of frequently used screening methods namely batch fermentation in Erlenmeyer flasks with batch fermentation in 24-well plates. The results demonstrate that the variance potentially can be reduced two and a half-fold using 24-well plates leading to improved resolution when testing the impact of varying fermentation parameters on product formation.

A real-time PCR assay for the specific identification of serotype O:9 of Yersinia enterocolitica.

A real-time PCR assay was developed based on a 181-bp fragment of the recently cloned per gene, including an internal amplification control (124 bp), for the detection of Yersinia enterocolitica O:9 (Ye O:9). The validation included 48 Ye O:9, 33 Y. enterocolitica non-O:9 and 35 other closely-related bacterial strains, containing per gene homologies. The assay was specific for the Ye O:9 tested, the detection limit was 1-10 genome copies of purified DNA and amplification efficiency was between 90.5-103%, indicating a linear regression throughout the detection window.

Towards an international standard for PCR-based detection of foodborne thermotolerant campylobacters: interaction of enrichment media and pre-PCR treatment on carcass rinse samples.

As part of a large EU project for standardisation of polymerase chain reaction (PCR), a systematic evaluation of the interaction of enrichment media, type of DNA polymerase and pre-PCR sample treatment for a PCR detecting thermotolerant campylobacters was carried out. The growth-supporting capacity and PCR compatibility of enrichment in Preston, Mueller-Hinton and Bolton broth (blood-containing and blood-free) were evaluated. The effect of resin-based DNA extraction and DNA extraction by boiling on the final PCR assay was investigated. The time-course studies indicated that a 20-h sample enrichment in blood-containing Bolton broth, followed by a simple resin-based extraction of DNA and a PCR amplification using Tth polymerase, resulted in strong and clear PCR amplicons for target (287 bp) and internal amplification control (IAC, 124 bp). The enrichment PCR-based method, tested on 68 presumably naturally contaminated poultry-rinse samples, showed a diagnostic sensitivity of 97.5% (39 PCR-positive/40 total positive samples) and a diagnostic specificity of 100% (28 PCR-negative/28 total negative samples; P=0.32) when compared to a standard bacteriological method (ISO 10272).

Validated 5' nuclease PCR assay for rapid identification of the genus Brucella.

A real-time, genus-specific 5' nuclease PCR assay for amplification of a 322-bp fragment of the per gene was developed for rapid (<2 h) identification of Brucella spp. from agar plates. The assay, including an internal amplification control (116 bp), identified Brucella strains (n = 23) and did not detect non-Brucella strains (n = 174), indicating its usefulness, particularly for laboratories with stringent quality assurance programs.

Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation.

As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.

Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: validation in a multicenter collaborative trial.

As part of a European research project, the performance of a PCR assay to detect food-borne thermotolerant campylobacters (Campylobacter jejuni, C. coli, and C. lari) was evaluated through an international collaborative trial involving 12 participating laboratories. DNA from 10 target and 8 nontarget strains was tested, and the results were reported as the presence of a positive signal after gel electrophoresis. The overall inclusivity (sensitivity) was 93.7%, and the exclusivity (specificity) was 100%. The results indicate that the assay can become an international standard and can be confidently applied in microbiological laboratories.

Preston and Park-Sanders protocols adapted for semi-quantitative isolation of thermotolerant Campylobacter from chicken rinse.

Human campylobacteriosis has become the major cause of foodborne gastrointestinal diseases in several European countries. In order to implement effective control measures in the primary production, and as a tool in risk assessment studies, it is necessary to have sensitive and quantitative detection methods.Thus, semi-quantitative detection of thermophilic Campylobacter spp. in 20 naturally contaminated chicken rinse samples was carried out using the two most common standard protocols: Preston and Park-Sanders, as proposed by Nordic Committee on Food Analysis (NMKL) and International Standard Organization (ISO), respectively. For both protocols, the chicken rinse samples were prepared in 500 ml buffered peptone water, as recommended in the ISO protocol no. 6887-2. The results indicated that the Preston protocol was superior to the Park-Sanders protocol in supporting growth of Campylobacter spp. In conclusion, the established semi-quantitative assessment using Preston broth could be useful in monitoring the outcome of control programs or quantitative risk assessments.

Characterisation of symbionts of entomopathogenic nematodes by universally primed-PCR (UP-PCR) and UP-PCR product cross-hybridisation.

This work introduces and demonstrates the applicability of universally primed-PCR (UP-PCR) for differentiating bacterial symbionts of entomopathogenic nematodes. Furthermore, typing by UP-PCR product cross-hybridisation was successfully introduced to cluster the bacterial strains. The work was initiated by isolating 10 isolates of Photorhabdus temperata (S172) from the nematode host Heterorhabditis sp. (DK172) and 12 isolates of Xenorhabdus bovienii (S1) from the nematode Steinernema feltiae (DK1). The isolates were compared by UP-PCR using different primers. The two bacterial species (P. temperata and X. bovienii) could be distinguished on the basis of the banding pattern whereas isolates isolated from the same nematode host had identical banding patterns. Three isolates obtained from DK172 and DK1, respectively, were then selected along with a number of reference strains (Hb, HP88, C1, K122, HSH2, HL81, T228, 61, AN6, Q58) and further characterised by UP-PCR product cross-hybridisation. The Xenorhabdus strains (Q58, AN6, 61, T228, S1) represented three species and these species were separated by the hybridisation technique. The Photorhabdus strains (Hb, HP88, C1, K122, HSH2, HL81, S172) represented two species and were also separated according to this in the cross-hybridisation. Within each species of Photorhabdus, two subgroups were formed as a result of intensity of the hybridisation signals. This grouping was in agreement with previous studies in other laboratories.

Identification of a universally primed-PCR-derived sequence-characterized amplified region marker for an antagonistic strain of Clonostachys rosea and development of a strain-specific PCR detection assay.

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.

Identification of Trichoderma strains from building materials by ITS1 ribotyping, UP-PCR fingerprinting and UP-PCR cross hybridization.

To study the role of Trichoderma in sick building syndrome, it is essential to be able to accurately identify species. Forty-four strains of Trichoderma spp. isolated from Danish buildings damaged by water leaks were identified using ITS1 ribotyping and universally primed PCR, UP-PCR. Ribotyping allowed the assignment of the strains into three distinct groups. High similarity of UP-PCR banding profiles of the strains allowed species designation for almost all strains (43 out of 44) when compared with the UP-PCR banding profiles obtained from reference strains of T. atroviride, T. citrinoviride, T. harzianum, T. longibrachiatum and T. viride. However, cross hybridization of UP-PCR products showed that the latter strain had high DNA homology to the ex-type strain of T. hamatum. The combined approach is a convenient way for reliable identification of Trichoderma strains.

Mechanisms of Induced Resistance in Barley Against Drechslera teres.

ABSTRACT Quantitative and qualitative histopathological methods and molecular analyses were used to study the mechanisms by which preinoculation with either of the nonbarley pathogens, Bipolaris maydis and Septoria nodorum, inhibited growth of Drechslera teres. Collectively, our data suggest that induced resistance is the principal mechanism responsible for impeding the pathogen. The enhancement of resistance in the host was primarily manifested during penetration by D. teres, and after penetration, where growth of D. teres ceased soon after development of infection vesicles. Thus, 24 h after pretreatment with B. maydis or S. nodorum, the penetration frequency from D. teres appressoria was reduced from 42.7% in the controls to 9.5 and 14.8%, respectively. The reductions were associated with increased formation of fluorescent papillae in induced cells (early defense reaction). The postpenetrational inhibition of D. teres completely stopped fungal growth and was apparently linked to an enhancement of multicellular hypersensitive responses in inducer-treated leaves (late defense reaction). Papillae formation and multicellular hypersensitive reactions were also observed in fully susceptible, noninduced control leaves, but they were inadequate to stop fungal progress. Northern blots from leaves treated with either inducer alone support the conclusion that induced resistance is involved in suppression of D. teres by increased formation of papillae and hypersensitive reactions. Thus, the blots showed strong expression of several defense response genes that are involved in these reactions in barley attacked by Erysiphe graminis f. sp. hordei.