Efficient Detection of Leptosphaeria maculans from Infected Seed lots of Oilseed Rape

An efficient DNA extraction protocol and polymerase chain reaction (PCR) assay for detecting Leptosphaeria maculans from infected seed lots of oilseed rape were developed. L. maculans, the causal agent of blackleg, a damaging disease in oilseeds rape/canola worldwide, was listed as a quarantine disease by China in 2009. China imports several millions of tons of oilseeds every year. So there is a high risk that this pathogen will be introduced to China via contaminated seeds. Seed contamination is one of the most significant factors in the global spread of phytopathogens. Detection of L. maculans in infected seed lots by PCR assay is difficult due to the low level of pathogen mycelium/spores on seeds and PCR inhibitors associated with the seeds of oilseed rape. In our study, these two major obstacles were overcome by the development of a two‐step extraction protocol combined with a nested PCR. This extraction protocol (kit extraction after CTAB method) can efficiently extract high‐quality DNA for PCR. Amplification results showed that the detection threshold for conventional PCR and nested PCR was, respectively, 1 ng and 10 fg of DNA per μl in mycelia samples. On contaminated seed lots of oilseed rape, the detection threshold of conventional and nested PCR was 709 fg/μl and 709 ag/μl of DNA, respectively. The DNA extraction protocol and PCR assay developed in this study can be used for rapid and reliable detection of L. maculans from infected seeds of oilseed rape .     

Detection of Yersinia enterocolitica in food by PCR amplification

A polymerase chain reaction (PCR) assay was developed for detection of pathogenic, virulent strains of Yersinia enterocolitica . By using both virulence loci virF and ail as markers for pathogenicity, detection of species with a virulence factor present was possible. DNA preparation in the presence of hexadecyl trimethy ammonium bromide (CTAB) was followed by two 44 cycle amplification reactions, one for each of the markers. As few as 10² Y. enterocolitica cells were detected in ground pork in the presence of 10⁵–10⁶ bacteria of other species. The described PCR assay provides a sensitive robust assay for the detection of virulent Y. enterocolitica in food.     

Detection of the defoliating and nondefoliating pathotypes of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in artificial and natural soils by nested PCR

In Spain, Verticillium wilt, caused by Verticillium dahliae, is the most important disease of cotton and olive. Isolates of V. dahliae infecting these crops can be classified into highly virulent, defoliating (D), and mildly virulent, nondefoliating (ND), pathotypes. Infested soil is the primary source of inoculum for Verticillium wilt epidemics in cotton and olive, and severity of disease relates to the prevailing V.dahliae pathotype. In this work we have adapted the use of previously developed primer pairs specific for D and ND V. dahliae for the detection of these pathotypes by nested PCR in artificial and natural soils. Success in the detection procedure depends upon efficiency in extracting PCR-quality DNA from soil samples. We developed an efficient DNA extraction method from microsclerotia infesting the soil that includes the use of acid washed sand during the grinding process and skimmed milk to avoid co-purification of Taq-polymerase inhibitors with DNA. The specific nested-PCR procedure effectively detected 10 or more microsclerotia per gram of soil. The detection procedure has proven efficient when used with a naturally infested soil, thus demonstrating usefullness of the diagnostic method for rapid and accurate assessment of soil contamination by V. dahliae pathotypes.     

PCR-sexing of bovine embryos: a simplified protocol

To make bovine embryo sexing under farm conditions more feasible we developed a simplified protocol utilizing manual biopsy and detection of the Y chromosome directly from polymerase chain reaction (PCR) reaction tubes. Twenty-four embryos (morulae and blastocysts) were biopsied manually into 2 to 4 samples. One sample of each original embryo was diagnosed for sex, based on restriction fragment length polymorphism of PCR-amplified DNA of the ZFX/ZFY locus. The remaining 44 samples were diagnosed using the tube detection assay. In this assay the biopsies were pipetted into 0.5 -ml reaction tubes containing lysis mixture, incubated 10 to 60 min at 37 degrees C and inactivated 10 min at 98 degrees C. Then the PCR mixture was added containing buffer, DNA polymerase, ethidium bromide and primers designed to amplify the highly repeated btDYZ-1 region of the bovine Y chromosome. After 50 cycles of PCR, the reaction tubes were examined under UV illumination for pink fluorescence indicating the presence of Y-chromosomal DNA. All sexing results from the replicates were in agreement with the ZFX/ZFY assay, with 12 of the original embryos diagnosed as females and 12 as males. We conclude that highly efficient and accurate PCR-sexing of embryos can be accomplished without the use of micromanipulators, control primers and electrophoresis. The 2 reaction mixtures needed for sex diagnosis can be stored at -20 degrees C and -196 degrees C, respectively. The tube detection assay minimizes the risk of carryover contamination by previously amplified products as there is no need to open the tubes following PCR.     

Detection of enterotoxigenic Clostridium perfringens in meat samples by using molecular methods

To prevent food-borne bacterial diseases and to trace bacterial contamination events to foods, microbial source tracking (MST) methods provide important epidemiological information. To apply molecular methods to MST, it is necessary not only to amplify bacterial cells to detection limit levels but also to prepare DNA with reduced inhibitory compounds and contamination. Isolates carrying the Clostridium perfringens enterotoxin gene (cpe) on the chromosome or a plasmid rank among the most important food-borne pathogens. Previous surveys indicated that cpe-positive C. perfringens isolates are present in only ～5% of nonoutbreak food samples and then only at low numbers, usually less than 3 cells/g. In this study, four molecular assays for the detection of cpe-positive C. perfringens isolates, i.e., ordinary PCR, nested PCR, real-time PCR, and loop-mediated isothermal amplification (LAMP), were developed and evaluated for their reliability using purified DNA. For use in the artificial contamination of meat samples, DNA templates were prepared by three different commercial DNA preparation kits. The four molecular assays always detected cpe when >103 cells/g of cpe-positive C. perfringens were present, using any kit. Of three tested commercial DNA preparation kits, the InstaGene matrix kit appeared to be most suitable for the testing of a large number of samples. By using the InstaGene matrix kit, the four molecular assays efficiently detected cpe using DNA prepared from enrichment culture specimens of meat samples contaminated with low numbers of cpe-positive C. perfringens vegetative cells or spores. Overall, the current study developed molecular assay protocols for MST to detect the contamination of foods with low numbers of cells, and at a low frequency, of cpe-positive C. perfringens isolates.     

Development of an F57 sequence-based real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk

A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M.avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratuberculosis isolates and 33 other bacterial strains. The LC-PCR assay has a broad linear range (2 x 10(1) to 2 x10(6) copies) for quantitative estimation of the number of M. avium subsp. paratuberculosis F57 target copies in positive samples. To maximize the assay's detection sensitivity, an efficient strategy for isolation of M. avium subsp. paratuberculosis DNA from spiked milk samples was also developed. The integrated procedure combining optimal M. avium subsp. paratuberculosis DNA isolation and real-time PCR detection had a reproducible detection limit of about 10 M. avium subsp. paratuberculosis cells per ml when a starting sample volume of 10 ml of M. avium subsp. paratuberculosis-spiked milk was analyzed. The entire process can be completed within a single working day and is suitable for routine monitoring of milk samples for M. avium subsp. paratuberculosis contamination. The applicability of this protocol for naturally contaminated milk was also demonstrated using milk samples from symptomatic M. avium subsp. paratuberculosis-infected cows, as well as pooled samples from a dairy herd with a confirmed history of paratuberculosis.     

Rapid and Sensitive Detection of Xanthomonas fragariae by Simple Alkaline DNA Extraction and the Polymerase Chain Reaction

Methods for DNA preparation from Xanthomonas fragariae in infected or artificially contaminated strawberry plants were compared in diagnostic assays using the polymerase chain reaction (PCR). The bacterium was detected using PCR with primers specific to a region of its hrp gene. Sensitivity of detection was 1.25 ×l 10³ CFU ml^-1 using DNA from bacterial suspensions prepared by an alkali extraction method. This was 10-fold more sensitive than DNA extraction by boiling, and was equal to that in which DNA was prepared by a more involved cetyltrimethylammonium bromide (CTAB) procedure. Sensitivity of detection from artificially contaminated strawberry tissues was 10-fold less than that from cell suspensions. The results indicated that a rapid and simple method of alkali DNA sample preparation is applicable for the sensitive and reliable detection of X. fragariae and possibly other plant pathogenic bacteria.     

Detection of Acidovorax valerianellae, the causing agent of bacterial leaf spots in corn salad [Valerianella locusta (L.) Laterr.], in corn salad seeds

Aim: The black leaf spot disease on corn salad caused by the bacterium Acidovorax valerianellae has been observed in Europe for several years and causes economic losses in corn salad cropping. Contaminated seeds or infested soil are considered as the major infection sources. The use of healthy seed material is the only way to prevent disease outbreaks. Therefore, a sensitive diagnostic method for seed testing should be developed. Methods and Results: Using a triple antibody sandwich ELISA with a high‐specific monoclonal antibody, a quick and reliable detection method for contamination of seed lots with the pathogen was developed. This method allowed to detect contaminated seed lots as well as contamination with A. valerianellae in single seeds. Furthermore, the occurrence and distribution of the pathogen could be shown in symptomatic corn salad leaves and in naturally infested seeds by transmission electron microscopy and immunogold labelling for the first time. Conclusion: Our results confirm the seed transmission of this corn salad disease. Pathogen load and distribution vary between positively tested seed lots. Significance and Impact of the study: With this method, not only routine testing of seed material to eliminate contaminated seed lots from production is possible but also the control of sanitation procedures to reduce contamination.     

Evaluation of PCR assays for quantifying seed-borne infection by Fusarium and Microdochium seedling blight pathogens

Aims:  To evaluate competitive PCR assays for quantifying seed-borne Microdochium and Fusarium seedling blight pathogen DNA and to determine test and year repeatability and sources of variability.
Methods and Results:  Relationships between DNA and plate counts were significant for Fusarium and Microdochium seedling blight pathogens in 152 seed batches from 3 years. Coefficient of determinations, however, differed greatly ( Fusarium ; R ² = 0·25, P  =   0·029, Microdochium ; R ² = 0·73, P  <   0·001). Significant differences between years were observed in the regression slopes for Microdochium . Pathogen DNA quantified in 16 extractions after sampling was highly correlated to results following storage for 1–2 years ( R ² > 0·90). Residual maximum likelihood analysis showed that the least and greatest variance components of the testing procedure were DNA extraction subsampling and PCR assay respectively.
Conclusions:  Amount of pathogen DNA is a useful estimator of seed batch contamination for Microdochium but not Fusarium seedling blight pathogens. Although reproducible over time, improvements to the testing procedure should focus on repeated PCR amplifications to reduce assay variability.
Significance and Impact of the Study:  Replacing plate counts with competitive PCR for determining the severity of seed batch contamination is feasible in areas where Microdochium seedling blight pathogens predominate.     

Detection of yeast by PCR in sucrose solutions using a sample preparation method based on filtration

An assay based on PCR was developed for the detection of yeast in naturally contaminated industrial sugar solutions. A number of characterized as well as non-characterized yeast strains, isolated from samples from a sugar refinery, were detected with the PCR assay. Specificity tests showed that the presence of neither bacteria nor mould resulted in false positive results. A concentration step, based on filtration, was employed prior to the PCR detection in order to reach a detection level of 0.1 c.f.u./ml of naturally contaminated sugar solution. The detection method based on PCR was found to be more rapid (<5 h) and easier to perform than the slower and more labour-intensive, traditional culturing techniques.     

Direct detection and isolation of plasmid-bearing virulent serotypes of Yersinia enterocolitica from various foods.

A procedure was developed for direct detection, isolation, and maintenance of plasmid-bearing virulent serotypes of Yersinia enterocolitica from different food sources. Plasmid-bearing virulent strains of Y. enterocolitica representing five serotypes were simultaneously detected and isolated from enriched swab samples of artificially contaminated pork chops, ground pork, cheese, and zucchini, using Congo red binding and low-calcium-response tests. The method was also effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Virulence of the strains isolated from these foods was confirmed by PCR, the expression of plasmid-associated phenotypes, and mouse pathogenicity.     

Rapid-Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in Environmental Samples

In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.     

Multiplexed, particle-based detection of DNA using flow cytometry with 3DNA dendrimers for signal amplification.

BACKGROUND: Complex mixtures of DNA may be found in environmental and medical samples. There is a need for techniques that can measure low concentrations of target DNAs. For a multiplexed, flow cytometric assay, we show that the signal-to-noise ratio for fluorescence detection may be increased with the use of 3DNA dendrimers. A single fluorescent DNA molecule per bead could be detected with conventional flow cytometry instrumentation. METHODS: The analyte consisted of single-stranded (ss) DNA amplicons that were hybridized to capture probes on the surface of fluorescent polystyrene microspheres (beads) and initially labeled with streptavidin-R-phycoerythrin (single-step labeling). These beads have a low reporter fluorescence background and high efficiency of DNA hybridization. The DNA/SA-RPE complex was then labeled with 3DNA dendrimers and SA-RPE. The bead complexes were detected with a Luminex 100 flow cytometer. Bead standards were developed to convert the intensity to the number of SA-RPE labels per bead and the number of dendrimers per bead. RESULTS: The dendrimer assay resulted in 10-fold fluorescence amplification compared with single-step SA-RPE labeling. Based on concentration curves of pure target ss-amplicons, the signal-to-noise ratio of the dendrimer assay was greater by a factor of 8.5 over single-step SA-RPE labeling. The dendrimer assay was tested on 16S ribosomal DNA amplified from filter retentates of contaminated groundwater. Multiplexed detection of a single dendrimer-labeled DNA molecule per bead was demonstrated. CONCLUSIONS: Multiplexed detection of DNA hybridization on a single molecule level per bead was achieved with conventional flow cytometry instrumentation. This assay is useful for detecting target DNAs at low concentrations.     

A novel DNA-based diagnostic test for the detection of annual and intermediate ryegrass contamination in perennial ryegrass

Perennial ryegrass (Lolium perenne L.) is a preferred choice for the turf grass industry due to its ability to provide a durable turf cover. Genetic or physical contamination of annual (L. multiflorum Lam.) or intermediate (L. hybridum) ryegrass species in perennial ryegrass is one of the major problems affecting the grass seed industry. At present, seedling root fluorescence (SRF), a biochemical marker, is used for the detection of annual ryegrass contamination. Due to the unreliability of the SRF test, the seed industry is seeking an alternative, more reliable and accurate detection method. Currently, there are no DNA tests available in ryegrass for detecting contamination with annual and intermediate ryegrass types. We developed a novel quantitative polymerase chain reaction (Q-PCR)-based DNA test for the detection of annual and/or intermediate ryegrass types in perennial ryegrass. This DNA test was designed using an insertion/deletion (InDel) site in the LpVRN2_2 (Vernalization 2) gene, which is one of the several genes controlling vernalization in ryegrass. The new DNA test is more reliable, accurate and cost-effective in detecting contamination, with a high sensitivity of 0.04% in a sample size of 5,000 seeds. Use of larger sample sizes (12.5-fold higher compared to SRF test) provided additional accuracy in detecting the level of contamination. The method has produced consistent results in 68 perennial, 26 annual and 14 intermediate ryegrass lines.     

Detection of Seed-Transmitted Brome Mosaic Virus by ELISA, Radial Immunodiffusion and Immunoelectroblotting Tests

The extent of contamination of wheat seedlots by brome mosaic virus (BMV), and the possible transmission of the virus through seed, were investigated by radial immunodiffusion (RID), enzyme linked immunosorbent assay (ELISA), and enzyme-assisted immunoelectroblotting (IEB) tests. BMV was purified from seed washings, and from plants grown from contaminated seed. All three serological tests proved of value in detecting contaminated seeds, while ELISA and IEB were especially useful in detecting virus in infected plants grown from such seed. Rhopalosiphum padi aphids were shown to increase the incidence of BMV infectLon in seedling batches containing a few seed-infected plants. The implications of these findings for wheat breeding schemes are discussed.     

Detection of multiple virulence-associated genes of Listeria monocytogenes by PCR in artificially contaminated milk samples.

The inhibitory effect of milk in the PCR detection of Listeria monocytogenes could be overcome by washing the contaminated milk sample with phosphate-buffered saline and concentrating the bacteria to 1/10 of the original volume. In order to avoid a possible failure in the detection of virulent L. monocytogenes, a one-step procedure which enabled demonstration of three virulence-associated genes, prfA, hlyA, and plcB, simultaneously in a single PCR mixture was developed.     

Development of a PCR assay combined with a short enrichment culture for detection of Campylobacter jejuni in estuarine surface waters

Abstract Two extraction procedures were examined, and it was found that DNA recovered from Campylobacter jejuni lysed by the cetyltrimethylammonium bromide (CTAB) method was more suitable for use as a PCR template than DNA released by the boiling method. The region targeted for PCR amplification was a 1.73-kb portion of the flagellin A gene of C. jejuni . The detection limit was lower than 30 cells per 100 ml in artificially contaminated waters. PCR assay and conventional culturing method had the same sensitivity, but results of the PCR technique were available within 48 h and so shortened the time necessary for detection by 48 h.     

Optimization of a radiolabel DNA-binding assay in cultured mammalian cells

An improved protocol for the radiolabel DNA-binding assay, which gives a high yield of highly pure DNA has been developed by use of mouse lymphoma cells. The critical difference from previously published methods is the use of enzymatic degradation of proteins in the later DNA purification steps rather than during the homogenisation procedure. Different DNA-purification methodologies were first compared and the protocol of choice was optimized later on; both steps were performed with [(35)S]-labelled amino acids for labelling of cellular protein, which enabled both the quantification of cellular protein contaminating the DNA sample and the distinction between cellular and enzyme-derived protein. The assay was later evaluated and shown to give reproducible results based on the data obtained with benzo[a]pyrene (B[a]P) and doxorubicin in two different laboratories. In addition, two further reference compounds, dopamine and diazepam and one proprietary AstraZeneca compound were also tested in mouse lymphoma cells in one laboratory. The two compounds B[a]P and doxorubicin were identified as suitable positive controls for routine testing in the presence and absence of S9, respectively. Exposing 90-100×10(6) cells to (14)C-labelled compound with a molar radioactivity of 2MBq/μmol, yields approximately 500μg DNA with <3% total protein contamination, of which approximately 7% is of cellular origin (<0.2%). The detection level is approximately 2adducts/10(8) dNTP.     

Loop-mediated isothermal amplification assay for detection and discrimination of Toxocara canis and Toxocara cati eggs directly from sand samples

We developed a novel and simple method, using loop-mediated isothermal amplification (LAMP), for the detection and discrimination of Toxocara canis and Toxocara cati eggs. The new method employs 4 steps: (1) concentration of Toxocara eggs in a small amount of sand; (2) dissolution of the proteinaceous membrane of eggs and simultaneously separation of them from the sand using NaClO treatment; (3) extraction of DNA using NaOH treatment; and (4) detection of T. canis / T. cati DNA using a LAMP assay. All these steps are fast, easy to perform, and do not require expensive equipment or reagents. The novel method was tested both experimentally and in a field study. In the laboratory, we could reliably detect as few as 3 T. canis eggs in artificially contaminated sand, if the experiment was repeated twice. In the field trial, we were able to detect T. cati DNA from 4 natural sandpits having moderate to heavy contamination, although not in a single lightly contaminated sandpit. All of the examined sandpits were found to be contaminated with eggs of T. cati, but none appeared to contain T. canis. Our new method could extract DNA from T. canis and T. cati eggs directly from sand samples as well as detect and distinguish these 2 species in a few easy steps, with markedly reduced time and expense.