PCR as a Screening Test for Invasive Aspergillosis in Haematological Patients: A Pilot Study

Invasive aspergillosis is a leading cause of morbidity and mortality in immunocompromised patients, particularly in individuals with haematological malignancy and in haematopoietic stem cell transplant recipients. Nowadays, the galactomannan (GM) assay has been widely used as an indication of invasive aspergillosis, even though the test is known to generate false-positive results. The aim of this study was to compare the performance of GM and real-time PCR (qPCR) to detected Aspergillus in blood samples obtained from high-risk haematological patients. Haematological patients were screened twice weekly with GM testing, which was performed by the Platelia ELISA kit. An additional sample of whole blood (4 ml) was obtained for the purpose of qPCR testing. Sixty-four samples from 12 patients with haematopoietic stem cell transplant or haematological malignancy were studied. The overall accordance between GM and qPCR tests was 96.9 % (62 samples). Only two samples showed contradictory results, with positive GM test and negative real-time PCR results. Based on the high concordance between GM and qPCR in terms of negative results, the main utility of qPCR could be in the confirmation of positive results seen with GM testing.     

Impact of gene stacking on gene flow: the case of maize

To respect the European labelling threshold for the adventitious presence of genetically modified organisms (GMOs) in food and feed, stakeholders mainly rely on real-time PCR analysis, which provides a measurement expressed as a percentage of GM-DNA. However, this measurement veils the complexity of gene flow, especially in the case of gene stacking. We have investigated the impact of gene stacking on adventitious GM presence due to pollen flow and seed admixture as well as its translation in terms of the percentage of GM-DNA in a non-GM maize harvest. In the case of varieties bearing one to four stacked events, we established a set of relationships between the percentage of GM kernels and the percentage of GM-DNA in a non-GM harvest as well as a set of relationships between the rate of seed admixture and the percentages of GM material in a non-GM harvest. Thanks to these relationships, and based on simulations with a gene flow model, we have been able to demonstrate that the number of events and the stacking structure of the emitting fields impact the ability of a non-GM maize producer to comply with given GM kernel or GM-DNA thresholds. We also show that a great variability in the rates of GM kernels, embryos and DNA results from seed admixture. Finally, the choice of a unit of measurement for a GM threshold in seed lots can have opposite effects on the ability of farmers to comply with a given threshold depending on whether they are crop or seed producers.     

Impact of genetic structures on haploid genome-based quantification of genetically modified DNA: theoretical considerations, experimental data in MON 810 maize kernels (Zea mays L.) and some practical applications

Real-time Polymerase Chain Reaction (PCR) based assays are widely used to estimate the content of genetically modified (GM) materials in food, feed and seed. It has been known that the genetic structures of the analyte can significantly influence the GM content expressed by the haploid genome (HG) % estimated using real-time PCR assays; this kind of influence is also understood as the impact of biological factors. The influence was first simulated at theoretical level using maize as a model. We then experimentally assessed the impact of biological factors on quantitative results, analysing by quantitative real-time PCR six maize MON 810 hybrid kernels with different genetic structures: (1) hemizygous from transgenic male parent, (2) hemizygous from transgenic female parent and (3) homozygous at the transgenic locus. The results obtained in the present study showed clear influences of biological factors on GM DNA quantification: 1% of GM materials by weight (wt) for the three genetic structures contained 0.39, 0.55 and 1.0% of GM DNA by HG respectively, from quantitative real-time PCR analyses. The relationships between GM wt% and GM HG% can be empirically established as: (1) in the case of the presence of a single GM trait: GM HG% = GM wt% × (0.5 ± 0.167Y), where Y is the endosperm DNA content (%) in the total DNA of a maize kernel, (2) in the case of the presence of multiple GM traits: GM HG% = N × GM wt% × (0.5 ± 0.167Y), where N is the number of GM traits (stacked or not) present in an unknown sample. This finding can be used by stakeholders related to GMO for empirical prediction from one unit of expression to another in the monitoring of seed and grain production chains. Practical equations have also been suggested for haploid copy number calculations, using hemizygous GM materials for calibration curves.     

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 .     

Development of a Real-time Polymerase Chain Reaction for the Detection of Fusarium oxysporum f. sp. basilici from Basil Seed and Roots

A real‐time polymerase chain reaction (PCR) assay using a TaqMan^® probe was developed to detect the causal agent of wilt and crown rot of basil from infected plants and seed in Italy. The aim of the study was to diminish testing time, previously performed using nested‐PCR, and to create the conditions for future automation. The sensitivity of the assay was shown to be similar to the detection limit of the available nested‐PCR procedure. The advantages of real‐time PCR system include halving of the testing time, as well as the ability to identify both internally and externally infected seed to the sensitivity of 1 pg of genomic DNA. The assay was able to detect the presence of the pathogen in infected seed up to a sensitivity of 24 (SD: ±10) CFU per 100 seeds.     

TaqMan real-time PCR versus four conventional PCR assays for detection of apple proliferation phytoplasma

A recently developed TaqMan real-time PCR assay for detection of apple proliferation phytoplasma was evaluated in comparison to four conventional PCR-based methods with the aim to assess its potential for research and routine applications. All five protocols were tested in parallel on the same DNA isolates obtained from orchard trees. The performance of the methods was evaluated by means of sensitivity, specificity, susceptibility to inhibition, handling effort, testing time, assay expenses, and potential risk for operator and environment. Compared to the conventional PCR methods, the TaqMan real-time PCR procedure combined the highest test sensitivity with the highest test specificity and was, above all, not susceptible to PCR inhibition. Furthermore, TaqMan real-time PCR had the simplest and fastest testing process, involving a minimum of handling steps. Its disadvantage is the high cost of consumables and reagents, exceeding that of a standard PCR procedure up to four-fold. However, the higher material costs could be compensated by considerably lower personnel costs and by saving expenses for hazardous waste disposal. Due to the simple testing procedure and the output of results as numeric data the TaqMan real-time PCR assay has a high potential for automation, and seems to represent the currently most suitable method for large-scale testing procedures.     

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock¹. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours^{2, 4}. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes⁵. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods⁶. This assay was based on a study in which PCR was used to measure the growth of bacteria⁷. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.     

Unexpected Diversity of Feral Genetically Modified Oilseed Rape (Brassica napus L.) Despite a Cultivation and Import Ban in Switzerland

Despite cultivation and seed import bans of genetically modified (GM) oilseed rape (Brassica napus L.), feral GM plants were found growing along railway lines and in port areas at four sites in Switzerland in 2011 and 2012. All GM plants were identified as glyphosate-resistant GM event GT73 (Roundup Ready, Monsanto). The most affected sites were the Rhine port of Basel and the St. Johann freight railway station in Basel. To assess the distribution and intra- and interspecific outcrossing of GM oilseed rape in more detail, we monitored these two sites in 2013. Leaves and seed pods of feral oilseed rape plants, their possible hybridization partners and putative hybrid plants were sampled in monthly intervals and analysed for the presence of transgenes by real-time PCR. Using flow cytometry, we measured DNA contents of cell nuclei to confirm putative hybrids. In total, 2787 plants were sampled. The presence of GT73 oilseed rape could be confirmed at all previously documented sampling locations and was additionally detected at one new sampling location within the Rhine port. Furthermore, we found the glufosinate-resistant GM events MS8xRF3, MS8 and RF3 (all traded as InVigor, Bayer) at five sampling locations in the Rhine port. To our knowledge, this is the first time that feral MS8xRF3, MS8 or RF3 plants were detected in Europe. Real-time PCR analyses of seeds showed outcrossing of GT73 into two non-GM oilseed rape plants, but no outcrossing of transgenes into related wild species was observed. We found no hybrids between oilseed rape and related species. GM plants most frequently occurred at unloading sites for ships, indicating that ship cargo traffic is the main entry pathway for GM oilseed rape. In the future, it will be of major interest to determine the source of GM oilseed rape seeds.     

Rapid detection and identification of the bacterium Pantoea stewartii in maize by TaqMan real-time PCR assay targeting the cpsD gene

Aims: The development and evaluation of a sensitive and specific TaqMan^® real-time polymerase chain reaction (PCR) for the detection and identification of Pantoea stewartii on maize. Methods and Results: A TaqMan^®-based real-time PCR assay targeting the cpsD gene enabling specific detection of P. stewartii in maize leaves and seeds was developed. Under optimal conditions, the selected primers and probe were specific for the detection of all 14 reference P. stewartii strains by real-time PCR. The 32 non-Panteoa and eight other Pantoea strains tested negative. The TaqMan^® PCR assay detected 1 pg of purified DNA and 10⁴P. stewartii colony forming units per millilitre (10 cells per reaction) in pure cultures consisting of 92·0% intact (viable) cells. Direct processing of leaf lesions and seeds by the real-time PCR detected 10 and 50 P. stewartii cells per reaction respectively. TaqMan^® real-time PCR results were validated by dilution plating of macerates and PCR-based subcloning followed by DNA sequencing. Conclusions: The real-time PCR assay described is a rapid, reliable and more sensitive tool for the detection of P. stewartii. Significance and Impact of the study: This real-time PCR assay would avoid false-negative results and reduce the time required for certifying maize seed shipments.     

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.     

Assessment of Real-time PCR Based Methods for Quantification of Pollen-mediated Gene Flow from GM to Conventional Maize in a Field Study

Maize is one of the main crops worldwide and an increasing number of genetically modified (GM) maize varieties are cultivated and commercialized in many countries in parallel to conventional crops. Given the labeling rules established e.g. in the European Union and the necessary coexistence between GM and non-GM crops, it is important to determine the extent of pollen dissemination from transgenic maize to other cultivars under field conditions. The most widely used methods for quantitative detection of GMO are based on real-time PCR, which implies the results are expressed in genome percentages (in contrast to seed or grain percentages). Our objective was to assess the accuracy of real-time PCR based assays to accurately quantify the contents of transgenic grains in non-GM fields in comparison with the real cross-fertilization rate as determined by phenotypical analysis. We performed this study in a region where both GM and conventional maize are normally cultivated and used the predominant transgenic maize Mon810 in combination with a conventional maize variety which displays the characteristic of white grains (therefore allowing cross-pollination quantification as percentage of yellow grains). Our results indicated an excellent correlation between real-time PCR results and number of cross-fertilized grains at Mon810 levels of 0.1–10%. In contrast, Mon810 percentage estimated by weight of grains produced less accurate results. Finally, we present and discuss the pattern of pollen-mediated gene flow from GM to conventional maize in an example case under field conditions.     

Evaluation of the efficacy of immunomagnetic separation for the detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds

Aims: To evaluate the effectiveness of the optimized immunomagnetic separation (IMS)‐plating protocol in relation to other culture, serological and molecular techniques currently used for Clavibacter michiganensis subsp. michiganensis in seed‐testing laboratories. Methods and results: Bacterial suspensions, tomato seed extracts spiked with the pathogen and naturally infected seeds were IMS‐plated for the detection of C. m. subsp. michiganensis. These results were compared with plating on general (YPGA) and semiselective (mSCM) media, double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA), immunofluorescent assay (IF) or polymerase chain reaction (PCR). Different seed lots and pathogen strains were also tested. IMS‐plating allowed the detection of less than 10 CFU ml^?1 of pathogen in all assayed samples. The mSCM medium provided positive results for 10 CFU ml^?1 in naturally infected seeds, but up to 14 days was necessary for the typical colonies of the target to be come visible. By serological techniques, 10³ and up to 10⁴ CFU ml^?1 were detected by IF and ELISA, respectively. DNA extraction was required to obtain positive results by PCR in seed extracts containing 10³ CFU ml^?1 or more. Conclusions: Among the evaluated methods, IMS‐plating provided the best results regarding sensitivity and specificity for C. m. subsp. michiganensis detection, allowing the recovery of viable bacteria from seed extracts. Significance and impact of the study: IMS‐plating increases isolation rates of C. m. subsp. michiganensis and could improve standard protocols currently used for routine analysis.     

The application of real-time PCR to the identification, detection and quantification of Pyrenophora species in barley seed

A real-time quantitative PCR technique has been used to develop a rapid and sensitive seed health test for Pyrenophora spp. on barley seed. Using the fluorescent reporter dye SYBR Green I for real-time detection of PCR amplification, pathogen DNA extracted from infected seed can be quantified to the picogram level. The amount of Pyrenophora DNA extracted from seed samples of an artificial infection level gradient, constructed by mixing infected and uninfected seed, correlated with good agreement ( r  = 0.931) to percentage infection levels of the same samples measured by agar plate testing. In addition, a correlation of r  = 0.883 was obtained between the two testing methods for naturally infected seed, ranging from 0% to 89% infection. Samples could be quantified to below the 2% voluntary threshold required for deciding on seed treatment. The proposed test was performed in three parts: (i) quantification of Pyrenophora spp. infection using Pyrenophora -specific PCR primers; (ii) test of any negative samples from (i) with barley-specific PCR primers to check the DNA extraction process; (iii) test of positive samples from (i) for the presence of Pyrenophora graminea using P. graminea -specific PCR primers. All PCRs were performed in the LightCycler™ instrument allowing each PCR run and analysis to be completed within 30 min. With the current daily receipt of samples (batches up to 16) the test can be completed in 8 h, compared to 7 days for the traditional agar plate test.     

Rapid Real-Time PCR Assay for Culture and Tissue Identification of <Emphasis Type="Italic">Geomyces destructans</Emphasis>: the Etiologic Agent of Bat Geomycosis (White Nose Syndrome)

Geomyces destructans is the etiologic agent of bat geomycosis, commonly referred to as white nose syndrome (WNS). This infection has caused severe morbidity and mortality in little brown bats (Myotis lucifugus) and has also spread to other bat species with significant decline in the populations. Currently, G. destructans infection is identified by culture, ITS–PCR, and histopathology. We hypothesized that a real-time PCR assay would considerably improve detection of G. destructans in bats. The 100 bp sequence of the Alpha-L-Rhamnosidase gene was validated as a target for real-time PCR. The assay sensitivity was determined from serial dilution of DNA extracted from G. destructans conidia (5 × 10⁻¹–5 × 10⁷), and the specificity was tested using DNA from 30 closely and distantly related fungi and 5 common bacterial pathogens. The real-time PCR assay was highly sensitive with detection limit of two G. destructans conidia per reaction at 40 PCR cycles. The assay was also highly specific as none of the other fungal or bacterial DNA cross-reacted in the real-time PCR assay. One hundred and forty-seven bat tissue samples, suspected of infection with G. destructans, were used to compare the real-time PCR assay to other methods employed for the detection of G. destructans. Real-time PCR was highly sensitive with 80 of 147 (55%) samples testing positive for G. destructans DNA. In comparison, histopathology examination revealed 64/147 (44%) positive samples. The internal transcribed spacer (ITS)–PCR yielded positive amplicon for G. destructans from 37 tissue samples (25%). The least sensitive assay was the fungal culture with only 17 tissue samples (12%) yielding G. destructans in culture. The data suggested that the real-time PCR assay is highly promising for rapid, sensitive, and specific identification of G. destructans. Further trials and inter-laboratory comparisons of this novel assay are recommended to improve the diagnosis of bat geomycosis.     

High level expression of a functionally active cholera toxin B: rabies glycoprotein fusion protein in tobacco seeds

A synthetic DNA construct containing cholera toxin B subunit, genetically fused to the surface glycoprotein of rabies virus was expressed in tobacco plants from a seed specific (legumin) promoter. Seed specific expression was monitored by real-time PCR, GM1-ELISA and Western blot analyses. The fusion protein accumulated in tobacco seeds at up to 1.22% of the total seed protein. It was functionally active in binding to the GM1-ganglioside receptors, suggesting its assembly into pentamers in seeds of the transgenic plants. Immunoblot analysis confirmed that the ~80.6 kDa monomeric fusion polypeptide was expressed in tobacco seeds and accumulated as a ~403 kDa pentamer. Evaluation of its immunoprotective ability against rabies and cholera is to be examined.     

Detection of airborne genetically modified maize pollen by real-time PCR

The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi-urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real-time PCR analysis. Our results suggest that this 'GM pollen monitoring by bioaerosol sampling and PCR screening' approach might represent an useful aid in the surveillance of GM-free areas, centres of origin and natural reserves.     

用实时荧光PCR方法鉴定转基因玉米T14/T25

本研究以实时荧光PCR技术鉴定商业化种植的转基因玉米T14/T25品系。根据转基因玉米T14/T25转入的外源基因质粒图谱,设计转基因引物和探针进行PCR和实时荧光PCR检测,建立了转基因玉米品系鉴定的实时荧光PCR方法。实验结果表明,用TaqMan探针可检测到T14/T25产生的荧光信号,而对其他玉米品系则检测不到荧光信号,为转基因产品的鉴定检测提供了新方法。Abstract: To identify genetically modified (GM) maize T14/T25 lines, a real-time fluorescent PCR (RTF PCR) assay was performed in this study. Primers and Taqman probes specific for inserted genes in the T14/T25 were used to conduct the real-time fluorenscent (RTF) PCR and PCR assays. The RTF PCR method was established to detect and identify GM maize lines. The results show that the TaqMan probe could identify T14/T25 maize used, while other GM and NO-GM maize didn’t be detected. The RTF PCR could be a new method for detecting other genetically modified organism.     

A novel real-time PCR assay for the detection of Helicobacter pullorum-like organisms in chicken products

A novel real-time PCR assay was developed for the direct detection in food of Helicobacter pullorum-like bacteria, which are occasionally associated with human enteric disease. Experiments using control strains showed that the realtime PCR assay was specific and reproducible, with a detection level of 1 colony-forming unit (CFU)/g. The assay was then applied to determine contamination rates in 30 samples of three types of chicken-meat products obtained from five retail outlets in Spain (Valencia); all of the samples were initially considered to be culture-negative for Helicobacter even after an enrichment period. H.pullorum-like DNA was detected in seven out of ten chicken carcasses and in one chicken-burger sample (without enrichment), as well as in one liver sample (after enrichment). Sequencing of three randomly selected PCR products confirmed concordance (99% homology) with the H. pullorum 16S rDNA gene. The advantages of real-time PCR over conventional PCR assays are the improved detection level, speed of testing, and validation of specificity by melting-point analysis. The fact that bacteria are frequently present in chicken carcasses sold in retail stores highlights the importance of more widely monitoring contamination rates. The novel assay described herein allows better assessment of potential human health risks posed by H. pullorum.     

Real-time polymerase chain reaction assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder

Real-time polymerase chain reaction (real-time PCR) is a laboratory technique based on PCR. This technique is able to detect sequence-specific PCR products as they accumulate in “real time” during the PCR amplification, and also to quantify the number of substrates present in the initial PCR mixture before amplification begins. In the present study, real-time PCR assay was employed for rapid and real-time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 5 to 10⁷ spores. DNA was isolated from spiked soil and talcum powder, using PBS containing 1 % Triton-X-100, followed by heat treatment. The isolated DNA was used as template for real-time PCR and PCR. Real-time PCR amplification was obtained in 60 min under the annealing condition at 60°C by employing primers targeting the pag gene of B. anthracis. In the present study, the detection limit of real-time PCR assay in soil was 10³ spores and10² spores in talcum powder, respectively, whereas PCR could detect 10⁴ spores in soil and 10³ spores in talcum powder, respectively.