The microbial detection array combined with random Phi29-amplification used as a diagnostic tool for virus detection in clinical samples

A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples.     

Towards unbiased parentage assignment: combining genetic, behavioural and spatial data in a Bayesian framework

Inferring the parentage of a sample of individuals is often a prerequisite for many types of analysis in molecular ecology, evolutionary biology and quantitative genetics. In all but a few cases, the method of parentage assignment is divorced from the methods used to estimate the parameters of primary interest, such as mate choice or heritability. Here we present a Bayesian approach that simultaneously estimates the parentage of a sample of individuals and a wide range of population-level parameters in which we are interested. We show that joint estimation of parentage and population-level parameters increases the power of parentage assignment, reduces bias in parameter estimation, and accurately evaluates uncertainty in both. We illustrate the method by analysing a number of simulated test data sets, and through a re-analysis of parentage in the Seychelles warbler, Acrocephalus sechellensis. A combination of behavioural, spatial and genetic data are used in the analyses and, importantly, the method does not require strong prior information about the relationship between nongenetic data and parentage.     

The study of ubiquitin-dependent increase in monoamine oxidase sensitivity to proteolysis and specific inhibitor, pargyline

Insertion of exogenous ubiquitin into rat brain mitochondria in the presence of ATP and the ATPregenerating system (creatine phosphate/creatine phosphokinase) results in the increase in: sensitivity of mitochondrial monoamine oxidases (MAO) A and B to inhibition by mechanism based inhibitor and incorporation of [³H]-pargyline, which was especially notable in the fraction obtained by immunoprecipitation of mitochondrial proteins with anti-ubiquitin antiserum and protein A Sepharose. This suggests that MAO is a potential substrate for ubiquitination in vitro. However, the content of the tritium label in this fraction was less than 0.1 % and not more than 0.25% of total radioactivity of [³H]-pargyline bound to control and ATP-ubiquitin treated mitochondria, respectively. Insertion of ubiquitin into mitochondria did not influence molecular masses of [³H]-pargyline labeled proteins. These results suggest that direct ubiquitination of MAO insignificantly contributes to marked changes in the sensitivity of MAO A and MAO B to proteolysis and specific inhibition found under these experimental conditions. It is possible that more complex processes are involved into realization of these effects during ATP-dependent ubiquitin incorporation into mitochondria.     

Development of a real-time PCR assay for detection of Mytilus species specific alleles: Application to a sampling survey in Scotland

Shellfish aquaculture is a growing industry in Scotland, dominated by the production of the mussel Mytilus edulis, the native species. Recently the discovery of Mytilus galloprovincialis and Mytilus trossulus together with M. edulis and all 3 hybrids in cultivation in some Scottish sea lochs led to questions regarding the distribution of mussel species in Scotland. The establishment of an extensive sampling survey, involving the collection of mussels at 34 intertidal sites and 10 marinas around Scotland, motivated the development of a high-throughput method for identification of Mytilus alleles from samples. Three Taqman®-MGB probes and one set of primers were designed, based on the previously described Me 15/16 primers targeting the adhesive protein gene sequence, and samples were screened for the presence of M. edulis, M. galloprovincialis and M. trossulus alleles using real-time PCR. Mytilus edulis alleles were identified in samples from all 44 sites. Mytilus galloprovincialis alleles were found together with M. edulis alleles extensively in northern parts of the west and east coasts. Mytilus trossulus alleles were identified in samples from 6 sites in the west and south-west of Scotland. Because M. trossulus is generally undesirable in cultivation and therefore preventing the geographical spread of this species across Scotland is considered beneficial by the shellfish aquaculture industry, these 6 samples were further analysed for genotype frequencies using conventional PCR. Although distribution of the non-native species M. galloprovincialis and M. trossulus have proven to be more widespread than previously thought, there is no evidence from our study of either M. trossulus or M. galloprovincialis acting as an invasive species in Scotland. The real-time PCR method developed in this study has proven to be a rapid and effective tool for the identification of M. edulis, M. galloprovincialis and M. trossulus alleles from samples and should prove useful in future surveys, ecological or aquaculture management related studies in both unispecific and mixed species areas of these species.     

Quantification of Fusarium graminearum in infected wheat by species specific real-time PCR applying a TaqMan Probe

A new real-time PCR based method was developed for the species-specific detection, identification and quantification of Fusarium graminearum in planta. It utilizes a TaqMan hybridisation probe targeting the beta-tubulin gene and a plasmid standard. The assay is highly specific giving no product with DNA of closely related species. It is very sensitive, detecting down to five gene copies per reaction, and is able to produce reliable quantitative data over a range of six orders of magnitude.     

Synthesis of encephalomyocarditis virus in a cell-free system: from DNA to RNA virus in one tube

Virus particles are used in vaccination, drug delivery, and material sciences. Here we devised a system where the RNA virus encephalomyocarditis virus (EMCV) is synthesized from DNA templates in vitro. When a plasmid or a PCR product harboring the full-length cDNA of EMCV in the T7 promoter/terminator unit was incubated in a HeLa cell extract supplemented with T7 RNA polymerase, EMCV was produced within 4 h at an efficiency of over 10-fold compared with the system programmed with the EMCV RNA. The EMCV RNA transcribed by the virally encoded RNA-dependent RNA polymerase was predominantly incorporated into the EMCV particle even in the presence of a larger amount of the EMCV RNA transcribed by T7 RNA polymerase from the plasmid.     

Microbes increase thermal sensitivity in the mosquito Aedes aegypti,with the potential to change disease distributions

The mosquito Aedes aegypti is the primary vector of many disease-causing viruses, including dengue (DENV), Zika, chikungunya, and yellow fever. As consequences of climate change, we expect an increase in both global mean temperatures and extreme climatic events. When temperatures fluctuate, mosquito vectors will be increasingly exposed to temperatures beyond their upper thermal limits. Here, we examine how DENV infection alters Ae. aegypti thermotolerance by using a high-throughput physiological ‘knockdown’ assay modeled on studies in Drosophila. Such laboratory measures of thermal tolerance have previously been shown to accurately predict an insect’s distribution in the field. We show that DENV infection increases thermal sensitivity, an effect that may ultimately limit the geographic range of the virus. We also show that the endosymbiotic bacterium Wolbachia pipientis, which is currently being released globally as a biological control agent, has a similar impact on thermal sensitivity in Ae. aegypti. Surprisingly, in the coinfected state, Wolbachia did not provide protection against DENV-associated effects on thermal tolerance, nor were the effects of the two infections additive. The latter suggests that the microbes may act by similar means, potentially through activation of shared immune pathways or energetic tradeoffs. Models predicting future ranges of both virus transmission and Wolbachia’s efficacy following field release may wish to consider the effects these microbes have on host survival.     

Techniques for detecting chytridiomycosis in wild frogs: comparing histology with real-time Taqman PCR

Chytridiomycosis is a lethal disease of amphibians associated with mass mortalities and population declines worldwide. An accurate, non-invasive technique for detecting chytridiomycosis is urgently needed to determine the current geographical distribution of the disease, and its prevalence in wild amphibian populations. Herein we evaluate a recently devised, rapid, non-invasive, swab-PCR assay. We sampled 101 wild juvenile Mixophyes iteratus by both a skin swab for use in PCR analysis, and a toe-clip for examination by histological methods. The swab-PCR assay detected chytridiomycosis infection in a minimum of 14.9% of frogs, whereas histology detected infection in no more than 6.9% of frogs. We conclude that the swab-PCR technique is the more reliable means of detecting chytridiomycosis in wild amphibians, and that it precludes the need for toe-clipping as a means of sampling for the presence of the disease in future surveys. Further, we document a significant negative relationship between a juvenile frog's snout-vent length and its likelihood of being infected with the disease.     

Erratum to: Development of SCAR markers for rapid and specific detection of Pseudomonas syringae pv. morsprunorum races 1 and 2, using conventional and real-time PCR

Applied Microbiology and Biotechnology -     

Ribonucleotide reductase subunit R1: a gene conferring sensitivity to valproic acid-induced neural tube defects in mice

Neural tube defects (NTDs), although prevalent and easily diagnosed, are etiologically heterogeneous, rendering mechanistic interpretation problematic. To date, there is evidence that mammalian neural tube closure (NTC) initiates and fuses intermittently at four discrete locations. Disruption of this process at any of these four sites may lead to a region-specific NTDs, possibly arising through closure site-specific genetic mechanisms. Although recent efforts have focused on elucidating the genetic components of NTDs, a void persists regarding gene identification in closure site-specific neural tissue. To this end, experiments were conducted to identify neural tube closure site-specific genes that might confer regional sensitivity to teratogen-induced NTDs. Using an inbred mouse strain (SWV/Fnn) with a high susceptibility to VPA- induced NTDs that specifically targets and disrupts NTC between the prosencephalon and mesencephalon region (future fore/midbrain; neural tube closure site II), we identified a VPA-sensitive closure site II-specific clone. Sequencing of this clone from an SWV neural tube cDNA library confirmed that it encodes the r1 subunit of the cell cycle enzyme ribonucleotide reductase (RNR). The abundance of rnr-r1 mRNA was significantly increased in response to VPA drug treatment. This upregulated expression was accompanied by a significant decrease in cellular proliferation in the closure site II neural tube region of the embryos, as determined by ELISA cellular proliferation assays performed on BrdU-pulsed neuroepithelial cells in vivo. We hypothesize that rnr-r1 plays a critical role in the development of VPA-induced exencephaly.     

An alternative real-time PCR method to detect the Bacillus cereus group in naturally contaminated food gelatine: a comparison study

Aims:  Comparison of an internally controlled real-time PCR assay with the standard plate-based assay (ISO 21871) for the detection of Bacillus cereus group cells in gelatine.
Methods and Results:  A comprehensive TaqMan probe was designed allowing the real-time PCR assay to be fully inclusive and exclusive. An internal amplification control was designed and implemented at 500 copies per reaction without impact on target detection. Specific and selective detection of target cells was achieved with a quick and simple DNA preparation procedure. No significant difference (κ = 0·99) was observed between the performance of the real-time PCR and the standard plate-based method on naturally contaminated gelatines ( n  = 197). Relative accuracy, relative sensitivity and relative specificity were ≥99%.
Conclusions:  The real-time PCR assay is a valid alternative of the standard plate-based assay.
Significance and Impact of the Study:  The real-time PCR assay decreased the time between sample collection and result from 2 days to 2 h, while analysis cost did not increase. The gelatine-producing industry can ensure gelatine safety and quality in a much faster way.     

Evaluation of a duplex real-time PCR assay to detect MRSA from broth culture,human sera seeded with MRSA and from patient's serum

The need for rapid methods in order to precisely detect methicillin-resistant Staphylococcus aureus (MRSA) is extensively acknowledged. This study evaluated a quantitative real-time PCR assay targeting mecA (encoding high level resistance to methicillin) and femB (a specific genomic marker for S. aureus) genes to detect MRSA from broth culture, from serum seeded with MRSA and straight from the patient''s serum. One hundred and thirty-five clinical isolates of MRSA strains and different species were utilised in this study. In addition, a pilot study with 9 patients'' serum samples was performed. The sensitivity and specificity values for this assay were 99% and 100% respectively. The detection limit for this method was 1.23×10² CFU/ml from the serum seeded with MRSA cells and the limiting concentration of DNA for detection was 18 fg, which equates to 5.14 genomic DNA copies. In addition, this assay detected MRSA from patient''s serum (7 out of 9) with sensitivity of 77.8%. Overall, the assay was rapid, efficient, sensitive and easy to perform.     

Development of a rapid and sensitive method combining a cellulose ester microfilter and a real-time quantitative PCR assay to detect Campylobacter jejuni and Campylobacter coli in 20 liters of drinking water or low-turbidity waters

Investigations of Campylobacter jejuni and Campylobacter coli in samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection of Campylobacter in 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) of Campylobacter per 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with a C. coli strain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence of C. jejuni and C. coli genomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination by Campylobacter.     

No evidence for DUP25 in patients with panic disorder using a quantitative real-time PCR approach

A duplication of chromosome 15q24-q26 (DUP25) has been reported to be associated with anxiety disorders. We tested for the presence of DUP25 in a sample of 50 patients with panic disorder and 50 controls using a quantitative real-time PCR approach. Contrary to the original finding, our results were compatible with the absence of DUP25, and no significant difference could be detected between patients and controls (P=1.0). Thus, our study does not support the hypothesis of an involvement of DUP25 in panic disorder.     

Human and Escherichia coli cyclophilins: sensitivity to inhibition by the immunosuppressant cyclosporin A correlates with a specific tryptophan residue

The human T-cell protein cyclophilin shows high affinity for and is the proposed target of the major immunosuppressant drug cyclosporin A (CsA). Cyclophilin also has peptidyl prolyl cis-trans isomerase activity that is inhibited by CsA with an IC50 of 6 nM, while by contrast a homologous PPIase from Escherichia coli has been found to be much less sensitive to CsA, shown here to be 500-fold less potent at an IC50 of 3000 nM. This E. coli rotamase lacks the single highly conserved tryptophan residue of eukaryotic cyclophilins, and we show here that mutation of the natural F112 to W112 enhances E. coli rotamase susceptibility to CsA inhibition by 23-fold. Correspondingly, the human W121 mutations to F121 or A121 yield cyclophilins with 75- and 200-fold decreased sensitivity to CsA, while kcat/Km values of rotamase activity in a tetrapeptide assay drop only 2- and 13-fold, respectively. This complementary gain and loss of CsA sensitivity to mutation to or from tryptophan validate the indole side chain as a major determinant in immunosuppressant drug recognition and the separation of PPIase catalytic efficiency from CsA affinity.     

Short communication: Single tube allele specific (STAS) PCR for direct determination of the mutation in the porcine ryanodine receptor gene associated with malignant hyperthermia

The ability to add or delete specific genes in swine will likely provide considerable benefits not just to agriculture but also to medicine, where pigs have potential as models for human disease and as organ donors. Here we have transferred nuclei from a genetically modified fibroblast cell line to porcine oocytes, matured in vitro under defined culture conditions, to create piglets expressing enhanced green fluorescent protein. The nuclear transfer-derived piglets were of normal size, although some mild symptoms of “large offspring syndrome” were evident. These experiments represent a next step towards creating swine with more useful genetic modifications.     

Restoration of gene function by homologous recombination: from PCR to gene expression in one step

We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete lambdaPR or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage lambda Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding beta-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.     

Use of both 16S rRNA and engineered functional genes with real-time PCR to quantify an engineered,PCB-degrading Rhodococcus in soil

A real-time PCR (RTm-PCR) assay using fluorescently labeled oligonucleotides (TaqMan probes) was used to detect and quantify the recombinant Rhodococcus sp. strain RHA1(fcb) in soil. One primer and probe set targeted a hypervariable region of the 16S rRNA gene unique to strain RHA1(fcb) and its phylogenetic relatives, and the other set targeted the recombinant 4-chlorobenzoate (4-CBA) degradation operon (fcb) and was strain-specific. The method had a 6-log dynamic range of detection (10(2)-10(7) cells ml(-1)) for both probes when DNA from pure cultures was used. Although the method was less sensitive in soil, the estimated number of cells in soil by real-time PCR corresponded to the measured number of RHA1(fcb) cells determined by colony-forming units.     

Quantitative real-time PCR assays to identify and quantify fecal Bifidobacterium species in infants receiving a prebiotic infant formula

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5' nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5' nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% +/- 9.8% to 73.4% +/- 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% +/- 1.92% and 8.11% +/- 4.12%, respectively, versus 0.15% +/- 0.11% and 1.38% +/- 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.