Improved detection of Rhodococcus coprophilus with a new quantitative PCR assay.

Agricultural practices, such as spreading liquid manure or the utilisation of land as animal pastures, can result in faecal contamination of water resources. Rhodococcus coprophilus is used in microbial source tracking to indicate animal faecal contamination in water. Methods previously described for detecting of R. coprophilus in water were neither sensitive nor specific. Therefore, the aim of this study was to design and validate a new quantitative polymerase chain reaction (qPCR) to improve the detection of R. coprophilus in water. The new PCR assay was based on the R. coprophilus 16S rRNA gene. The validation showed that the new approach was specific and sensitive for deoxyribunucleic acid from target host species. Compared with other PCR assays tested in this study, the detection limit of the new qPCR was between 1 and 3 log lower. The method, including a filtration step, was further validated and successfully used in a field investigation in Switzerland. Our work demonstrated that the new detection method is sensitive and robust to detect R. coprophilus in surface and spring water. Compared with PCR assays that are available in the literature or to the culture-dependent method, the new molecular approach improves the detection of R. coprophilus.

Identification des dermatophytes causant des teignes de la peau glabre (Tinea glabra) et du cuir chevelu (Tinea capitis) par PCR

Contexte: Le nombre de teignes du cuir chevelu et de la peau glabre étant en nette augmentation, l'identification du pathogène qui est indispensable pour un traitement ciblé, a, par conséquence, un grand intérêt pour la santé publique. Dans divers cas, un animal de compagnie peut être identifié en tant que source du pathogène. La fréquence de cultures restant stériles est particulièrement élevée en cas de prétraitement antifongique. Objectif: Le but de ce travail est de mettre au point une méthode rapide d'identification du dermatophyte pathogène in situ par PCR/séquençage dans les cas de teignes du cuir chevelu et/ou de la peau glabre. Matériel et méthodes : De l'ADN a été extrait de squames (N=5) et cheveux (N=21) dont l'examen direct démontrait une infection fongique (N=26) ou se révèlait négatif (N=1). Ensuite, une première PCR du segment 28s de l'ADN ribosomale fongique a été effectuée, suivie par une PCR nichée intérieure à ce segment. L'amplicon a été séquencé et le champignon est identifié par alignement. Résultats : Seule la PCR enchainée a permis d'obtenir une quantité suffisante d'amplicon pour permettre le séquençage. Dans 4 cas sur 5 de tinea pedis, 10 sur 12 de tinea glabra, respectivement 4 sur 4 de tinea capitis, dans lesquels un dermato- phyte a été identifié en culture, le même dermatophyte a été identifié par PCR/séquençage. Une fois sur 27 prélèvements, un autre dermatophyte a été identifié par PCR/séquençage. Ce résultat pourrait être dû à une fausse identification du champignon en culture. Dans un cas de tinea pedis et un cas de tinea corporis, la culture est restée stérile, mais un dermatophyte a pu être identifié par PCR et séquençage. Conclusions : La méthode décrite est à la fois rapide (24 h au lieu de deux semaines pour la culture), sensible et très spécifique. Elle est particulièrement utile dans les cas de teigne du cuir chevelu, dans lesquels le traitement est différent selon l'espèce de dermatophyte et où il s'agit d'un traitement systémique lourd, souvent chez l'enfant.

Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean

The objective of this work was to validate, by quantitative PCR in real time (RT-qPCR), genes to be used as reference in studies of gene expression in soybean in drought-stressed trials. Four genes commonly used in soybean were evaluated: Gmβ-actin, GmGAPDH, GmLectin and GmRNAr18S. Total RNA was extracted from six samples: three from roots in a hydroponic system with different drought intensities (0, 25, 50, 75 and 100 minutes of water stress), and three from leaves of plants grown in sand with different soil moistures (15, 5 and 2.5% gravimetric humidity). The raw cycle threshold (Ct) data were analyzed, and the efficiency of each primer was calculated for an overall analysis of the Ct range among the different samples. The GeNorm application was used to evaluate the best reference gene, according to its stability. The GmGAPDH was the least stable gene, with the highest mean values of expression stability (M), and the most stable genes, with the lowest M values, were the Gmβ-actin and GmRNAr18S, when both root and leaves samples were tested. These genes can be used in RT-qPCR as reference gene for expression analysis.

Development of a duplex real-time PCR for the detection of Rickettsia spp. and typhus group rickettsia in clinical samples.

Molecular diagnosis using real-time polymerase chain reaction (PCR) may allow earlier diagnosis of rickettsiosis. We developed a duplex real-time PCR that amplifies (1) DNA of any rickettsial species and (2) DNA of both typhus group rickettsia, that is, Rickettsia prowazekii and Rickettsia typhi. Primers and probes were selected to amplify a segment of the 16S rRNA gene of Rickettsia spp. for the pan-rickettsial PCR and the citrate synthase gene (gltA) for the typhus group rickettsia PCR. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing human DNA and 22 pathogens or skin commensals. Real-time PCR was applied to 16 clinical samples. Rickettsial DNA was detected in the skin biopsies of three patients. In one patient with severe murine typhus, the typhus group PCR was positive in a skin biopsy from a petechial lesion and seroconversion was later documented. The two other patients with negative typhus group PCR suffered from Mediterranean and African spotted fever, respectively; in both cases, skin biopsy was performed on the eschar. Our duplex real-time PCR showed a good analytical sensitivity and specificity, allowing early diagnosis of rickettsiosis among three patients, and recognition of typhus in one of them.

Real-time quantitative PCR assay for measurement of bird telomeres

We present the application of a real-time quantitative PCR assay, previously developed to measure relative telomere length in humans and mice, to two bird species, the zebra finch Taeniopygia guttata and the Alpine swift Apus melba. This technique is based on the PCR amplification of telomeric (TTAGGG)(n) sequences using specific oligonucleotide primers. Relative telomere length is expressed as the ratio (T/S) of telomere repeat copy number (T) to control single gene copy number (S). This method is particularly useful for comparisons of individuals within species, or where the same individuals are followed longitudinally. We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a single control gene. In both species, we validated our PCR measurements of relative telomere length against absolute measurements of telomere length determined by the conventional method of quantifying telomere terminal restriction fragment (TRF) lengths using both the traditional Southern blot analysis (Alpine swifts) and in gel hybridization (zebra finches). As found in humans and mice, telomere lengths in the same sample measured by TRF and PCR were well correlated in both the Alpine swift and the zebra finch.. Hence, this PCR assay for measurement of bird telomeres, which is fast and requires only small amounts of genomic DNA, should open new avenues in the study of environmental factors influencing variation in telomere length, and how this variation translates into variation in cellular and whole organism senescence.

Identificação por PCR dos alelos-S associados à compatibilidade gametofítica em ameixeira japonesa

O objetivo deste trabalho foi identificar e caracterizar molecularmente os alelos-S de cultivares de ameixeira japonesa e verificar a compatibilidade gametofítica entre estes. Foram utilizados dois pares de iniciadores específicos na amplificação dos alelos via PCR, em 18 cultivares: Pluma 7, Gulf Rubi, Blood Plum, Wickson, América, Santa Rosa, Rosa Mineira, Estrela Púrpura, Amarelinha, The First, Harry Pieckstone, Santa Rita, Seleção 16, Seleção A26 (Burbank), Seleção 21, Seleção 19, Methley e Simka. As condições da PCR e as combinações de oligonucleotídeos iniciadores utilizadas permitiram a identificação de alelos-S nas cultivares, bem como a indicação dos polinizadores mais compatíveis. As cultivares América e Santa Rosa, bem como Blood Plum, Wickson, Rosa Mineira, Estrela Púrpura e Seleção 21 apresentam incompatibilidade total entre si, por compartilharem os mesmos alelos.

eggNOG v4.0: nested orthology inference across 3686 organisms

With the increasing availability of various 'omics data, high-quality orthology assignment is crucial for evolutionary and functional genomics studies. We here present the fourth version of the eggNOG database (available at http://eggnog.embl.de) that derives nonsupervised orthologous groups (NOGs) from complete genomes, and then applies a comprehensive characterization and analysis pipeline to the resulting gene families. Compared with the previous version, we have more than tripled the underlying species set to cover 3686 organisms, keeping track with genome project completions while prioritizing the inclusion of high-quality genomes to minimize error propagation from incomplete proteome sets. Major technological advances include (i) a robust and scalable procedure for the identification and inclusion of high-quality genomes, (ii) provision of orthologous groups for 107 different taxonomic levels compared with 41 in eggNOGv3, (iii) identification and annotation of particularly closely related orthologous groups, facilitating analysis of related gene families, (iv) improvements of the clustering and functional annotation approach, (v) adoption of a revised tree building procedure based on the multiple alignments generated during the process and (vi) implementation of quality control procedures throughout the entire pipeline. As in previous versions, eggNOGv4 provides multiple sequence alignments and maximum-likelihood trees, as well as broad functional annotation. Users can access the complete database of orthologous groups via a web interface, as well as through bulk download.

Premenopausal serum androgens and breast cancer risk: a nested case-control study.

ABSTRACT: INTRODUCTION: Prospective epidemiologic studies have consistently shown that levels of circulating androgens in postmenopausal women are positively associated with breast cancer risk. However, data in premenopausal women are limited. METHODS: A case-control study nested within the New York University Women's Health Study was conducted. A total of 356 cases (276 invasive and 80 in situ) and 683 individually-matched controls were included. Matching variables included age and date, phase, and day of menstrual cycle at blood donation. Testosterone, androstenedione, dehydroandrosterone sulfate (DHEAS) and sex hormone-binding globulin (SHBG) were measured using direct immunoassays. Free testosterone was calculated. RESULTS: Premenopausal serum testosterone and free testosterone concentrations were positively associated with breast cancer risk. In models adjusted for known risk factors of breast cancer, the odds ratios for increasing quintiles of testosterone were 1.0 (reference), 1.5 (95% confidence interval (CI), 0.9 to 2.3), 1.2 (95% CI, 0.7 to 1.9), 1.4 (95% CI, 0.9 to 2.3) and 1.8 (95% CI, 1.1 to 2.9; Ptrend = 0.04), and for free testosterone were 1.0 (reference), 1.2 (95% CI, 0.7 to 1.8), 1.5 (95% CI, 0.9 to 2.3), 1.5 (95% CI, 0.9 to 2.3), and 1.8 (95% CI, 1.1 to 2.8, Ptrend = 0.01). A marginally significant positive association was observed with androstenedione (P = 0.07), but no association with DHEAS or SHBG. Results were consistent in analyses stratified by tumor type (invasive, in situ), estrogen receptor status, age at blood donation, and menopausal status at diagnosis. Intra-class correlation coefficients for samples collected from 0.8 to 5.3 years apart (median 2 years) in 138 cases and 268 controls were greater than 0.7 for all biomarkers except for androstenedione (0.57 in controls). CONCLUSIONS: Premenopausal concentrations of testosterone and free testosterone are associated with breast cancer risk. Testosterone and free testosterone measurements are also highly reliable (that is, a single measurement is reflective of a woman's average level over time). Results from other prospective studies are consistent with our results. The impact of including testosterone or free testosterone in breast cancer risk prediction models for women between the ages of 40 and 50 years should be assessed. Improving risk prediction models for this age group could help decision making regarding both screening and chemoprevention of breast cancer.

Identification of parasitoid species using PCR amplification and restriction enzyme digestion

Spodoptera frugiperda is a pest of great economic importance in the Americas. It is attacked by several species of parasitoids, which act as biological control agents. Parasitoids are morphologically identifiable as adults, but not as larvae. Laboratory rearing conditions are not always optimal to rear out parasitic wasps from S. frugiperda larvae collected from wild populations, and it frequently happens that parasitoids do not complete their life cycle and stop developing at the larval stage. Therefore, we explored ways to identify parasitoid larvae using molecular techniques. Sequencing is one possible technique, yet it is expensive. Here we present an alternate, cheaper way of identifying seven species of parasitoids (Cotesia marginiventris, Campoletis sonorensis, Pristomerus spinator, Chelonus insularis, Chelonus cautus, Eiphosoma vitticolle and Meteorus laphygmae) using PCR amplification of COI gene followed by a digestion with a combination of four restriction endonucleases. Each species was found to exhibit a specific pattern when the amplification product was run on an agarose gel. Identifying larvae revealed that conclusions on species composition of a population of parasitic wasps can be biased if only the emerging adults are taken into account.

New diagnostic real-time PCR for specific detection of Parachlamydia acanthamoebae DNA in clinical samples

Given the low sensitivity of amoebal coculture, we developed a specific real-time PCR for the detection of Parachlamydia. The analytical sensitivity was high, and the inter- and intrarun variabilities were low. When the PCR was applied to nasopharyngeal aspirates, it was positive for six patients with bronchiolitis. Future studies should assess the role of Parachlamydia in bronchiolitis.

EXTRAÇÃO DE DNA GENÔMICO DE Passiflora spp. PARA ANÁLISES PCR-RAPD

A identificação e caracterização da diversidade genética de plantas por meio de técnicas moleculares envolvem a avaliação de vários indivíduos, necessitando-se, portanto, de métodos rápidos e precisos de extração do DNA. O co-isolamento de polissacarídeos, fenóis e compostos secundários é o principal problema encontrado no isolamento e purificação de DNA vegetal. Folhas das diversas espécies de Passiflora possuem níveis variados desses compostos que podem comprometer este procedimento. O presente estudo foi realizado com o objetivo de avaliar a qualidade e quantidade de DNA de folhas de variedades de Passiflora spp., utilizando-se de três métodos de extração. Os três métodos forneceram DNA em qualidade e quantidade suficientes para a realização da técnica PCR-RAPD.

Discovery of a 29-gene panel in peripheral blood mononuclear cells for the detection of colorectal cancer and adenomas using high throughput real-time PCR.

Colorectal cancer (CRC) is the second leading cause of cancer-related death in developed countries. Early detection of CRC leads to decreased CRC mortality. A blood-based CRC screening test is highly desirable due to limited invasiveness and high acceptance rate among patients compared to currently used fecal occult blood testing and colonoscopy. Here we describe the discovery and validation of a 29-gene panel in peripheral blood mononuclear cells (PBMC) for the detection of CRC and adenomatous polyps (AP). Blood samples were prospectively collected from a multicenter, case-control clinical study. First, we profiled 93 samples with 667 candidate and 3 reference genes by high throughput real-time PCR (OpenArray system). After analysis, 160 genes were retained and tested again on 51 additional samples. Low expressed and unstable genes were discarded resulting in a final dataset of 144 samples profiled with 140 genes. To define which genes, alone or in combinations had the highest potential to discriminate AP and/or CRC from controls, data were analyzed by a combination of univariate and multivariate methods. A list of 29 potentially discriminant genes was compiled and evaluated for its predictive accuracy by penalized logistic regression and bootstrap. This method discriminated AP >1cm and CRC from controls with a sensitivity of 59% and 75%, respectively, with 91% specificity. The behavior of the 29-gene panel was validated with a LightCycler 480 real-time PCR platform, commonly adopted by clinical laboratories. In this work we identified a 29-gene panel expressed in PBMC that can be used for developing a novel minimally-invasive test for accurate detection of AP and CRC using a standard real-time PCR platform.

Selection of internal control genes for real-time quantitative PCR in ovary and uterus of sows across pregnancy

Reproductive traits play a key role in pig production in order to reduce costs and increase economic returns. Among others, gene expression analyses represent a useful approach to study genetic mechanisms underlying reproductive traits in pigs. The application of reverse-transcription quantitative PCR requires the selection of appropriate reference genes, whose expression levels should not be affected by the experimental conditions, especially when comparing gene expression across different physiological stages.

Malaria real-time PCR: correlation with clinical presentation.

Among 112 patients infected only by Plasmodium falciparum, WHO criteria of severity were compared with parasite load assessed by microscopy and quantitative PCR. Clinical severity was significantly correlated with higher parasite load as determined by microscopy (p < 0.001) and by PCR (p < 0.001). Hence, quantitative PCR might be useful to predict outcome.