Sensitive detection of African swine fever virus using real-time PCR with a 5 ' conjugated minor groove binder probe

The design of a 5' conjugated minor groove binder (MGB) probe real-time PCR assay is described for the rapid, sensitive and specific detection of African swine fever virus (ASFV) DNA. The assay is designed against the 9GL region and is capable of detecting 20 copies of a DNA standard. It does not detect any of the other common swine DNA viruses tested in this study. The assay can detect ASFV DNA in a range of clinical samples. Sensitivity was equivalent to the Office International des Epizooties (OIE) recommended TaqMan assay. In addition the assay was found to have a detection limit 10-fold more sensitive than the conventional PCR recommended by the OIE. Linear range was ten logs from 2 x 10(1) to 2 x 10(10). The assay is rapid with an amplification time just over 2 h. The development of this assay provides a useful tool for the specific diagnosis of ASF in statutory or emergency testing programs or for the detection of ASFV DNA in research applications. (C) 2010 Elsevier B.V. All rights reserved.

Improving molecular detection of Candida DNA in whole blood:comparison of seven fungal DNA extraction protocols using real-time PCR

The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.

Global down-regulation of HOX gene expression in PML-RARalpha + acute promyelocytic leukemia identified by small-array real-time PCR

Acute promyelocytic leukemia (APL) is associated with a reciprocal and balanced translocation involving the retinoic acid receptor-alpha (RARalpha). All-trans retinoic acid (ATRA) is used to treat APL and is a potent morphogen that regulates HOX gene expression in embryogenesis and organogenesis. HOX genes are also involved in hematopoiesis and leukemogenesis. Thirty-nine mammalian HOX genes have been identified and classified into 13 paralogous groups clustered on 4 chromosomes. They encode a complex network of transcription regulatory proteins whose precise targets remain poorly understood. The overall function of the network appears to be dictated by gene dosage. To investigate the mechanisms involved in HOX gene regulation in hematopoiesis and leukemogenesis by precise measurement of individual HOX genes, a small-array real-time HOX (SMART-HOX) quantitative polymerase chain reaction (PCR) platform was designed and validated. Application of SMART-HOX to 16 APL bone marrow samples revealed a global down-regulation of 26 HOX genes compared with normal controls. HOX gene expression was also altered during differentiation induced by ATRA in the PML-RARalpha(+) NB4 cell line. PML-RARalpha fusion proteins have been reported to act as part of a repressor complex during myeloid cell differentiation, and a model linking HOX gene expression to this PML-RARalpha repressor complex is now proposed.

Rapid quantification of microRNAs in plasma using a fast real time PCR system

The ability to rapidly detect circulating small RNAs, in particular microRNAs (miRNAs), would further increase their already established potential as biomarkers in a range of conditions. One rate-limiting factor is the time taken to perform quantitative real time PCR amplification. We therefore evaluated the ability of a novel thermal cycler to perform this step in less than 10 minutes. Quantitative PCR was performed on an xxpress® thermal cycler (BJS Biotechnologies, Perivale, UK), which employs a resistive heating system and forced air cooling to achieve thermal ramp rates of 10 °C/s, and a conventional peltier-controlled LightCycler 480 system (Roche, Basel, Switzerland) ramping at 4.8 °C/s. The threshold cycle (Ct) for detection of 18S rDNA from a standard genomic DNA sample was significantly more variable across the block (F-test, p=2.4x10-25) for the xxpress (20.01±0.47SD) than the LightCycler (19.87±0.04SD). RNA was extracted from human plasma, reverse transcribed and a panel of miRNAs amplified and detected using SYBR green (Kapa Biosystems, Wilmington, Ma, USA). The sensitivity of both systems was broadly comparable and both detected a panel of miRNAs reliably and indicated similar relative abundances. The xxpress thermal cycler facilitates rapid qPCR detection of small RNAs and brings point-of care diagnostics based upon circulating miRNAs a step closer to reality.

Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus , is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the Msp I satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus , up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation.

Quantification of ruminal Clostridium proteoclasticum by real-time PCR using a molecular beacon approach

Aims: All members of the ruminal Butyrivibrio group convert linoleic acid (cis-9,cis-12-18 : 2) via conjugated 18 : 2 metabolites (mainly cis-9,trans-11-18 : 2, conjugated linoleic acid) to vaccenic acid (trans-11-18 : 1), but only members of a small branch, which includes Clostridium proteoclasticum, of this heterogeneous group further reduce vaccenic acid to stearic acid (18 : 0, SA). The aims of this study were to develop a real-time polymerase chain reaction (PCR) assay that would detect and quantify these key SA producers and to use this method to detect diet-associated changes in their populations in ruminal digesta of lactating cows. Materials and Results: The use of primers targeting the 16S rRNA gene of Cl. proteoclasticum was not sufficiently specific when only binding dyes were used for detection in real-time PCR. Their sequences were too similar to some nonproducing strains. A molecular beacon probe was designed specifically to detect and quantify the 16S rRNA genes of the Cl. proteoclasticum subgroup. The probe was characterized by its melting curve and validated using five SA-producing and ten nonproducing Butyrivibrio-like strains and 13 other common ruminal bacteria. Analysis of ruminal digesta collected from dairy cows fed different proportions of starch and fibre indicated a Cl. proteoclasticum population of 2-9% of the eubacterial community. The influence of diet on numbers of these bacteria was less than variations between individual cows. Conclusion: A molecular beacon approach in qPCR enables the detection of Cl. proteoclasticum in ruminal digesta. Their numbers are highly variable between individual animals. Signifance and Impact of the Study: SA producers are fundamental to the flow of polyunsaturated fatty acid and vaccenic acid from the rumen. The method described here enabled preliminary information to be obtained about the size of this population. Further application of the method to digesta samples from cows fed diets of more variable composition should enable us to understand how to control these bacteria in order to enhance the nutritional characteristics of ruminant-derived foods, including milk and beef.

The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue

A LightCycler(R) real-time PCR hybridization probe-based assay that detects a conserved region of the 16S rRNA gene of pathogenic but not saprophytic Leptospira species was developed for the rapid detection of pathogenic leptospires directly from processed tissue samples. In addition, a differential PCR specific for saprophytic leptospires and a control PCR targeting the porcine beta-actin gene were developed. To assess the suitability of these PCR methods for diagnosis, a trial was performed on kidneys taken from adult pigs with evidence of leptospiral infection, primarily a history of reproductive disease and serological evidence of exposure to pathogenic leptospires (n = 180) and aborted pig foetuses (n = 24). Leptospire DNA was detected by the 'pathogenic' specific PCR in 25 tissues (14%) and the control beta-actin PCR was positive in all 204 samples confirming DNA was extracted from all samples. No leptospires were isolated from these samples by culture and no positives were detected with the 'saprophytic' PCR. In a subsidiary experiment, the 'pathogenic' PCR was used to analyse kidney samples from rodents (n = 7) collected as part of vermin control in a zoo, with show animals with high microagglutination titres to Leptospira species, and five were positive. Fifteen PCR amplicons from 1 mouse, 2 rat and 14 pig kidney samples, were selected at random from positive PCRs (n = 30) and sequenced. Sequence data indicated L. interrogans DNA in the pig and rat samples and L. inadai DNA, which is considered of intermediate pathogenicity, in the mouse sample. The only successful culture was from this mouse kidney and the isolate was confirmed to be L. inadai by classical serology. These data suggest this suite of PCRs is suitable for testing for the presence of pathogenic leptospires in pig herds where abortions and infertility occur and potentially in other animals such as rodents. Crown Copyright (C) 2007 Published by Elsevier Ltd. All rights reserved.

Detection of hantaviruses in Brazilian rodents by SYBR-Green-based real-time RT-PCR

Current knowledge of the pathogenic hantavirus indicates that wild rodents are its primary natural reservoir. Specific primers to detect the presence of viral genomes were developed using an SYBR-Green-based real-time RT-PCR protocol. One hundred sixty-four rodents native to the Atlantic Forest biome were captured in So Paulo State, Brazil, and their tissues were tested. The presence of hantavirus RNA was detected in sixteen rodents: three specimens of Akodon montensis, three of Akodon cursor, two of Necromys lasiurus, one of Juliomys sp., one of Thaptomys nigrita, five of Oligoryzomys nigripes, and one of Oryzomys sp. This SYBR Green real-time RT-PCR method for detection of hantavirus may be useful for surveying hantaviruses in Brazil.

Inflammatory cytokine mRNA detection by real time PCR in chorioamniotic membranes from pregnant women with preterm premature rupture of membranes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Development of a real-time PCR method for rapid sexing of human preimplantation embryos

Genes on the X chromosome are known to be responsible for more than 200 hereditary diseases. After IVF, the simple selection of embryo sex before uterine transfer can prevent the occurrence of affected offspring among couples at risk for these genetic disorders. The aim of this investigation was to develop a rapid method of preimplantation genetic diagnosis (PGD) using real-time polymerase chain reaction (PCR) for the sexing of human embryos, and to compare it to the fluorescence in-situ hybridization technique, considered to be the gold standard. After biopsies were obtained from 40 surplus non-viable embryos for transfer, a total of 98 blastomeres were analysed. It was possible to analyse 24 embryos (60%) by both techniques, generating a total of 70 blastomeres (35 per technique), white 28 blastomeres from 16 embryos (40%) were analysed only by real-time PCR. A rapid and safe method was developed in the present study for the sexual diagnosis of a single human cell (blastomere and buccal cell) using the emerging technology of real-time PCR. (C) 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Quantification of bovine cytokine gene expression using real-time RT-PCR methodology

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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

O objetivo deste trabalho foi validar, pela técnica de PCR quantitativo em tempo real (RT-qPCR) genes para serem utilizados como referência em estudos de expressão gênica em soja, em ensaios de estresse hídrico. Foram avaliados quatro genes comumente utilizados em soja: Gmβ-actin, GmGAPDH, GmLectin e GmRNAr18S. O RNA total foi extraído de seis amostras: três amostras de raízes em sistema de hidroponia com diferentes intensidades de déficit hídrico (0, 25, 50, 75 e 100 minutos de estresse hídrico), e três amostras de folhas de plantas cultivadas em areia com diferentes umidades do solo (15, 5 e 2,5% de umidade gravimétrica). Os dados brutos do intervalo cycle threshold (Ct) foram analisados, e a eficiência de cada iniciador foi calculada para uma analise da Ct entre as diferentes amostras. A aplicação do programa GeNorm foi utilizada para a avaliação dos melhores genes de referência, de acordo com a estabilidade. O GmGAPDH foi o gene menos estável, com o maior valor médio de estabilidade de expressão (M), e os genes mais estáveis, com menor valor de M, foram o Gmβ-actin e GmRNAr18S, tanto nas amostras de raízes como nas de folhas. Estes genes podem ser usados em RT-qPCR como gens de referência para análises de expressão gênica.

Quantitative real-time PCR identifies a critical region of deletion on 22q13 related to prognosis in oral cancer

Quantitative real time PCR was performed on genomic DNA from 40 primary oral carcinomas and the normal adjacent tissues. The target genes ECGFB, DIA1, BIK, and PDGFB and the microsatellite markers D22S274 and D22S277, mapped on 22q13, were selected according to our previous loss of heterozygosity findings in head and neck tumors. Quantitative PCR relies on the comparison of the amount of product generated from a target gene and that generated from a disomic reference gene (GAPDH-housekeeping gene). Reactions have been performed with normal control in triplicates, using the 7700 Sequence Detection System (PE Applied Biosystems). Losses in the sequences D22S274 (22q13.31) and in the DIA1 (22q13.2-13.31) gene were detected in 10 out of 40 cases (25%) each. Statistically significant correlations were observed for patients with relative copy number loss of the marker D22S274 and stages T3-T4 of disease (P=0.025), family history of cancer (P = 0.001), and death (P = 0.021). Relative copy number loss involving the DIA1 gene was correlated to family history of cancer (P<0.001), death (P=0.002), and consumption of alcohol (P=0.026). Log-rank test revealed a significant decrease in survival (P=0.0018) for patients with DIA1 gene loss. Relative copy number losses detected in these sequences may be related to disease progression and a worse prognosis in patients with oral cancer.

Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)