Prevalence of beak and feather disease virus in wild Platycercus elegans: comparison of three tissue types using a probe-based real-time qPCR test

The detection of avian viruses in wild populations has considerable conservation implications. For DNA-based studies, feathers may be a convenient sample type for virus screening and are, therefore, an increasingly common technique. This is despite recent concerns about DNA quality, ethics, and a paucity of data comparing the reliability and sensitivity of feather sampling to other common sample types such as blood. Alternatively, skeletal muscle tissue may offer a convenient sample to collect from dead birds, which may reveal viraemia. Here, we describe a probe-based quantitative real-time PCR for the relative quantification of beak and feather disease virus (BFDV), a pathogen of serious conservation concern for parrots globally. We used this method to test for BFDV in wild crimson rosellas (Platycercus elegans), and compared three different sample types. We detected BFDV in samples from 29 out of 84 individuals (34.5%). However, feather samples provided discordant results concerning virus presence when compared with muscle tissue and blood, and estimates of viral load varied somewhat between different sample types. This study provides evidence for widespread infection of BFDV in wild crimson rosellas, but highlights the importance of sample type when generating and interpreting qualitative and quantitative avian virus data.

Accessing Planktothrix species diversity and associated toxins using quantitative real-time PCR in natural waters

Tese de Doutoramento em Biologia apresentada à Faculdade de Ciências da Universidade do Porto, 2015.

Comparative validation using quantitative real-time PCR (qPCR) and conventional PCR of bovine semen centrifuged in continuous density gradient

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

Development and application of a real-time TaqMan((R)) qPCR assay for detection and quantification of 'Candidatus Mycoplasma haemolamae' in South American camelids

Two alpacas from a herd in southwest Switzerland died for unknown reasons. Necropsy revealed chronic weight loss and pale mucous membranes. Infection with hemotropic mycoplasmas was suspected and subsequently confirmed by molecular methods. In order to investigate the epidemiological situation in this herd, a real-time TaqMan((R)) qPCR assay for the specific detection and quantification of hemoplasma infection in South American camelids was developed. This assay was based on the 16S rRNA gene and amplified 'Candidatus Mycoplasma haemolamae' DNA, but not DNA from other hemoplasmas or non-hemotropic mycoplasma species. The lower detection limit was one copy/PCR, and the amplification efficiency was 97.4%. In 11 out of 24 clinically healthy herd mates of the two infected alpacas, 'Candidatus M. haemolamae' infection was confirmed. No correlation was found between bacterial load and clinical signs or anemia. The assay described herein enables to detect and quantify 'Candidatus M. haemolamae' and may be used in future studies to investigate the prevalence, pathogenesis and treatment follow-up of hemoplasma infections in South American camelids.

Absolute quantitation of Marek's disease virus and Herpesvirus of turkeys in chicken lymphocyte, feather tip and dust samples using real-time PCR

The further development of Taqman quantitative real-time PCR (qPCR) assays for the absolute quantitation of Marek's disease virus serotype 1 (MDV1) and Herpesvirus of turkeys (HVT) viruses is described and the sensitivity and reproducibility of each assay reported. Using plasmid DNA copies, the lower limit of detection was determined to be 5 copies for the MDV1 assay and 75 copies for the HVT assay. Both assays were found to be highly reproducible for Ct values and calculated copy numbers with mean intra- and inter-assay coefficients of variation being less than 5% for Ct and 20% for calculated copy number. The genome copy number of MDV1 and HVT viruses was quantified in PBL and feather tips from experimentally infected chickens, and field poultry dust samples. Parallelism was demonstrated between the plasmid-based standard curves, and standard curves derived from infected spleen material containing both viral and host DNA, allowing the latter to be used for absolute quantification. These methods should prove useful for the reliable differentiation and absolute quantitation of MDV1 and HVT viruses in a wide range of samples.

Real-time PCR microfluidic devices with concurrent electrochemical detection

Electrochemistry-based detection methods hold great potential towards development of hand-held nucleic-acid analyses instruments. In this work, we demonstrate the implementation of in situ electrochemical (EC) detection method in a microfluidic flow-through EC-qPCR (FTEC-qPCR) device, where both the amplification of the target nucleic-acid sequence and subsequent EC detection of the PCR amplicon are realized simultaneously at selected PCR cycles in the same device. The FTEC-qPCR device utilizes methylene blue (MB), an electroactive DNA intercalator, for electrochemical signal measurements in the presence of PCR reagent components. Our EC detection method is advantageous, when compared to other existing EC methods for PCR amplicon analysis, since FTEC-qPCR does not require probe-modified electrodes, or asymmetric PCR, or solid-phase PCR. Key technical issues related to surface passivation, electrochemical measurement, PCR inhibition by metal electrode, bubble-free PCR, were investigated. By controlling the concentration of MB and the exposure of PCR mixture to the bare metal electrode, we successfully demonstrated electrochemical measurement of MB in solution-phase, symmetric PCR by amplifying a fragment of lambda phage DNA.

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.

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.

Polymerase chain reaction and real-time PCR for diagnosing of Leishmania infantum chagasi in dogs

A importância do cão como reservatório de L. infantum chagasi no meio urbano tem estimulado a realização de inúmeros trabalhos de avaliação de técnicas de diagnóstico, uma vez que este procedimento, quando realizado corretamente, torna-se um importante passo na prevenção da doença em humanos. Dentre os métodos de diagnóstico, as técnicas moleculares têm adquirido destaque. Objetivou-se neste trabalho verificar o desempenho da Reação em Cadeia da Polimerase (PCR) e da PCR em tempo real (qPCR) para diagnóstico da Leishmaniose Visceral Canina (LVC) utilizando diferentes amostras biológicas. Para tanto foram utilizados 35 cães provenientes de uma área endêmica para LVC, onde foram utilizados para o diagnóstico molecular, aspirado de medula óssea, fragmentos de linfonodo e baço. Neste estudo a qPCR foi capaz de detectar um maior número de animais positivos quando comparada com a PCR. Já entre as diferentes amostras biológicas utilizadas não foi observada diferença significativa na detecção de DNA de L. infantumchagasi por meio da PCR e qPCR. Mesmo assim, considerando a facilidade de obtenção, o linfonodo pode ser considerada como a melhor amostra para diagnóstico molecular da infecção por L. infantum chagasi.

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.

Validation of a reference control for an SYBR-Green fluorescence assay-based real-time PCR for detection of bovine herpesvirus 5 in experimentally exposed bovine embryos

The objective of this study was to optimize an internal control to improve SYBR-Green-based qPCR to amplify/detect the BoHV-5 US9 gene in bovine embryos produced invitro and experimentally exposed to the virus. We designed an SYBR-Green-based binding assay that is quick to perform, reliable, easily optimized and compares well with the published assay. Herein we demonstrated its general applicability to detect BoHV-5 US9 gene in bovine embryos produced invitro experimentally exposed to BoHV-5. In order to validate the assay, three different reference genes were tested; and the histone 2a gene was shown to be the most adequate for normalizing the qPCR reaction, by considering melting and standard curves ( p<0.05). On the other hand, no differences were found in the development of bovine embryos invitro whether they were exposed to BoHV-5 reference and field strains comparing to unexposed embryos. The developed qPCR assay may have important field applications as it provides an accurate BoHV-5 US9 gene detection using a proven reference gene and is considerably less expensive than the TaqMan qPCR currently employed in sanitary programs. © 2013 Elsevier Ltd.

Assessment of a quantitative 5' nuclease real-time polymerase chain reaction using the nicotinamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) for Bartonella species in domiciled and stray cats in Brazil

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

Validation of absolute quantitative real-time PCR for the diagnosis of streptococcus agalactiae in fish

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

Brazilian spotted fever: Real-time PCR for diagnosis of fatal cases

Suspicion of Brazilian spotted fever (BSF) should occur in endemic regions upon surveillance of the acute febrile icteric hemorrhagic syndrome (AFIHS). However, limitations associated with currently available laboratory tests pose a challenge to early diagnosis, especially in fatal cases. Two real-time PCR (qPCR) protocols were evaluated to diagnose BSF in 110 fatal AFIHS cases, collected in BSF-endemic regions in 2009-2010. Of these, 24 were positive and 86 negative by indirect immunofluorescence (IFA) assay (cutoff IgG and/or IgM >= 128). DNA from these samples was used in the qPCR protocols: one to detect Rickettsia spp. (Citrate synthase gene) and another to determine spotted fever group (SFG) Rickettsia species (OmpA gene). Of the 24 IFA-positive samples, 5 (21%) were positive for OmpA and 9 (38%) for citrate synthase. In the IFA-negative group (n = 86), OmpA and citrate synthase were positive in 23 (27%) and 27 (31%), respectively. These results showed that the 2 qPCR protocols were about twice as sensitive as the IFA test alone (93% concordance). In conclusion, qPCR is a sensitive method for the diagnosis of fatal BSF cases and should be considered for routine surveillance of AFIHS in places like Brazil, where spotted fever-related lethality is high and other endemic diseases like dengue and leptospirosis can mislead diagnosis. (C) 2012 Elsevier GmbH. All rights reserved.

Suitability of real-time quantitative PCR to estimate the relative telomere length in European Hake (Merluccius merluccius Linnaeus, 1758)

Telomere length measurement has been proposed as a promising tool to estimate the age of individuals in natural populations. We used real-time quantitative PCR (qPCR) to measure relative telomere length in four tissues (brain, kidney, liver and muscle) of European hake (Merluccius merluccius) in different groups based upon body length an otolith age estimate. We observed a high level of inter-individual differences in the measurements of relative telomere length in hakes of similar age and body length groups. The results of qPCR analysis showed a great variability in all measures and a lack of repeatability and reproducibility with significant statistical differences in the results of the different assays. The paper discusses the technical reasons for the variability in qPCR obtained in this work and by other authors.