Evaluation and comparison of SYBR Green I Real-Time PCR and TaqMan Real-Time PCR methods for quantitative assay of Listeria monocytogenes in nutrient broth and milk

Specific traditional plate count method and real-time PCR systems based on SYBR Green I and TaqMan technologies using a specific primer pair and probe for amplification of iap-gene were used for quantitative assay of Listeria monocytogenes in seven decimal serial dilution series of nutrient broth and milk samples containing 1.58 to 1.58×107 cfu /ml and the real-time PCR methods were compared with the plate count method with respect to accuracy and sensitivity. In this study, the plate count method was performed using surface-plating of 0.1 ml of each sample on Palcam Agar. The lowest detectable level for this method was 1.58×10 cfu/ml for both nutrient broth and milk samples. Using purified DNA as a template for generation of standard curves, as few as four copies of the iap-gene could be detected per reaction with both real-time PCR assays, indicating that they were highly sensitive. When these real-time PCR assays were applied to quantification of L. monocytogenes in decimal serial dilution series of nutrient broth and milk samples, 3.16×10 to 3.16×105 copies per reaction (equals to 1.58×103 to 1.58×107 cfu/ml L. monocytogenes) were detectable. As logarithmic cycles, for Plate Count and both molecular assays, the quantitative results of the detectable steps were similar to the inoculation levels.

Evaluation of Quantitative RT-PCR Using Nonamplified and Amplified RNA

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a standard assay in molecular medicine for gene expression analysis. Samples from incisional/needle biopsies, laser-microdissected tumor cells and other biologic sources, normally available in clinical cancer studies, generate very small amounts of RNA that are restrictive for expression analysis. As a consequence, an RNA amplification procedure is required to assess the gene expression levels of such sample types. The reproducibility and accuracy of relative gene expression data produced by sensitive methodology as qRT-PCR when cDNA converted from amplified (A) RNA is used as template has not yet been properly addressed. In this study, to properly evaluate this issue, we performed 1 round of linear RNA amplification in 2 breast cell lines (C5.2 and HB4a) and assessed the relative expression of 34 genes using cDNA converted from both nonamplified (NA) and A RNA. Relative gene expression was obtained from beta actin or glyceraldehyde 3-phosphate dehydrogenase normalized data using different dilutions of cDNA, wherein the variability and fold-change differences in the expression of the 2 methods were compared. Our data showed that 1 round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples. The use of cDNA that is converted from both A and NA RNA in a single qRT-PCR experiment clearly creates bias in relative gene expression data.

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)

Piezoelectric biosensors for real-time monitoring of hybridization and detection of hepatitis C virus

The piezoelectric quartz crystal resonators modified with oligonucleotide probes were used for detection of hepatitis C virus (HCV) in serum. The gold electrodes on either rough or smooth surface crystals were modified with a self-assembled monolayer of cystamine. After activation with glutaraldehyde, either avidin or streptavidin were immobilized and used for attachment of biotinylated DNA probes (four different sequences). Piezoelectric biosensors were used in a flow-through setup for direct monitoring of DNA resulting from the reverse transcriptase-linked polymerase chain reaction (RT-PCR) amplification of the original viral RNA. The samples of patients with hepatitis C were analyzed and the results were compared with the standard RT-PCR procedure (Amplicor test kit of Roche, microwell format with spectrophotometric evaluation). The piezoelectric hybridization assay was completed in 10 min and the same sensing surface was suitable for repeated use. (C) 2004 Elsevier B.V. All rights reserved.

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)

Cloning, sequencing and expression analysis of the equine hepcidin gene by real-time PCR

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

Anti-poliovirus activity of Baccharis dracunculifolia and propolis by cell viability determination and real-time PCR

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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.

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)

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.

Allele frequency of hereditary equine regional dermal asthenia in American Quarter horses in Brazil determined by quantitative real-time PCR with high resolution melting analysis

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

The detection of Cryptosporidium serpentis in snake fecal samples by real-time PCR

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

Diagnosis of gastric cryptosporidiosis in birds using a duplex real-time PCR assay

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