Detecção do herpes vírus felino tipo 1 (HVF-1) pela técnica de PCR em tempo real e sua associação com sinais oculares em uma poipulação de gatos domésticos

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

Real-time polymerase chain-reaction detection of pathogens is feasible to supplement the diagnostic sequence for urinary tract infections

To evaluate, in a prospective pilot study, the feasibility of identifying pathogens in urine using real-time polymerase chain reaction (PCR), and to compare the results with the conventional urine culture-based procedures.

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.

Comparison of fecal culture and F57 real-time polymerase chain reaction for the detection of Mycobacterium avium subspecies paratuberculosis in Swiss cattle herds with a history of paratuberculosis

BACKGROUND: Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP. RESULTS: By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%). CONCLUSIONS: There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease

New methods for quantification and analysis of quantitative real-time polymerase chain reaction data

Quantitative real-time polymerase chain reaction (qPCR) is a sensitive gene quantitation method that has been widely used in the biological and biomedical fields. The currently used methods for PCR data analysis, including the threshold cycle (CT) method, linear and non-linear model fitting methods, all require subtracting background fluorescence. However, the removal of background fluorescence is usually inaccurate, and therefore can distort results. Here, we propose a new method, the taking-difference linear regression method, to overcome this limitation. Briefly, for each two consecutive PCR cycles, we subtracted the fluorescence in the former cycle from that in the later cycle, transforming the n cycle raw data into n-1 cycle data. Then linear regression was applied to the natural logarithm of the transformed data. Finally, amplification efficiencies and the initial DNA molecular numbers were calculated for each PCR run. To evaluate this new method, we compared it in terms of accuracy and precision with the original linear regression method with three background corrections, being the mean of cycles 1-3, the mean of cycles 3-7, and the minimum. Three criteria, including threshold identification, max R2, and max slope, were employed to search for target data points. Considering that PCR data are time series data, we also applied linear mixed models. Collectively, when the threshold identification criterion was applied and when the linear mixed model was adopted, the taking-difference linear regression method was superior as it gave an accurate estimation of initial DNA amount and a reasonable estimation of PCR amplification efficiencies. When the criteria of max R2 and max slope were used, the original linear regression method gave an accurate estimation of initial DNA amount. Overall, the taking-difference linear regression method avoids the error in subtracting an unknown background and thus it is theoretically more accurate and reliable. This method is easy to perform and the taking-difference strategy can be extended to all current methods for qPCR data analysis.^

Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid `real-time' polymerase chain reaction

We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples. This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction. The time from sample collection to result was less than 1h. RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment.

A 5 '-nuclease real-time reverse transcriptase-polymerase chain reaction assay for the detection of a broad range of influenza A subtypes, including H5N1

A 5'-nuclease real-time reverse transcriptase-polymerase chain reaction assay was developed for the detection of influenza type A and was validated using a range of influenza A subtypes, including avian strains, and 126 nasopharyngeal aspirate samples. The results show the assay is suitable for screening for influenza A infections, particularly in regions where avian strains may be circulating. (c) 2005 Elsevier Inc. All rights reserved.

O uso da PCR em tempo real para o estudo da carga parasitária e dos níveis transcricionais durante a infecção experimental por Trypanosoma cruzi

Trypanosoma cruzi is causative agent of Chagas disease, one of most neglected tropical diseases. Estimated that about 11 million people worldwide are infected by T. cruzi and about 6 to 7 million people are at risk in endemic areas. During the process of invasion of host and parasite interact enabling signal transduction and gene expression modulation in response to invasion. The diversity of activated proteins and pathways to repair the damage by disruption of the plasma membrane interest to us and thus present study developed a new form of detection and quantitation by polymerase chain reaction in real time (qPCR) of parasitic load T. cruzi and quantified transcriptional levels relative (RT-qPCR) of dysferlin, Sphingomyelin acid esferase (ASM), transcription factor EB (TFEB) Galectins 1 and 3 and Annexin A2. This study demonstrated that quantification by real time PCR using primers P21fw and P21rv was specific and sensitive for detection of T. cruzi in vivo and in vitro, as well as transcriptional levels of genes related to cytoskeletal organization and repair plasma membrane are modulated in response to damage generated by parasite.

Minimal residual disease monitoring in multiple myeloma: a comparison between allelic-specific oligonucleotide real-time quantitative polymerase chain reaction and flow cytometry.

BACKGROUND AND OBJECTIVES: Minimal residual disease (MRD) studies are useful in multiple myeloma (MM). However, the definition of the best technique and clinical utility are still unresolved issues. The aim of this study was to analyze and compare the clinical utility of MRD studies in MM with two different techniques: allelic-specific oligonucleotide real-time quantitative PCR (ASO-RQ-PCR), and flow cytometry (FCM). DESIGN AND METHODS: Bone marrow samples from 32 MM patients who had achieved complete response after transplantation were evaluated by ASO-RQ-PCR, using TaqMan technology, and multiparametric FCM. RESULTS: ASO-RQ-PCR was only applicable in 75% of patients for a variety of technical reasons, while FCM was applicable in up to 90%. Therefore, simultaneous PCR/FCM analysis was possible in only 24 patients. The number of residual tumor cells identified by both techniques was very similar (mean=0.29%, range=0.001-1.61%, correlation coefficient=0.861). However, RQ-PCR was able to detect residual myelomatous cells in 17 patients while FCM only did so in 11; thus, 6 cases were FCM negative but PCR positive, all of them displaying a very low number of clonal cells (median=0.014%, range=0.001-0.11). Using an MRD threshold of 0.01% (10(-4)) two risk groups with significantly different progression-free survival could be identified by either PCR (34 vs. 15m, p=0.04) or FCM (27 vs. 10m, p=0.05). INTERPRETATION AND CONCLUSIONS: Although MRD evaluation by ASO-RQ-PCR is slightly more sensitive and specific than FCM, it is applicable in a lower proportion of MM patients and is more time-consuming, while both techniques provide similar prognostic information.

A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.