Diagnosis of rabies and eastern and western equine viral encephalitides in equids by multiplex Hemi-Nested RT-PCR technique

Financial support: CNPq and Pasteur Institute of São Paulo

Analisi quantitativa dell'espressione genica mediante real-time rt-pcr.

Il primo capitolo di questo lavoro di tesi introduce i concetti di biologia necessari per comprendere il fenomeno dell’espressione genica. Il secondo capitolo descrive i metodi e le tecniche di laboratorio utilizzate per ottenere il cDNA, il materiale genetico che verrà amplificato nella real-time PCR. Nel terzo capitolo si descrive la tecnica di real-time PCR, partendo da una descrizione della PCR convenzionale fino a delineare le caratteristiche della sua evoluzione in real-time PCR. Si prosegue con la spiegazione del principio fisico alla base della tecnica e delle molecole necessarie (fluorofori e sonde) per realizzarla; infine si descrive l’hardware e il software dello strumento. Il quarto capitolo presenta le tecniche di analisi del segnale che utilizzano metodi di quantificazione assoluta o relativa. Infine nel quinto capitolo è presentato un caso di studio, cioè un’analisi di espressione genica con real-time PCR condotta durante l’esperienza di tirocinio presso il laboratorio ICM. e delle molecole necessarie (fluorofori e sonde) per realizzarla; infine si descrive l’hardware e il software dello strumento. Il quarto capitolo presenta le tecniche di analisi del segnale che utilizzano metodi di quantificazione assoluta o relativa. Infine nel quinto capitolo è presentato un caso di studio, cioè un’analisi di espressione genica con real-time PCR condotta durante l’esperienza di tirocinio presso il laboratorio ICM.

Virus isolation vs RT-PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions?

This study compared the results of reverse transcription-polymerase chain reaction (RT-PCR) and traditional virus isolation on cell culture in detection of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). RT-PCR was used for 172 tissue sample pools (total of 859 fish) originating from a field survey on the occurrence of VHSV and IHNV in farmed and wild salmonids in Switzerland. These samples represented all sites with fish that were either identified as virus-positive by means of virus isolation (three sites, four positive tissue sample pools) and/or demonstrated positive anti-VHSV-antibody titres (83 sites, 121 positive blood samples) in a serum plaque neutralization test (SPNT). The RT-PCR technique confirmed the four VHSV-positive tissue sample pools detected by virus isolation and additionally identified one VHSV-positive sample that showed positive anti-VHSV-AB titres, but was negative in virus isolation. With IHNV, RT-PCR detected two positive samples not identified by virus isolation while in these fish the SPNT result had been questionable. One of the IHNV-positive samples represents the first detection of IHNV-RNA in wild brown trout in Switzerland. Compared to SPNT, the RT-PCR method detected, as with virus isolation, a much lower number of positive cases; reasons for this discrepancy are discussed. Our results indicate that RT-PCR can not only be successfully applied in field surveys, but may also be slightly more sensitive than virus isolation. However, in a titration experiment under laboratory conditions, the sensitivity of RT-PCR was not significantly higher when compared with virus isolation.

Entwicklung einer PCR zum Nachweis der EAV-Infektion

The goal of this work was the development of suitable (real-time) RT-PCR techniques for fast and sensitive diagnosis of EAV and for molecular-epidemiological characterisation of viral strains, as an alternative to virus isolation. To this purpose two conventional RT-PCR methods and one real-time RT-PCR were adapted to detect the broadest possible spectrum of viral strains. Several dilutions with Bucyrus strain showed a 100-fold higher sensitivity of real-time RT-PCR and heminested RT-PCR compared to simple RT-PCR. Making use of 11 cell culture supernatants of different EAV isolates and 7 semen samples of positive stallions, the suitability of the techniques could be shown. Phylogenetic analysis of sequences of the newly analysed samples compared with known sequences indicated that more EAV-lineages exist than presently described.

Entwicklung einer real-time RT-PCR zum Nachweis von equinem Influenzavirus

Equine Influenza ist eine durch Influenza A-Viren verursachte, kontagiöse Respirationserkrankung beim Pferd. In dieser Arbeit wurde eine real-time RT-PCR in einem konservierten Abschnitt des Matrix-Segments des viralen Genoms für die schnelle und sensitive Diagnose von equinen Influenzaviren (EIV) und je eine RT-PCR Methode im Matrix- und im HA-Segment für die molekular-epidemiologische Charakterisierung der Viren entwickelt. Die Primer der real-time RT-PCR sind zu 99.4% der bekannten EIV-Sequenzen und zu 97.7% aller Influenza A-Sequenzen homolog. Die Homologie der Minor Groove Binder (MGB)-Sonde lag bei 99.3% und 99.6%. Diese hohen Werte ermöglichen die Anwendung des Assays für Influenzaviren bei anderen Spezies. Die diagnostische Eignung der Methode wurde mit Hilfe von 20 equinen, 11 porcinen sowie 2 aviären Proben verifiziert. Eine hohe Spezifität für Influenzaviren wurde experimentell und mittels Software-Simulation gezeigt. Die analytische Sensitivität des Tests lag bei 102–103 RNA-Kopien und 100–101 DNA-Kopien, was den Virusnachweis auch bei geringer Virusausscheidung ermöglicht. Alle amplifizierten EIV-Sequenzen konnten phylogenetisch den bekannten Linien zugeordnet werden.

Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy (R) or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to I infected in 800 samples with pepper but never detecting more than I infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

Detection sensitivity and quantitation of Plasmodium falciparum var gene transcripts by real-time RT-PCR in comparison with conventional RT-PCR

Antigenic variation in Plasmodium falciparum erythrocyte membrane protein 1, caused by a switch in transcription of the encoding var gene, is an important feature of malaria. In this study, we quantified the relative abundance of var gene transcripts present in P. falciparum parasite clones using real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional RT-PCR combined with cloning and sequencing, with the aim of directly comparing the results obtained. When there was sufficient abundance of RNA for the real-time RT-PCR assay to be operating within the region of good reproducibility, RT-PCR and real-time RT-PCR tended to identify the same dominant transcript, although some transcript-specific issues were identified. When there were differences in the estimated relative amounts of minor transcripts, the RT-PCR assay tended to produce higher estimates than real-time RT-PCR. These results provide valuable information comparing RT-PCR and real-time RT-PCR analysis of samples with small quantities of RNA as might be expected in the analysis of field or clinical samples.

Specific detection of enterovirus 71 directly from clinical specimens using real-time RT-PCR hybridization probe assay

Enterovirus 71 (EV71) is one of the main causative agents of hand, foot and mouth disease (HFMD) in young children. Infections caused by EV71 could lead to many complications, ranging from brainstem encephalitis to pulmonary oedema, resulting in high mortality. Thus, rapid detection of the virus is required to enable measures to be implemented in preventing widespread transmission. Based on primers and probes targeting at the VP1 region, a real-time reverse-transcriptase polymerase chain reaction (RT-PCR) hybridization probe assay was developed for specific detection of EV71 from clinical specimens. Quantitative analysis showed that the assay was able to detect as low as 5 EV71 viral copies and EV71 was detected from 46 of the 55 clinical specimens obtained from pediatric patients suffering from HFMD during the period from 2000 to 2003 in Singapore. This study showed that the single tube real-time RT-PCR assay developed in this study can be applied as a rapid and sensitive method for specific detection of EV71 directly from clinical specimens. (c) 2005 Elsevier Ltd. All rights reserved.