Development of a TaqMan real-time RT-PCR assay for the detection of rabies virus

Wacharapluesadee, Supaporn; Sutipanya, Jittapawan; Damrongwatanapokin, Sudarat; Phumesin, Patta; Chamnanpood, Pornchai; Leowijuk, Chaweewan; Hemachudha, Thiravat

doi:10.1016/j.jviromet.2008.05.004

Cited by 33 publications

(30 citation statements)

References 9 publications

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“…For this purpose, sensitive, specific-but-broad-spectrum assays such as the one described here should be applied. Other published real-time RT-PCR assays have been validated using small numbers of RABV strains only (29,20,27,36), thus their use for international reference laboratories needs additional evaluation.…”

Section: Discussionmentioning

confidence: 99%

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy

Hoffmann

Freuling

Wakeley³

et al. 2010

J Clin Microbiol

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To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs, an improved diagnostic sensitivity and reliability can be ascertained for postmortem and intra vitam real-time RT-PCR analyses in rabies reference laboratories.

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Section: Discussionmentioning

confidence: 99%

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy

Hoffmann

Freuling

Wakeley³

et al. 2010

J Clin Microbiol

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“…They are based on nested and real-time reverse transcription (RT)-PCR (5,9,19,30) or nucleic acid sequence-based amplification (NASBA) (36,37). Other protocols have also been developed to be used in animal diagnosis (3,20,35,38,39). However, all presently available methods have showed some limitations.…”

mentioning

confidence: 99%

“…Moreover, due to the variety of the members of the genus Lyssavirus, it is essential for a reliable molecular assay to be able to detect but also differentiate among existent rabies and rabies-related viruses. However, almost all of the molecular techniques available (9,19,30,35,36,38) require further sequencing of PCR products for lyssavirus typing, adding on an extra day for completing and interpreting results. The requirement for further genomic sequencing in order to clearly define the species involved in the infection increases the time and final cost of the complete analysis.…”

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confidence: 99%

Lyssavirus Detection and Typing Using Pyrosequencing

et al. 2011

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Rabies is a fatal zoonosis caused by a nonsegmented negative-strand RNA virus, namely, rabies virus (RABV). Apart from RABV, at least 10 additional species are known as rabies-related lyssaviruses (RRVs), and some of them are responsible for occasional spillovers into humans. More lyssaviruses have also been detected recently in different bat ecosystems, thanks to the application of molecular diagnostic methods. Due to the variety of the members of the genus Lyssavirus, there is the necessity to develop a reliable molecular assay for rabies diagnosis able to detect and differentiate among the existing rabies and rabies-related viruses. In the present study, a pyrosequencing protocol targeting the 3 terminus of the nucleoprotein (N) gene was applied for the rapid characterization of lyssaviruses. Correct identification of species was achieved for each sample tested. Results from the pyrosequencing assay were also confirmed by those obtained using the Sanger sequencing method. A pan-lyssavirus one-step reverse transcription (RT)-PCR was developed within the framework of the pyrosequencing procedure. The sensitivity (Se) of the one-step RT-PCR assay was determined by using in vitro-transcribed RNA and serial dilutions of titrated viruses. The assay demonstrated high analytical and relative specificity (Sp) (98.94%) and sensitivity (99.71%). To date, this is the first case in which pyrosequencing has been applied for lyssavirus identification using a cheaper diagnostic approach than the one for all the other protocols for rapid typing that we are acquainted with. Results from this study indicate that this procedure is suitable for lyssavirus detection in samples of both human and animal origin.

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“…Although these assays were sensitive, often detecting single RNA molecules (31), the lack of sequence homology between the various species has been cited as the main reason for their failure as universal real-time PCR assays, and thus, a separate primer-probe set was required for each species (11). The chemistry of all of the above-mentioned assays has been hydrolysis probes (often called TaqMan probes) due to their relative flexibility, i.e., they allow a certain degree of mismatching (up to four mismatches) between the target and the probe without affecting overall detection efficiency (30). Africa is host to wide diversity of lyssaviruses (4 different known species) that also display high intragenotypic variation, some of which were demonstrated only recently (19), and currently described diagnostic methods may not detect this high diversity.…”

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confidence: 99%

Improved PCR Methods for Detection of African Rabies and Rabies-Related Lyssaviruses

et al. 2010

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Eleven different lyssavirus species, four of which occur on the African continent, are presently recognized. These viruses cause rabies, the burden of which is highest in the developing world, where routine laboratory diagnosis is often not available. From an epidemiological and control perspective, it is necessary that diagnostic methods detect the diversity of lyssaviruses present in different regions of the world. A published and widely used heminested reverse transcription-PCR (hnRT-PCR) was evaluated for its ability to detect a panel of diverse African lyssaviruses. Due to the limitations experienced for this assay, an alternative hnRT-PCR was developed. The new assay was found to be accurate and sensitive in the detection of African lyssavirus RNA in a variety of clinical specimens. The assay was further adapted to a real-time PCR platform to allow rapid, one-step, quantitative, and single-probe detection, and an internal control for the verification of sample preparation was included. The limit of detection of the real-time PCR assay was 10 RNA copies per reaction, with inter-and intra-assay variability below 4%. Subsequently, in demonstrating utility, both assays were successfully applied to antemortem rabies diagnosis in humans. We believe that the quantitative real-time PCR assay could find application as a routine confirmatory test for rabies diagnosis in the future and that it will serve as a valuable research tool in the biology of African lyssaviruses. Alternatively, the hnRT-PCR assay can be used in laboratories that do not have access to expensive real-time PCR equipment for sensitive diagnosis of lyssaviruses.

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Development of a TaqMan real-time RT-PCR assay for the detection of rabies virus

Cited by 33 publications

References 9 publications

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy

Lyssavirus Detection and Typing Using Pyrosequencing

Improved PCR Methods for Detection of African Rabies and Rabies-Related Lyssaviruses

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