A rapid RT-PCR method to differentiate six established genotypes of rabies and rabies-related viruses using TaqMan™ technology

Black, Elizabeth M; Lowings, J. P.; Smith, Jemma; Heaton, Paul R.; McElhinney, Lorraine M.

doi:10.1016/s0166-0934(02)00062-9

Cited by 54 publications

(30 citation statements)

References 22 publications

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“…An optimized RT-PCR protocol for the detection of rabies virus genomic RNA performed on saliva combined with immunofluorescence assay of skin biopsy samples detected 9 of 9 patients with confirmed rabies by postmortem examination and 0 of 19 patients with suspected rabies who were later determined not to have rabies (61). Rapid RT-PCR with TaqMan genotype-specific probes can distinguish among the six established rabies virus and rabies virus-related genotypes (24).…”

Section: Other Viruses (I) Human Immunodeficiency Virusmentioning

confidence: 99%

Molecular Methods for Diagnosis of Viral Encephalitis

2004

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Hundreds of viruses cause central nervous system (CNS) disease, including meningoencephalitis and postinfectious encephalomyelitis, in humans. The cerebrospinal fluid (CSF) is abnormal in >90% of cases; however, routine CSF studies only rarely lead to identification of a specific etiologic agent. Diagnosis of viral infections of the CNS has been revolutionized by the advent of new molecular diagnostic technologies to amplify viral nucleic acid from CSF, including PCR, nucleic acid sequence-based amplification, and branched-DNA assay. PCR is ideally suited for identifying fastidious organisms that may be difficult or impossible to culture and has been widely applied for detection of both DNA and RNA viruses in CSF. The technique can be performed rapidly and inexpensively and has become an integral component of diagnostic medical practice in the United States and other developed countries. In addition to its use for identification of etiologic agents of CNS disease in the clinical setting, PCR has also been used to quantitate viral load and monitor duration and adequacy of antiviral drug therapy. PCR has also been applied in the research setting to help discriminate active versus postinfectious immune-mediate disease, identify determinants of drug resistance, and investigate the etiology of neurologic disease of uncertain cause. This review discusses general principles of PCR and reverse transcription-PCR, including qualitative, quantitative, and multiplex techniques, with comment on issues of sensitivity, specificity, and positive and negative predictive values. The application of molecular diagnostic methods for diagnosis of specific infectious entities is reviewed in detail, including viruses for which PCR is of proven efficacy and is widely available, viruses for which PCR is less widely available or for which PCR has unproven sensitivity and specificity, and nonviral entities which can mimic viral CNS disease

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Section: Other Viruses (I) Human Immunodeficiency Virusmentioning

confidence: 99%

Molecular Methods for Diagnosis of Viral Encephalitis

2004

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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.…”

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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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“…The most widely employed is heminested reverse transcription-PCR (hnRT-PCR) (21). However, this method has been shown in previous studies to have several inherent disadvantages, such as a low dynamic range, low sensitivity, and high risk of contamination (2,21,31). Furthermore, the first universal hnRT-PCR assay developed for the detection of six species of lyssaviruses (9), including 22 lyssavirus isolates from Africa, was developed over a decade ago.…”

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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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A rapid RT-PCR method to differentiate six established genotypes of rabies and rabies-related viruses using TaqMan™ technology

Cited by 54 publications

References 22 publications

Molecular Methods for Diagnosis of Viral Encephalitis

Molecular Methods for Diagnosis of Viral Encephalitis

Lyssavirus Detection and Typing Using Pyrosequencing

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

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