Comparative Analysis of Rabies Virus Reverse Transcription-PCR and Virus Isolation Using Samples from a Patient Infected with Rabies Virus

Panning, Marcus; Baumgarte, Sigrid; Pfefferle, Susanne; Maier, Tanja; Martens, Andreas; Drosten, Christian

doi:10.1128/jcm.00728-10

Cited by 19 publications

(13 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A number of real-time RT-PCRs have been designed for specifically detecting individual strains or lineages of RABV 2 and also to detect lyssaviruses across the genus 3,4,5,6,7,8,9 . All assays will have a limit of detection dependent on how conserved the primer (and if necessary, the probe) sequences are across the genus.…”

Section: Introductionmentioning

confidence: 99%

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Marston

Jennings

MacLaren

et al. 2019

JoVE

View full text Add to dashboard Cite

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to confirm rabies diagnosis but have only recently been accepted by the OIE (World Organisation for Animal Health) as a primary method to detect rabies infection. Real-time RT-PCR assays provide real-time data, and are closed-tube systems, minimizing the risk of contamination during setup. DNA intercalating fluorochrome real-time RT-PCR assays do not require expensive probes, minimizing the cost per sample, and when the primers are designed in conserved regions, assays that are specific across virus genera rather than specific to just one virus species are possible. Here we describe a pan-lyssavirus SYBR real-time RT-PCR assay that detects lyssaviruses across the Lyssavirus genus, including the most divergent viruses IKOV, WCBV and LLEBV. In conjunction with dissociation curve analysis, this assay is sensitive and specific, with the advantage of detecting all lyssavirus species. The assay has been adopted in many diagnostic laboratories with quality assured environments, enabling robust, rapid, sensitive diagnosis of animal and human rabies cases.

show abstract

Section: Introductionmentioning

confidence: 99%

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Marston

Jennings

MacLaren

et al. 2019

JoVE

View full text Add to dashboard Cite

show abstract

“…Furthermore, repeated sampling of skin biopsies is not practical for improvement of sensitivity of the FAT (10,14,15). To overcome the limits of FAT, several conventional and hemi-nested reverse transcription polymerase chain reaction assays (RT-PCR) targeting the N or L gene, have been developed (16,17,18,19,20,21,22) and widely applied for the intra-vitam diagnosis of human rabies routine diagnosis. Indeed, the N gene is the most conserved region among the RABV genome which is the reason for its frequent use as target for rabies virus diagnostic assays (23).…”

Section: Introductionmentioning

confidence: 99%

Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus

Faye

Dacheux

Weidmann

et al. 2017

Journal of Virological Methods

View full text Add to dashboard Cite

Rabies virus (RABV) remains one of the most important global zoonotic pathogens. RABV causes rabies, an acute encephalomyelitis associated with a high rate of mortality in humans and animals and affecting different parts of the world, particularly in Asia and Africa. Confirmation of rabies diagnosis relies on laboratory diagnosis, in which molecular techniques such as detection of viral RNA by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used. In this study, two real-time quantitative RT-PCR assays were developed for large-spectrum detection of RABV, with a focus on African isolates. The primer and probe sets were targeted highly conserved regions of the nucleoprotein (N) and polymerase (L) genes. The results indicated the absence of non-specific amplification and cross-reaction with a range of other viruses belonging to the same taxonomic family, i.e. Rhabdoviridae, as well as negative brain tissues from various host species. Analytical sensitivity ranged between 100 to 10 standard RNA copies detected per reaction for N-gene and L-gene assays, respectively. Effective detection and high sensitivity of these assays on African isolates showed that they can be successfully applied in general research and used in diagnostic process and epizootic surveillance in Africa using a double-check strategy.

show abstract

“…Superiority of the real time RT‐PCR over the conventional virus isolation methods has been reported for many other viruses such as influenza virus [Pregliasco et al, ; Steininger et al, ], respiratory syncytial virus [Reis et al, ], Rabies virus [Panning et al, ], Dengue virus [Azhar et al, ], and the human metapneumovirus [Matsuzaki et al, ]. Extensive work by several authors on diagnosis of the human metapneumovirus also showed that virus isolation was only successful in 33–50% of cases in which samples were diagnosed to be positive by RT‐PCR [Williams et al, ; Ebihara et al, ; Kahn, ; Collins and Crowe, ].…”

Section: Discussionmentioning

confidence: 99%

Comparison of RT‐PCR assay and virus isolation in cell culture for the detection of alkhumra hemorrhagic fever virus

Madani

Abuelzein

Azhar

et al. 2013

Journal of Medical Virology

View full text Add to dashboard Cite

Alkhumra hemorrhagic fever virus (AHFV) is an emerging flavivirus that was isolated originally from Saudi Arabia in 1994-1995. The main tests used for the detection of AHFV are the real time (rt) RT-PCR and virus isolation in cell culture. In the present study the detection of AHFV by rtRT-PCR was compared with virus isolation in BHK-21, HEp-2, and LLC-MK2 cell lines. AHFV suspensions grown in BHK-21, HEp-2, and LLC-MK2 cell lines were serially diluted 10-fold from 10(-1) to 10(-11) . Samples from each dilution were used to inoculate four cell culture tubes and were also examined by the rtRT-PCR for AHFV RNA. Fifteen non-inoculated cell culture samples (five from each cell line) were included blindly in both tests. Thus, a total of 132 AHFV-positive and 15 negative control samples were tested. The rtRT-PCR could detect the viral RNA in all diluted specimens up to and including the 10(-10) dilution (40 specimens for each cell line), whereas, cell cultures were positive in 70% of specimens for BHK-21, 65% for LLC-MK2, and 45% for HEp-2 at this dilution. None of the three cell cultures nor the rtRT-PCR was positive at 10(-11) dilution. The specificity and positive predictive values of virus isolation compared to rtRT-PCR were each 100%, whereas the negative predictive values were 29.4% for BHK-21, 26.3% for LLC-MK2, and 18.5% for HEp-2. In conclusion, the rtRT-PCR is more sensitive than virus isolation for detecting AHFV.

show abstract

Comparative Analysis of Rabies Virus Reverse Transcription-PCR and Virus Isolation Using Samples from a Patient Infected with Rabies Virus

Cited by 19 publications

References 15 publications

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus

Comparison of RT‐PCR assay and virus isolation in cell culture for the detection of alkhumra hemorrhagic fever virus

Contact Info

Product

Resources

About