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Real-Time RT-PCR for the Detection of Lyssavirus Species.

Deubelbeiss A, Zahno ML, Zanoni M, Bruegger D, Zanoni R - J Vet Med (2014)

Bottom Line: For the detection of species, seven probes were developed.Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown.Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology and Immunology, 3012 Berne, Switzerland.

ABSTRACT
The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

No MeSH data available.


Related in: MedlinePlus

Amplification of a CVS-11 serial dilution using hnRT-PCR. Ethidium bromide stained gel after amplification of the CVS-11 strain using dilutions from 10−1 to 10−8 (A–H). The amplification product of 582 bp is clearly visible up to a dilution of 10−5 (lane E). Ladder = standard for determination of amplification product size.
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fig1: Amplification of a CVS-11 serial dilution using hnRT-PCR. Ethidium bromide stained gel after amplification of the CVS-11 strain using dilutions from 10−1 to 10−8 (A–H). The amplification product of 582 bp is clearly visible up to a dilution of 10−5 (lane E). Ladder = standard for determination of amplification product size.

Mentions: For the comparison with infectious titres, RNA of the classical rabies virus strain CVS-11 was extracted from cell culture supernatants (BHK-21 cells or neuroblastoma cells MNA 42/13) and serially diluted from 10−1 to 10−8. The infectious titre was determined according to the Spearman-Karber formula [49, 50] in fluorescent focus forming doses 50% (FFD50). In the heminested RT-PCR (hnRT-PCR), the amplification product of 582 bp was visible up to a dilution of 10−5 corresponding to a detection limit of 0.4 FFD50 (10−0.4 FFD50; Figure 1). In the real-time RT-PCR (NDWD) the serial dilution was performed in triplicate. The limit of detection was defined as the last dilution at which at least 2 out of 3 replicates were positive. Using this definition, CVS-11 was detectable up to a dilution of 10−7 (once at a dilution of 10−8). The amplification plot showed regular intervals of the curves with CT-values from 18.1 (dilution 10−1) to 40.0 (dilution 10−7). Relating to infectious titre real-time RT-PCR reached a detection limit of 0.003 FFD50 (10−2.6) for CVS-11.


Real-Time RT-PCR for the Detection of Lyssavirus Species.

Deubelbeiss A, Zahno ML, Zanoni M, Bruegger D, Zanoni R - J Vet Med (2014)

Amplification of a CVS-11 serial dilution using hnRT-PCR. Ethidium bromide stained gel after amplification of the CVS-11 strain using dilutions from 10−1 to 10−8 (A–H). The amplification product of 582 bp is clearly visible up to a dilution of 10−5 (lane E). Ladder = standard for determination of amplification product size.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4590848&req=5

fig1: Amplification of a CVS-11 serial dilution using hnRT-PCR. Ethidium bromide stained gel after amplification of the CVS-11 strain using dilutions from 10−1 to 10−8 (A–H). The amplification product of 582 bp is clearly visible up to a dilution of 10−5 (lane E). Ladder = standard for determination of amplification product size.
Mentions: For the comparison with infectious titres, RNA of the classical rabies virus strain CVS-11 was extracted from cell culture supernatants (BHK-21 cells or neuroblastoma cells MNA 42/13) and serially diluted from 10−1 to 10−8. The infectious titre was determined according to the Spearman-Karber formula [49, 50] in fluorescent focus forming doses 50% (FFD50). In the heminested RT-PCR (hnRT-PCR), the amplification product of 582 bp was visible up to a dilution of 10−5 corresponding to a detection limit of 0.4 FFD50 (10−0.4 FFD50; Figure 1). In the real-time RT-PCR (NDWD) the serial dilution was performed in triplicate. The limit of detection was defined as the last dilution at which at least 2 out of 3 replicates were positive. Using this definition, CVS-11 was detectable up to a dilution of 10−7 (once at a dilution of 10−8). The amplification plot showed regular intervals of the curves with CT-values from 18.1 (dilution 10−1) to 40.0 (dilution 10−7). Relating to infectious titre real-time RT-PCR reached a detection limit of 0.003 FFD50 (10−2.6) for CVS-11.

Bottom Line: For the detection of species, seven probes were developed.Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown.Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology and Immunology, 3012 Berne, Switzerland.

ABSTRACT
The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

No MeSH data available.


Related in: MedlinePlus