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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

Efficiency of the real-time RT-PCR (NDWD). The linear regression of the CT-values (ordinate) and the titres of serially diluted CVS-11 (abscissa) exhibited a slope coefficient of −3.46 corresponding to an efficiency of 94.5% (E = 10(−1/slope  coefficient) − 1).
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fig2: Efficiency of the real-time RT-PCR (NDWD). The linear regression of the CT-values (ordinate) and the titres of serially diluted CVS-11 (abscissa) exhibited a slope coefficient of −3.46 corresponding to an efficiency of 94.5% (E = 10(−1/slope  coefficient) − 1).

Mentions: The efficiency of the real-time RT-PCR (NDWD) was determined using linear regression of the CT-values and the titres of the samples (Figure 2). On the basis of the slope coefficient, the efficiency can be inversely derived as E = 10(−1/slope  coefficient) − 1. The efficiency for CVS-11 was 94.5% with a slope coefficient of −3.46.


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

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

Efficiency of the real-time RT-PCR (NDWD). The linear regression of the CT-values (ordinate) and the titres of serially diluted CVS-11 (abscissa) exhibited a slope coefficient of −3.46 corresponding to an efficiency of 94.5% (E = 10(−1/slope  coefficient) − 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Efficiency of the real-time RT-PCR (NDWD). The linear regression of the CT-values (ordinate) and the titres of serially diluted CVS-11 (abscissa) exhibited a slope coefficient of −3.46 corresponding to an efficiency of 94.5% (E = 10(−1/slope  coefficient) − 1).
Mentions: The efficiency of the real-time RT-PCR (NDWD) was determined using linear regression of the CT-values and the titres of the samples (Figure 2). On the basis of the slope coefficient, the efficiency can be inversely derived as E = 10(−1/slope  coefficient) − 1. The efficiency for CVS-11 was 94.5% with a slope coefficient of −3.46.

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