%0 Journal Article %K lyssavirus %K rabies %K real-time RT-PCR %K pyro-sequencing %K SYBRGreen %K molecular diagnostics %B Berliner und Münchener Tierärztliche Wochenschrift %C Hannover %D 2012 %G English %I Schlütersche Verlagsgesellschaft mbH & Co. KG %P 264-271 %R 10.2376/0005-9366-125-264 %T Perspectives on molecular detection methods of lyssaviruses %V 125 %1 {"oldId":70229,"title":"Perspectives on molecular detection methods of lyssaviruses","topline":"","teaserText":"","content":"

Summary<\/span>
Lyssaviruses belong to the family Rhabdoviridae<\/span> within the order Mononegavirales<\/span>. Their genomes consist of a negative stranded linear RNA. Although dog transmitted rabies causes the majority of the estimated 55 000 worldwide fatalities per year, bat lyssaviruses have also caused human cases. Based on a broad range of different host species, their geographical distribution and the evolutionary age lyssaviruses display a high degree of genetic variability. Thus the development of molecular diagnostics for the reliable detection and identification of members of the genus Lyssavirus<\/span> (pan-lyssavirus assay) is a challenge, but is valuable for the fight against rabies worldwide. In this study different published PCR-systems or primers for the detection of a wide range of lyssaviruses were tested as real-time RT-PCRs with different one-step and two-step assays. Comparison of different two-step assays by varying the RT-chemistry revealed differences in sensitivity. Nevertheless, most of the tested one-step systems provided an improved performance, including a reduced assay time and a reduced risk of cross-contaminations, when compared to the optimized two-step assay. Finally, we also provide an overview of additional state-of-the-art molecular methods to detect and differentiate lyssavirus species in general.

Keywords:<\/span>
lyssavirus, rabies, real-time RT-PCR, pyro-sequencing, SYBRGreen, molecular diagnostics


Zusammenfassung<\/span>
Lyssaviren geh\u00f6ren der Ordnung Mononegavirales<\/span> innerhalb der Familie Rhabdoviridae<\/span> an. Es handelt sich um einzelstr\u00e4ngige RNA-Viren mit einem linearen, negativ orientierten Genom. Obwohl die durch Hundebisse \u00fcbertragene Tollwut den Gro\u00dfteil der etwa 55 000 weltweiten Todesf\u00e4lle pro Jahr darstellt, k\u00f6nnen auch fast alle Fledermaus-Lyssaviren diese Krankheit beim Menschen hervorrufen. Basierend auf einem breiten Wirtsspektrum, ihrer geografischen Verteilung und dem evolution\u00e4ren Alter besitzen Lyssaviren einen erheblichen Grad genetischer Variabilit\u00e4t. Daher ist die Entwicklung molekular-diagnostischer Verfahren zum verl\u00e4sslichen Nachweis und zur Identifikation der Mitglieder des Genus Lyssavirus<\/span> (pan-lyssavirus assay) eine Herausforderung, andererseits aber sehr n\u00fctzlich im Kampf gegen die Tollwut und Tollwut-\u00e4hnliche Erkrankungen weltweit. In dieser Arbeit wurden unterschiedliche publizierte PCR Systeme oder Primer zum Nachweis einer gro\u00dfen Bandbreite von Lyssaviren als real-time RT-PCR mit verschiedenen one-step und two-step Systemen getestet. Ein Vergleich verschiedener two-step Systeme mittels Variation der RT-Chemie zeigte Unterschiede in der Sensitivit\u00e4t. Dennoch zeigten die meisten der getesteten one-step Methoden bessere Ergebnisse bei gleichzeitiger Verk\u00fcrzung der Versuchsdauer und Reduktion des Risikos von Kreuzkontaminationen im Vergleich zum optimierten two-step System. Schlie\u00dflich geben wir einen \u00dcberblick \u00fcber weitere aktuelle molekulare Methoden zum Lyssavirus-Nachweis sowie zur Differenzierung der unterschiedlichen Spezies.

Schl\u00fcsselw\u00f6rter:<\/span>
Lyssaviren, Tollwut, real-time RT-PCR, Pyro-Sequenzierung, SYBRGreen, molekulare Diagnostik <\/p>","categories":["Open Access","Tier\u00e4rztliche Wochenschrift","Abostufe BMTW","Fachartikel","Abostufe frei"],"fromDate":"May 4, 2012 12:00:00 AM","toDate":"Dec 31, 2050 12:00:00 AM","oldUrls":["http:\/\/vetline.de\/lyssavirus-rabies-real-time-rt-pcr-pyro-sequencing-sybrgreen-molecular-diagnostics\/150\/3130\/70229","http:\/\/vetline.de\/lyssavirus-rabies-real-time-rt-pcr-pyro-sequencing-sybrgreen-molecular-diagnostics\/150\/3216\/70229"],"doiLanguage":"englisch","doiProductFormat":"Online","doiPublisher":"Schl\u00fctersche Verlagsgesellschaft mbH & Co. KG","doiSerialWorkTitle":"Berl. M\u00fcnch. Tier\u00e4rztl. Wschr.","doiDocumentUri":"http:\/\/www.vetline.de\/lyssavirus-rabies-real-time-rt-pcr-pyro-sequencing-sybrgreen-molecular-diagnostics\/150\/3130\/70229","doiSource":"Berl. M\u00fcnch. Tier\u00e4rztl. Wschr. 125: 5-6, 264-271 (2012)","doiissn":"0005-9366","doiNr":"10.2376\/0005-9366-125-264","doiFirstPage":"264","doiLastPage":"271","doiTransmitted":true,"doiAuthor":"Fischer M, Hoffmann B, Freuling CM, M\u00fcller T, Beer M","pdf":{"path":"http:\/\/data\/bmtw_2012_05_0264.pdf","title":"bmtw - Perspectives on molecular detection methods of lyssaviruses ","description":""},"authors":[{"firstName":"M","middleName":"","lastName":"Fischer"},{"firstName":"B","middleName":"","lastName":"Hoffmann"},{"firstName":"C","middleName":"M","lastName":"Freuling"},{"firstName":"T","middleName":"","lastName":"M\u00fcller"},{"firstName":"M","middleName":"","lastName":"Beer"}],"contentOptimised":"

Summary<\/strong>
Lyssaviruses belong to the family Rhabdoviridae<\/em> within the order Mononegavirales<\/em>. Their genomes consist of a negative stranded linear RNA. Although dog transmitted rabies causes the majority of the estimated 55 000 worldwide fatalities per year, bat lyssaviruses have also caused human cases. Based on a broad range of different host species, their geographical distribution and the evolutionary age lyssaviruses display a high degree of genetic variability. Thus the development of molecular diagnostics for the reliable detection and identification of members of the genus Lyssavirus<\/em> (pan-lyssavirus assay) is a challenge, but is valuable for the fight against rabies worldwide. In this study different published PCR-systems or primers for the detection of a wide range of lyssaviruses were tested as real-time RT-PCRs with different one-step and two-step assays. Comparison of different two-step assays by varying the RT-chemistry revealed differences in sensitivity. Nevertheless, most of the tested one-step systems provided an improved performance, including a reduced assay time and a reduced risk of cross-contaminations, when compared to the optimized two-step assay. Finally, we also provide an overview of additional state-of-the-art molecular methods to detect and differentiate lyssavirus species in general.

Keywords:<\/strong>
lyssavirus, rabies, real-time RT-PCR, pyro-sequencing, SYBRGreen, molecular diagnostics


Zusammenfassung<\/strong>
Lyssaviren geh\u00f6ren der Ordnung Mononegavirales<\/em> innerhalb der Familie Rhabdoviridae<\/em> an. Es handelt sich um einzelstr\u00e4ngige RNA-Viren mit einem linearen, negativ orientierten Genom. Obwohl die durch Hundebisse \u00fcbertragene Tollwut den Gro\u00dfteil der etwa 55 000 weltweiten Todesf\u00e4lle pro Jahr darstellt, k\u00f6nnen auch fast alle Fledermaus-Lyssaviren diese Krankheit beim Menschen hervorrufen. Basierend auf einem breiten Wirtsspektrum, ihrer geografischen Verteilung und dem evolution\u00e4ren Alter besitzen Lyssaviren einen erheblichen Grad genetischer Variabilit\u00e4t. Daher ist die Entwicklung molekular-diagnostischer Verfahren zum verl\u00e4sslichen Nachweis und zur Identifikation der Mitglieder des Genus Lyssavirus<\/em> (pan-lyssavirus assay) eine Herausforderung, andererseits aber sehr n\u00fctzlich im Kampf gegen die Tollwut und Tollwut-\u00e4hnliche Erkrankungen weltweit. In dieser Arbeit wurden unterschiedliche publizierte PCR Systeme oder Primer zum Nachweis einer gro\u00dfen Bandbreite von Lyssaviren als real-time RT-PCR mit verschiedenen one-step und two-step Systemen getestet. Ein Vergleich verschiedener two-step Systeme mittels Variation der RT-Chemie zeigte Unterschiede in der Sensitivit\u00e4t. Dennoch zeigten die meisten der getesteten one-step Methoden bessere Ergebnisse bei gleichzeitiger Verk\u00fcrzung der Versuchsdauer und Reduktion des Risikos von Kreuzkontaminationen im Vergleich zum optimierten two-step System. Schlie\u00dflich geben wir einen \u00dcberblick \u00fcber weitere aktuelle molekulare Methoden zum Lyssavirus-Nachweis sowie zur Differenzierung der unterschiedlichen Spezies.

Schl\u00fcsselw\u00f6rter:<\/strong>
Lyssaviren, Tollwut, real-time RT-PCR, Pyro-Sequenzierung, SYBRGreen, molekulare Diagnostik <\/p>","primaryLanguage":"englisch","summary":"Lyssaviruses belong to the family Rhabdoviridae<\/em> within the order Mononegavirales<\/em>. Their genomes consist of a negative stranded linear RNA. Although dog transmitted rabies causes the majority of the estimated 55 000 worldwide fatalities per year, bat lyssaviruses have also caused human cases. Based on a broad range of different host species, their geographical distribution and the evolutionary age lyssaviruses display a high degree of genetic variability. Thus the development of molecular diagnostics for the reliable detection and identification of members of the genus Lyssavirus<\/em> (pan-lyssavirus assay) is a challenge, but is valuable for the fight against rabies worldwide. In this study different published PCR-systems or primers for the detection of a wide range of lyssaviruses were tested as real-time RT-PCRs with different one-step and two-step assays. Comparison of different two-step assays by varying the RT-chemistry revealed differences in sensitivity. Nevertheless, most of the tested one-step systems provided an improved performance, including a reduced assay time and a reduced risk of cross-contaminations, when compared to the optimized two-step assay. Finally, we also provide an overview of additional state-of-the-art molecular methods to detect and differentiate lyssavirus species in general.","keywords":["lyssavirus","rabies","real-time RT-PCR","pyro-sequencing","SYBRGreen","molecular diagnostics"],"zusammenfassung":"Lyssaviren geh\u00f6ren der Ordnung Mononegavirales<\/em> innerhalb der Familie Rhabdoviridae<\/em> an. Es handelt sich um einzelstr\u00e4ngige RNA-Viren mit einem linearen, negativ orientierten Genom. Obwohl die durch Hundebisse \u00fcbertragene Tollwut den Gro\u00dfteil der etwa 55 000 weltweiten Todesf\u00e4lle pro Jahr darstellt, k\u00f6nnen auch fast alle Fledermaus-Lyssaviren diese Krankheit beim Menschen hervorrufen. Basierend auf einem breiten Wirtsspektrum, ihrer geografischen Verteilung und dem evolution\u00e4ren Alter besitzen Lyssaviren einen erheblichen Grad genetischer Variabilit\u00e4t. Daher ist die Entwicklung molekular-diagnostischer Verfahren zum verl\u00e4sslichen Nachweis und zur Identifikation der Mitglieder des Genus Lyssavirus<\/em> (pan-lyssavirus assay) eine Herausforderung, andererseits aber sehr n\u00fctzlich im Kampf gegen die Tollwut und Tollwut-\u00e4hnliche Erkrankungen weltweit. In dieser Arbeit wurden unterschiedliche publizierte PCR Systeme oder Primer zum Nachweis einer gro\u00dfen Bandbreite von Lyssaviren als real-time RT-PCR mit verschiedenen one-step und two-step Systemen getestet. Ein Vergleich verschiedener two-step Systeme mittels Variation der RT-Chemie zeigte Unterschiede in der Sensitivit\u00e4t. Dennoch zeigten die meisten der getesteten one-step Methoden bessere Ergebnisse bei gleichzeitiger Verk\u00fcrzung der Versuchsdauer und Reduktion des Risikos von Kreuzkontaminationen im Vergleich zum optimierten two-step System. Schlie\u00dflich geben wir einen \u00dcberblick \u00fcber weitere aktuelle molekulare Methoden zum Lyssavirus-Nachweis sowie zur Differenzierung der unterschiedlichen Spezies.","schluesselwoerter":["Lyssaviren","Tollwut","real-time RT-PCR","Pyro-Sequenzierung","SYBRGreen","molekulare Diagnostik"],"translatedTitle":"","abstractE":"Lyssaviruses belong to the family Rhabdoviridae within the order Mononegavirales. Their genomes consist of a negative stranded linear RNA. Although dog transmitted rabies causes the majority of the estimated 55 000 worldwide fatalities per year, bat lyssaviruses have also caused human cases. Based on a broad range of different host species, their geographical distribution and the evolutionary age lyssaviruses display a high degree of genetic variability. Thus the development of molecular diagnostics for the reliable detection and identification of members of the genus Lyssavirus (pan-lyssavirus assay) is a challenge, but is valuable for the fight against rabies worldwide. In this study different published PCR-systems or primers for the detection of a wide range of lyssaviruses were tested as real-time RT-PCRs with different one-step and two-step assays. Comparison of different two-step assays by varying the RT-chemistry revealed differences in sensitivity. Nevertheless, most of the tested one-step systems provided an improved performance, including a reduced assay time and a reduced risk of cross-contaminations, when compared to the optimized two-step assay. Finally, we also provide an overview of additional state-of-the-art molecular methods to detect and differentiate lyssavirus species in general.","date":{"year":2012,"date":"05\/2012","accepted":"2012-05-04"},"volume":"125","openAccess":true,"journal":"Berliner und M\u00fcnchener Tier\u00e4rztliche Wochenschrift","titleImageId":944,"pages":"264-271","redirects":["lyssavirus-rabies-real-time-rt-pcr-pyro-sequencing-sybrgreen-molecular-diagnostics\/150\/3130\/70229","lyssavirus-rabies-real-time-rt-pcr-pyro-sequencing-sybrgreen-molecular-diagnostics\/150\/3216\/70229"],"tierartCategories":[],"artikelartCategories":["Open Access","Tier\u00e4rztliche Wochenschrift","Abostufe BMTW","Fachartikel","Abostufe frei"]} %8 05/2012