Detection and quantitation of akabane and aino viruses by multiplex real-time reverse-transcriptase PCR
| Autor: | Larisa Kuznetzova, Dalia Chai, Merisol Guini, Jackob Brenner, Hagai Yadin, Yehuda Stram, Ruth Meirom, Arie Rogel |
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| Rok vydání: | 2004 |
| Předmět: |
biology
Base Sequence Reverse Transcriptase Polymerase Chain Reaction Akabane virus Bunyaviridae Molecular Sequence Data RNA-Directed DNA Polymerase Amplicon biology.organism_classification Virology Molecular biology Reverse transcription polymerase chain reaction Complementary DNA Multiplex polymerase chain reaction TaqMan Animals RNA Viral Multiplex Cattle DNA Primers |
| Zdroj: | Journal of virological methods. 116(2) |
| ISSN: | 0166-0934 |
| Popis: | A multiplex, quantitive reverse-transcriptase real-time PCR, using MGB TaqMan chemistry, for detecting akabane virus (AKAV) and aino virus (AINV) is described. Each specific probe was labeled with a different fluorescent dye--VIC for detecting AKAV and 6-carboxy-fluorescein (FAM) for detecting AINV. All available sequences of viral S RNA were aligned and primers and probes were designed so that AKAV primers and probes would recognize all AKAVs but not AINV, and vice versa. The parameters for multiplex reactions enabled the detection of both viruses in one tube reaction with similar efficiency. To quantitate the viruses, cDNA amplicons containing the real-time amplicon were prepared using forward primers carrying the T7 promoter sequences. The cDNAs were used directly as templates for run-off transcription and 10-fold dilutions of the products served as standards to quantitate unknown viral samples. Using this system had shown that it could detect approximately 3-30 copies of viral S genome. |
| Databáze: | OpenAIRE |
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