Serotype-specific detection of African horsesickness virus by real-time PCR and the influence of genetic variations.
نویسنده
چکیده
Real-time PCR hybridization probe sets were tested for the specific detection of amplified genome segment 2 cDNA from all nine serotypes of African horsesickness virus (AHSV). The hybridization probes were derived from the sequences of genome segments 2 of the nine reference strains of the virus and were designed to have clearly distinguishable peak melting temperatures. Viral dsRNA from each of the serotypes was specifically detected after reverse transcription, real-time PCR and melting curve analysis. The method was used to successfully serotype a range of field isolates, although most of the these showed peak melting temperature shifts. These shifts could be related to nucleotide substitutions in the regions that are targeted by the probes. Sensitivity was demonstrated to be sufficient for use with dsRNA isolated directly from infected organ samples, making it potentially useful as a rapid diagnostic tool.
منابع مشابه
A Comparison of Different PCR-Based Methods for the Detection of African Horsesickness Virus
Detection of African horsesickness virus (AHSV) by three different reverse transcription polymerase chain reaction (RT-PCR) based methods were compared. Primers were complementary to sequences contained in the gene encoding non-structural protein 2 of AHSV serotype 3. All three assays were group-specific for AHSV and detected all nine serotypes but not related orbiviruses. A dilution range was ...
متن کاملDetection of African horsesickness virus and discrimination between two equine orbivirus serogroups by reverse transcription polymerase chain reaction.
A reverse transcription polymerase chain reaction (RT-PCR), based on the gene encoding the NS2 protein of African horsesickness virus (AHSV), was developed for rapid serogroup-specific detection of AHSV. The specificity of RT-PCR products was confirmed by Southern blot hybridization using a radioactively labelled cDNA probe specific for the NS2 gene. This RT-PCR could discriminate between all k...
متن کاملPolymerase chain reaction for the detection and differentiation of Marek’s disease virus strains MDV-1 and HVT
Marek’s disease (MD) is a lymphoproliferative disease of chickens characterized by lymphocyticinfiltration of various organs. The present study was an attempt to use polymerase chain reaction (PCR) tooptimize a rapid and reliable assay for detection of MDV genome. Detection of serotype 1 of MDV (MDV-1)was confirmed by presence of a 200 bp DNA fragment as a PCR product. Differentiation of MDV-1 ...
متن کاملDevelopment of SYBR Green I Based Real-Time RT-PCR Assay for Specific Detection of Watermelon silver mottle Virus
Background: Watermelon silver mottle virus (WSMoV), which belongs to the genus Tospovirus, causes significant loss in Cucurbitaceae plants. Objectives: Development of a highly sensitive and reliable detection method for WSMoV. Materials and Methods: Recombinant plasmids for targeting the sequence of nucleocapsid protein gene of WSMoV were constructed. SYBR Green I real-time PCR was established...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Journal of virological methods
دوره 154 1-2 شماره
صفحات -
تاریخ انتشار 2008