Improvement and Optimization of a Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Detection and Typing of Vesicular Stomatitis Virus

Abstract

Abstract. An improvement to a previously reported real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay for the detection of Vesicular stomatitis virus (VSV) is described. Results indicate that the new assay is capable of detecting a panel of genetically representative strains of VSV present in North, Central, and South America. The assay is specific for VSV and allows for simultaneous differentiation between Vesicular stomatitis Indiana virus and Vesicular stomatitis New Jersey virus. This realtime RT-PCR is able to detect current circulating strains of VSV and can be used for rapid diagnosis of VSV and differentiation of VSV from other vesicular diseases, such as foot-and-mouth disease.

Rapid Molecular Detection of Genetically Diverted Foot and Mouth Disease Virus Serotype O During the Outbreak of 2012 in Egypt

“…In this study, we used IRES and 3D regions as targets to perform an assay of sensitive, fast and accurate detection of FMDV. The exploitation of the two RT-qPCR techniques either routine or high speed protocol could assess the sensitivity and accuracy of FMD diagnosis of the field samples Using of variety of samples and tissues isolated from different animal species in Egypt was considered an important need and good tool for detection of a real-life situation of FMDV observed in areas of endemic infections (Hole et al, 2010). Suitable laboratory methods are so-called rapid-cycle or high-speed PCR assays; results are provided in less than 1 h (Wittwer et al, 2001), those PCR systems have been described recently for various pathogens such as Vibrio cholera, group B Streptococcus bacteria, Influenza virus or adenoviruses (Fujimoto et al, 2010;Wilson et al, 2010;Sakurai et al, 2011;Koskela et al, 2009;Molsa et al, 2012).…”

Venereal Transmission of Vesicular Stomatitis Virus by Culicoides sonorensis Midges

“…RNA was precipitated using isopropanol, washed in 75% ethanol, and eluted in 50 µL of nuclease-free water. RNA extracts were analyzed using TaqMan Fast Virus 1-Step MasterMix (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a RT-qPCR targeting the L segment [53]: forward primer VSVNJ7274: 5 -TGATTCAATATAATTATTTTGGGAC-3; reverse primer VSVNJ7495: 5 -AGG CTCAGAGGCATGTTCAT-3 ; probe: FAM-TTGCACACCAGAACATTCAA-3 -BHQ1. For amplification, the following temperature profile was used: Reverse-transcription 1 cycle at 50 • C for 5 min, denaturing and polymerase activation at 95 • C for 20 s, and amplification: 40 cycles of 95 • C for 15 s and 60 • C for 60 s. Samples were initially tested in pools containing RNA of 5 individual midges followed by testing of individual samples from positive pools to determine the exact number of positive individuals.…”

Application of the real-time PCR in veterinary practice

“…В частности, проблема дифференциальной диагностики возникает в ряде эпизоотических ситуаций, и существуют реально сложившиеся комплексы манифестно сходных инфекций среди КРС (например, убиквитарные факторные болезни молодняка с пневмоэнтеритным синдромом), свиней (африканская, классическая чума свиней и другие болезни с геморрагическим синдромом), птицы (ньюкаслская болезнь, птичий грипп, парамиксовирусная и другие острые инфекции), заболевания с нейрологическими расстройствами (бешенство, болезнь Ауески, листериоз, прионные болезни). Решением в таком случае является ранняя лабораторная диагностика, в частности, с помощью ПЦР-РВ [6].…”

“…Данные значения служат инструментом для количественного определения инфекционного агента, которое выражают в стандартных единицах: количестве копий ДНК/мл, копий ДНК/г, копий ДНК/фиксированное количество клеток или титр возбудителя в десятичных логарифмах (lg ТЦД 50 /мл) [2,6,10]. При этом исследователи отмечают, что количество возбудителя заболевания, в частности, вирусов, с помощью ПЦР-РВ можно оценивать в диапазоне концентраций от 10 1-2 до 10 9 единиц [7,8] за счет широкого динамического диапазона кинетики накопления ампликонов.…”