Entomopoxvirüslerin Viral Titresinin Kantitatif Gerçek Zamanlı PCR (qPCR) ile Belirlenmesi

Year 2024, , 570 - 576, 31.12.2024

Erkan Anıl Aydin , Cihan İnan

https://doi.org/10.35229/jaes.1559848

Abstract

Poksvirüsler, hem insanları hem hayvanları hem de böcekleri enfekte eden büyük çift sarmallı DNA (dsDNA) virüsleridir. Bu virüslerin en bilinen üyesi, çiçek hastalığına neden olan ve aşılama ile ortadan kaldırılan Variola virüsüdür. Diğer üyeler hala konakçılarını enfekte ederken, bu virüsleri tespit etmek, izole etmek ve onlarla savaşmak önemlidir. Bu amaçla virüslerin titrelerini belirlemek değerlidir. Virüs titreleri aşı geliştirme, antiviral aktivite, genetik mühendisliği, gen terapisi ve hastalık tedavi çalışmaları için kullanılır. Tüm bu kullanımlara rağmen, virüs titresini belirlemek için kullanılan yöntemler sıkıcı ve zaman alıcıdır. Bu çalışmada titre belirlemenin hızını ve verimliliğini artırmak için kantitatif gerçek zamanlı PCR kullanan bir yöntem geliştirilmiştir. Bu yöntemde, AMEV virüsünden bilinen tek kopyalı bir gen kullanılarak, bir plazmite klonlandı ve standart grafikler oluşturuldu. Titresi bilinmeyen virüsü standart grafik kullanarak analiz edildi. Sonuç olarak Poxvirüslerin titresi basit, hızlı ve etkili bir şekilde belirlenebileceği bir metod geliştirildi.

Keywords

qPCR, Poksvirus, AMEV, Virus Titresi, DNA

Determination of the Entomopoxviruses Viral Titer by Quantitative Real Time PCR (qPCR)

Abstract

Poxviruses are large double-stranded DNA (dsDNA) viruses that infects both human, animals and insects. Most known member of this viruses is Variola virus that caused pox disease and eradicated by vaccination. While other members still infects their hosts it is important to detect, isolate and fight with this viruses. For this purpose determining the titers of viruses is important. Virus titers are used for vaccine development, antiviral activity, genetic engineering, gene therapy and disease treatment studies. Despite all these uses, the methods used to determine virus titer are tedious and time-consuming. We developed a method using quantitative real-time PCR to improve the speed and efficiency of titer determination. In this method, we used a known single-copy gene from AMEV virus, cloned it into a plasmid and generated standard graphs. We measured the virus whose titer was unknown using the standard graph. As a result, a method was developed that can be used to determine the titer of Poxviruses in a simple, fast and effective way.

Keywords

qPCR, Poxvirus, AMEV, Virus Titer, DNA

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