Optimization of Real-time PCR method to quantify the measles viral particles by using F gene

Subject Areas : Molecular Microbiology

Tahereh Zarei ¹ , Khosrow Aghaiypour ² , Farshid Kafilzadeh ³ , Abdolhamid Shoshtari ⁴

1 - Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran
2 - Department of Biotecnology, Razi Vaccine &Serum Research Institute, Karaj, Iran
3 - Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran
4 - Department of Research and Detection of Pultri Diseas, Razi Vaccine &Serum Research Institute, Karaj, Iran

Received: 2009-12-16 Accepted : 2010-01-15 Published : 2010-01-01

Keywords: Real-time PCR, Measles virus, F gene,

Abstract :

Background and objective: Although measles disease has controlled measles disease worldwide, there are some reports of the viral infection in vaccinated populations. Therefore, accurate and timely diagnosis is essential for the virus. Most of new molecular methods for virus detection cannot quantify the virus load. The purpose of this study was to set up Real-time PCR method using measles virus F gene. Materials and methods: Firstly, two primers and a probe from the F gene cloned into plasmid pET-22b (+)(Novagen) were designed. A ten fold serial dilution of standard plasmid containing the gene was prepared and a standard curve was plotted .The project was carried out on the 7500 Real-Time PCR detection System (Applied Biosystems) with Taq man kit (Applied Biosystems). Result: At least 30 viral particles per reaction could be determined and the dilutions between 10-4 to 10-9 equivalent 3×101 to 3×106 copies of the standard curve) were linear at best. Conclusion: Real-time PCR results showed that the technique can be used as highly specific and rapid method to detect and to determine the number of measles viral particles from the samples containing the virus.

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