Development of 1a and 3a HCV genotyping based on 5-UTR region by Hybridization probe Realtime-PCR

Document Type : Research Article

Authors

• Ali Nazemi ¹
• seyed edalat pishkar ²

¹ The Partment of Biology, Faculty of Basic Sciences ,Tonekabon Branch, Islamic Azad University,Tonekabon, Iran

² Islamic Azad University Tonekabon Branch: Tonekabon, mazandaran, IR

Abstract

HCV with millions of patients is one of the leading causes of liver cell cancer and liver cirrhosis. Based on the nucleotide differences, HCV has several types that play an important role in response to treatment. this study aims to develop a fast and accurate way to identify the most common genotypes of this virus in Iran based on the 5´-UTR region using probe hybridization method. In this study, 45 positive serum samples with a Titer of more than 1000U/mL, all of which were infected with genotypes 1a and 3a, were prepared from Golestan hospital. After designing and synthesizing the primers and probes, the system hybridization of the probe and the extraction of viral RNA with High Pure RNA Viral Extraction kit (Roche, Germany) the reaction optimization was performed. The results of the resulting genotyping were then compared with the initial results of the samples. The results obtained on the device showed that genotypes 1a and 3a have different melting point peaks with a difference of about 2 degrees. Also, the results of the initial genotyping, which was based on the sequence determination of the 5´-UTR region, are consistent with the results obtained in this study. Although the hybridization method of the probe developed in this study is not perfect and is not able to identify 7 types of HCV, but despite the high frequency of 1a and 3a Genotypes in Iran (about 90%) and the speed of reaction and its single-stage can be Use it for initial screening.

Keywords

• Hepatitis C
• HCC
• Liver Cirrhosis
• Probe Hybridization
• RT-PCR

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