Development of a new- fast and sensitive one step/one tube nested RT-PCR method in a closed system by double-joint primer for detecting HCV

Objective: Polymerase chain reaction (PCR) is one of the most important progress in the field of molecular biology and diagnosis. Despite simplicity in concept, the reaction needs complex interaction between target sequence, primers, dNTPs and DNA polymerase for a successful amplification and diagnosis. For the detection of RNA viruses highly sensetive and specific technique is required. Hence amplification based on Nested PCR would improve sensitivity, and also use of one step reaction would decrease probable contaminations as reported previously in several studies. The aim of the current study was to develop a new, rapid and sensitive one step– one tube Nested PCR in a closed system, by using two novel coherent primers. Materials and Methods: In this study, a novel and special primer development method was used for one step– one tube reaction. After development and optimization, the assay was evaluated with known positive and negative controls. Results: The developed assay was performed on ۵۰ HCV positive samples and ۱۰ negative controls and ۵ samples from each HIV, HBV, TTV (Torque Teno Virus) and GBV-C (Hepatitis G Virus: HGV). Based on the obtained results, sensitivity and specifity was calculated. ۴۸ out of ۵۰ HCV positive samples showed expected band while none of the negative controls gave any band. Conclusion: Based on the specific primer design system which has been used in the current study; the inner primer was synthesized as complementary of routine PCR primers and was bound to the outer primers. Therefore, there is no probability for false priming in the both rounds of the reaction, hence there would be no nonspecific amplifications. Other advantages of this assay system were prevention of contamination which was due to one step- one tube reaction, decrease in duration and the cost of the reaction.

کلیدواژه ها:

Nested PCR ، HCV ، Nested PCR ، One Step/One Tube PCR ، Fast PCR ، ویروس هپاتیت C ، PCR یک لولهای- یک مرحلهای ، PCR سریع