[Evaluation of touchdown nested PCR to circumvent spurious priming and increase specificity during HIV and GBV-C gene amplification]

Primer-Template hybridization temperature is one of the important parameters in Nested PCR optimization. Unlike instant temperature for sequence amplification in routine PCR process, Touchdown PCR is a modified form of standard PCR that employs a range of annealing temperature. This study intended to develop a Touchdown Nested PCR in order to circumvent spurious priming and enhancing specify during gene amplification. This is an experimental study conducted at Tarbiat Modarres University of Tehran during 2008-2009. Study samples were collected from Digestive Diseases Research Centre at Shariati Hospital and HIV research center-Imam Khomeini Hospital. After extracting the nucleic acid, primer designing for HIV and GBV-C and c-DNA synthesis; Nested PCR was performed on negative and positive samples using standard and touchdown protocols. The intended band was observed in all positive samples. No band was observed in any human and viral negative control samples. After electrophoresis of PCR products, non specific band were seen in HIV and GBV-C samples during standard PCR. Using the touchdown protocol, undesirable bands were omitted or significantly decreased. In the present study, despite the formation of uncalled bands in standard reaction; using the touchdown method led to omission of non-specific bands without any significant effect on the final products. As for its simplicity, cost and time saving, it seems that using this method is a rational and economical way for fast optimization of PCR reactions

Detection and prevalence of polyoma virus BK among Iranian kidney transplant patients by a novel nested-PCR

BK virus is an increasingly recognized pathogen in transplant recipient patients associated with nephropathy and emerged as a cause of allograft failure linked to immunosuppressive regimens in renal transplant recipients. This study develops a sensitive PCR method to detect the viremia and viruria as well as the incidence of BK virus infection in renal transplant recipients. A nested PCR method was developed and a total of 45 paired serum and urine samples from renal transplant recipient patients were collected and tested with the developed assay. The threshold of the developed detection assay was 10 copies/ul of BKV DNA in samples. Our results also indicated that about 40% of the urine and 26.7% of serum samples were positive for BKV in renal transplant patients in this study. This Nested-PCR method was found a specific, sensitive and simple procedure for detection of viruria and viremia of BK virus in renal transplant recipients