novel sensitive multiplex RT-PCR ELISA method for detection of HIV-1/HCV co-infection

Despite sensitive antibody based blood donor screening, infection can be transmitted during window period. Therefore sensitive methods based on nucleic acid test [NATs] have been considered. The aim of this project was to design a more sensitive method for detection of PCR products and diagnosis of HIV-1/HCV co- infection accurately.After designing specific primers and probes, the Multiplex RT-PCR method was optimized and the PCR products were labeled with Digoxigenin. The PCR product was denatured under alkaline condition and was hybridized with the specific probe that had a biotin at 5' end, and then was added to streptavidin coated wells. After washing an antibody against DIG, conjugated with alkaline phosphates enzyme was used, following second washing, the substrate [ABTS] solution was added to each reaction well. Development of green color shows the positive where as no color shows negative results. 35 samples were tested with the developed method including 27 positive samples, 8 confirmed negative and 4 standard panels. False negative or positive reactions were not observed. This method had acceptable sensitivity and specificity for detecting HCV and HIV-1 infections during window period, also the method can be quantified which can be used for the flow-up and treatment of patients. In addition to the very high sensitivity of the test, it is cost effective and takes less time to perform

Development of a sensitive quantitative competitive PCR assay for detection of human cytomegalovirus DNA

Accurate and rapid diagnosis of human cytomegalovirus [HCMV] disease in immunocompromised patients has remained as a challenge. Quantitative competitive PCR [QC-PCR] methods for detection of HCMV in these individuals have improved the positive and negative predictive values of PCR for diagnosis of HCMV disease. In this study we used QC-PCR assay, using a co-amplified DNA standard, to quantitate the HCMV glycoprotein B [gB] gene in different samples. A DNA internal standard [IS] was designed by replacing HCMV primer binding site at 5' ends of primers that amplifies a 156-bp fragment of lambda genome, and a 200 bp amplicon was produced. Two DNA fragments of 257 bp wild type and 200-bp [IS] were co-amplified with the same oligonucleotide primer sets, analyzed by gel electrophoresis and used for construction of a standard curve. From this, the copy number of the gB gene present in different samples could be determined. Co-amplification with 1,000 copies of IS, allowed quantitation of 10-100,000 of HCMV DNA in a single PCR. This rapid assay avoids using radioactive components and other less efficient quantitative systems. It has the potential for early identification of patients at high risk of development of HCMV disease, and is useful for therapeutic monitoring