Journal of Arak University of Medical Sciences

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Development and application of a sensitive multiplex real-time RT-PCR for simultaneously detection of HIV-1 and HCV in plasma samples

Mahdi Paryan, Mahdieh Mondanizadeh, Samira Mohammadi-Yeganeh , Behzad Khansarinejad,
Volume 14, Issue 5 (11-2011)

Abstract

Background: HIV-1 and HCV are two of the most important blood-borne infectious agents. Hence, reliable, precise, and sensitive detection of these viruses in infected patients and donated blood units is highly important. Noticing the limitations of serological assays in detection of these infectious agents, this study was to use fast and sensitive molecular assays like real-time PCR. Materials and Methods: In this trial, a home-brewed SYBR green-based multiplex real time PCR, on the basis of melting curve analysis, was developed for the single or simultaneous detection of HCV and HIV-1 infections in plasma samples. Data were analyzed using SPSS software version 16. Results: The results obtained from different reactions on several clinical samples showed that the analytical sensitivities of the developed assay for HIV-1 and HCV were 200 and 100 copies/ml, respectively. It was also shown that the primers designed for each virus had no interaction with each other and other interfering agents. Conclusion: Noticing the good level of sensitivity and specificity, easy handling, relatively low cost, and rapid analysis of samples, this method can be a useful and rapid approach for simple and effective detection of HCV and HIV-1 in plasma samples.

Rapid detection of HIV-1 viral RNA by real-time transcription mediated amplification assay

Mahdi Paryan, Samira Mohammadi Yeganeh, Behzad Khansari Nejad, Mahdieh Mondanizadeh, Saeed Paryan,
Volume 15, Issue 4 (September 2012)

Abstract

Background: Several different molecular methods have been developed that are capable of detecting HIV-1 in clinical specimens with different levels of sensitivity and specificity. This article describes the results of a reliability study on the development and application of a new real-time TMA method for isothermal detection of HIV-1. Materials and Methods: In this ex Primental study, the molecular beacon primer and probe set were designed for a 176-base-pair region of HIV-1 pol gene using a specialized software. Logarithmic serial dilutions from 10-107 copies of an in-vitro transcribed RNA were used for determination of the analytical sensitivity of the assay. Clinical specimens that had previously been evaluated positive or negative by a valid commercial assay were used for assessing the clinical sensitivity and specificity of the assay. Results: The analytical and clinical sensitivities of the assay were determined 500 copies/ml and 93.3%, respectively. The primers and the probe were HIV-1 specific and no cross-reaction was observed with other blood-borne viruses and human genome bioinformatically. The clinical specificity of the developed real-time TMA assay was examined experimentally using 20 negative samples and determined to be 100%. Conclusion: The developed real-time TMA assay can be used as an appropriate tool for the rapid and isothermal detection of HIV-1 in patients' blood and plasma samples.

Application of Real–Time PCR for Rapid Detection of Rifampicin Resistant Mycobacterium Tuberculosis

Behnaz Taheri, Siyamak Samiee, Mehdi Paryan, Ehsanollah Ghaznavi-Rad,
Volume 16, Issue 5 (8-2013)

Abstract

Background: The emergence of drug-resistant strain of M.tuberculosis is one of the most critical issues facing TB researchers and clinicians. Rapid diagnosis of drug-resistant tuberculosis is essential for the prompt initiation of effective second-line therapy to improve treatment outcome and limit transmission of this obstinate disease. The aim of this study is to develop a Real-time PCR assay for the detection of mutations in RRDR (rifampcin resistance determinant region) of rpoB which conferring rifampicin resistance in Mycobacterium tuberculosis.

Materials and Methods: In this experimental study, the primer and probe set were designed for a RRDR region of rpoB gene using a specialized software. Clinical specimens that had previously been evaluated resistant or sensitive by using convential method, were used for assessing the clinical sensitivity and specificity of the assay.

Results: The clinical sensitivity of the assay was determined 100%. The primers and the probes were rpoB specific and no cross-reaction was observed with other microorganisms and human genome bioinformatically. The clinical specificity of developed Real-time PCR assay was examined experimentally using 25 negative samples and determined to be 100%.

Conclusion: The developed real-time PCR assay can be used as an appropriate and efficient tool for the rapid detection of rifampicin-resistant Mycobacterium tuberculosis.

Bioinformatic Prediction of miRNAs Targeting NOTCH1 and HBx Genes in Chronic Hepatitis B-Induced Hepatocellular Carcinoma

Niloofar Moradi, Mehdi Paryan, Behzad Khansarinejad, Mohammad Rafiei, Mahdieh Mondanizadeh,
Volume 19, Issue 12 (3-2017)

Abstract

Abstract

Background: Hepatocellular carcinoma (HCC) is the third major cause of cancer death worldwide. Hepatitis B virus (HBV) and HBx gene play an important role in the development of HCC by influencing signaling pathways. Since there is no detectable symptom in the early phase of HCC, there is need to find new HCC-specific markers with high sensitivity for early detection and diagnosis of HCC. On the other hand, by the advent and development of bioinformatic sciences, it is now possible to predict miRNAs as biomarkers, and their targets. Therefore, in the present study, based on the results of the bioinformatic software applications with different algorithm, we selected the miRNA targeting HBx and NOTCH1 mRNAs according to higher score, suitable connection with target gene and confirming them in more softwares.

Materials and Methods: First, the sequences of NOTCH1 and HBx genes were retrieved from NCBI. Afterwards, several software applications such as TargetScan, mirWalk, miRBase, Miranda, PicTar, miRVir, and DIANA were applied to predict miRNAs.

Results: Based on the high scoring by bioinformatics softwares and suitable targeting, miR-34a were selected to target NOTCH1 and miR-6510, miR-5193 and miR-214 were chosen to targetHBX gene.

Conclusion: Because of tumor suppression roles of miR-214 and miR-34a, they probably could be used as therapeutic strategy in cancer researches. It is also seems that the miR-5193 could act as a specific marker in Hepatocellular carcinoma.

Preparation of Mono Specific Antiserum against Salmonella O and H antigens for Application in Agglutination and ELISA Tests

Mrs. Mahnaz Ghahramani Til, Mrs. Rezvaneh Sadat Fatemi, Dr. Rahman Shokri, Dr. Mahdi Banitalebi Dehkordi, Dr. Mahdi Paryan,
Volume 25, Issue 4 (October & November 2022)

Abstract

Introduction: Salmonella infection (salmonellosis) is a common bacterial disease that affects the intestinal tract. Several methods like Multiplex or real-time PCR, ELISA, and Agglutination are used to identify these bacteria. However, normally rapid, cost effective and easy diagnostic methods such as agglutination test is recommended. In Iran, positive control antiserums used in diagnostic kits work based on polyvalent agglutination and are against O and H antigens. The purpose of this research was to produce specific anti-sera against O and H antigens for using in agglutination and ELISA kits.
Methods: New Zealand white rabbits were immunized by intravenous injections of inactivated bacterial O and H antigens adjusted to a cell density equivalent to a turbidity of a McFarland number 3 standard. Serum collection was performed 7 days after the last injection. Collected Antisera were tested with positive human specimens as well as cross-reaction antibodies. Absorption method was used to obtain specific anti-sera against O and H antigens. Produced Anti-O and Anti-H antibodies were mixed with bacterial H and O antigens respectively and incubated for 1 hours in 37˚c. The Mixture was centrifuged and the supernatant was collected. Furthermore, in order to use these antisera in specific kits such as ELISA, Immunofluorescence etc., purification methods like Ammonium sulphate precipitation, tangential Flow Filtration and Chromatography were performed. This study was approved by the ethics committee of Pasteur Institute of Iran (Code: IR.PII.REC.1399.006).
Results: Results of agglutination test before and after adsorption showed cross-reaction before adsorption and no cross-reaction with H and O antigens with monospecific antisera against O and H after adsorption, respectively. Moreover, high quality and quantity of mono-specific antibody was obtained after purification.
Conclusions: Serum-based assays are recommended for the timely diagnosis of the disease since these assays are specific, sensitive, inexpensive, and rapid. Therefore, the produced antiserum in the present research can be used in primary screening of salmonella infections based on agglutination tests which are cost effective and simple. In addition, purified anti-sera can be used in the development of ELISA and Immunofluorescence assays.

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