[Detection of HCMV infection in glioma brain tumors]

Human cytomegalovirus [HCMV] is a beta-herpesvirus that causes persistent infection in humans, as well as severe disease in fetuses and immunocompromised individuals. Although HCMV is not currently causally implicated in human cancer, emerging evidence suggests that HCMV infection may be specifically associated with malignancies such as gliomas. Gliomas are one of the most common brain tumors that affect humans. It is classified into four grades. In this study, we have developed and used a real-time PCR method for the detection and diagnosis of HCMV infection in glioma brain tumor samples. Paraffin-embedded tumor samples were chosen from patients who referred to Imam Khomeini Hospital Neurosurgery Ward. DNA was extracted from paraffinembedded tissues by a DNA extraction kit. After designing specific primers for the HCMV US28 region, a real-time PCR method was developed for detection of HCMV US28. The results of qualitative real-time PCR on 4/18 patients [22.2%] were positive. Two patients with positive HCMV results died. This is the first study that has monitored HCMV genes in samples from glioma patients in Iran. Considering the results of this study and controversies associated with other studies, a more comprehensive study using this and other diagnostic methods is suggested.

Rapid low-cost detection of hepatitis C virus RNA in HCV-infected patients by real-time RT-PCR using SYBR green I

We intend to design and validate a low-cost assay for the detection of hepatitis C virus [HCV] RNA using rapid-cycle RT-PCR. The procedure is performed in a closed system with little risk of contamination allowing PCR and product identification to be performed within one or two hours. A SYBR Green-based real-time RT-PCR for rapid detection of HCV. Amplicon synthesis was monitored continuously by SYBR Green I, which binds to double stranded DMA during PCR. The PCR products were identified by melting curve analysis. Standard sera with known concentrations of HCV RNA and 150 clinical samples were used to validate our assay. The minimum detection level of our assay was less than 50 ID/mL. The results on 100 plasma samples were comparable with commercial assays. This method is useful for rapid qualitative detection of HCV infection and particularly suitable for routine diagnostic applications

use of a sensitive RT-Nested PCR method for detection of Hepatitis C virus

Hepatitis C virus is the major cause of viral hepatitis and its diagnosis in suspected specimens is of great importance. The risk of transfusion- transmitted virus infection is primarily the result of failure in serological screening tests to detect recently infected donors in the pre-seroconversion window period of infection. Therefore, sensitive and accurate diagnosis of HCV prior to antibody production to reduce window period is necessary. In the present study, a sensitive and specific RT-Nested PCR method for detection of a conserved HCV 5 UTR sequence was developed. Two pairs of primers for amplification of the target sequence in two rounds of PCR were selected. The developed RT- Nested PCR assay was performed on HCV-antibody confirmed positive samples as well as negative controls and standard samples. In order to compare the results, One Step RT-PCR kit was used in this study. 25 HCV-positive plasma samples whose positivity were confirmed by ELISA and Western Blot tests, also as well as 10 fold dilutions of a high viral load plasma sample obtained from a HCV-positive patient as standard samples and 25 negative control plasmas from healthy blood donors were collected and tested by this assay. In all of positive samples a 175bp band was observed on agarose gel electrophoresis, but no band could be detected in negative control plasma. Results from developed RT-PCR assay and One Step RT-PCR kit showed a good correlation. According to the results of this study, the developed RT-Nested PCR assay has a good sensitivity and specificity for diagnosis of HCV infection. It has the advantage of viral genome detection prior to seroconversion and can be used to detect HCV infection during window period