DNA was added to a 25 PCR mixture containing 12.5 μL of TaqMan universal PCR master mix, 300 nM of each primer, and 200 nM of probe. Real-time PCR was performed with TaqMan universal master mix (Takara Bio, Shiga, Japan). All primers and probes were purchased from Bioneer Co., Ltd. The primers consisted of the following sequences: forward primer: 5′-CAGAGCCATCTATTGCTTAC-3′, reverse primer: 5′-CATGGTGTCTGTTTGAGGTT-3′, and probe: 5′-JOE-ACACAACTGTGTTCACTAGC-BHQ1-3′ ( 34). Internal control of DNA extraction and PCR amplification was conducted by amplification of the human β globin gene. These primers and probe amplified the 137 bp segment of the UL83 gene in HCMV. The sequence of the forward primer was 5′-TGAACATCCCCAGCATCAACG-3′, of the reverse primer was 5′-CAGTCCCGAGACMGTGAGAC-3′, and of the probe was 5′-FAM TGCCACATCTGCTTGCCCGACGC-BHQ1-3´. The extractions were carried out according to the manufacturer’s instructions.Įxtracted DNA of plasma samples was subjected to TaqMan® real-time PCR targeting the UL83 gene, with the primers and probe proposed by Khansarinejad et al. Plasma samples (200 µL) were used for DNA extraction using the QIAamp DNA blood mini kit (Qiagen, USA). All of the selected patients completed questionnaires and informed consent forms for participation in this study. The specimens were collected at two different stages of the disease: early (before GCV was initiated) and late (after six months of GCV therapy). Clinical isolates from 48 bone marrow transplant patients and 39 renal transplant patients with suspected HCMV infections were identified and sent to the Keyvan virology specialty laboratory (KVSL) for molecular examination of the UL97 gene. The purpose of the present study was the examination of GCV-resistant HCMV in Iranian immunosuppressed patients, before and after treatment.Ī total of 87 specimens from Iranian patients were targeted in the present study. Therefore, there is an increasing need for early detection of the emerging mutants ( 31, 32). However, these tests are burdensome, time-consuming, and labor-intensive ( 30). In the last two decades, several methods based on phenotypic testing have been described for the detection of HCMV-resistant mutations, such as the plaque reduction assay, ELISA, and DNA hybridization assays ( 27- 29). Although mutations conferring GCV resistance have been mapped in both genes, the majority of the mutations appear in the UL97 gene, rather than as UL54 gene mutations ( 26). HCMV resistance to GCV is mainly associated with the presence of mutations within UL97, DNA polymerase, or both viral genes ( 12, 18- 25). The UL97 kinase, a virus-encoded product, activates the drug by monophosphorylation ( 12- 17). Ganciclovir, a nucleoside analogue, must be phosphorylated to exert antiviral activity as an inhibitor of viral DNA polymerase UL54. Resistance to GCV has been evaluated by many researchers because this agent is used as the first-line treatment in 90% of HCMV patients ( 11). This can give rise to resistant strains of HCMV ( 7- 10). ![]() Human cytomegalovirus infections usually require long-term treatment with antiviral agents for efficacy against the infective viruses. These agents inhibit HCMV DNA synthesis by suppressing the DNA polymerase of the causative virus. Currently, the most common antiviral agents include ganciclovir (GCV), foscarnet, and cidofovir. ![]() Antiviral agents are used in the management of HCMV infections. Hence, early diagnosis for the accurate treatment of HCMV is especially necessary to avoid poor clinical outcomes ( 3- 6). Susceptible groups include renal transplant recipients, bone marrow recipients, and AIDS patients ( 1, 2). Human cytomegalovirus (HCMV) infections are a major cause of morbidity and mortality among immunocompromised patients. UL97 Protein Human Cytomegalovirus Ganciclovir Iran 1. The findings of the present study can be utilized to detect GCV resistance patterns among Iranian immunocompromised patients and to treat HCMV infections accordingly.
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