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Updated: May 2, 2026
Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB
Published on: August 6, 2011
Design and Development of a Multiplex Real-Time PCR Method for the Detection of Herpes Family Viruses in Transplant
Reyhaneh Kalhor1, Hassan Noorbazargan2, Hourieh Kalhor3
1Department of Genetics, College of Science, Kazerun Branch, Islamic Azad University, Kazerun, Iran.
Abstract:
Early detection of viral infections is crucial for combating their spread in transplant patients, as it is significantly contributing to the success of organ transplantation. In transplant patients, infections motivated by herpes family viruses are common and pose serious barriers to successful organ engulfment. This study aimed to design and develop a Multiplex Real-time PCR-based method for accurate identification of these viruses, including Herpes Simplex Virus (HSV), Epstein-Barr Virus (EBV), and Varicella-Zoster Virus (VZV), in transplant patients. Primer pairs and probes targeting conserved regions of each virus genome were designed using bioinformatics software. Plasmid standards, detection limit, clinical, analytical, and sensitivity characteristics were determined using the designed primers and probes. Finally, simultaneous detection of the three herpes family viruses using Multiplex Real-time PCR was done on 22 blood specimens from transplant patients. The analytical sensitivity for HSV (6.25 copies per microliter) and VZV/EBV (25 copies per microliter) was 100% positive in all repetitions. The high copy number of one virus did not significantly affect the detection of the other two viruses with low copy numbers. Clinical sensitivity was determined to be 95% based on the analysis of 22 samples. Evaluation of the assay's specificity reached 100% using various viral samples and human genome. Reproducibility of the reaction was assessed at the intra-assay and inter-assay levels, demonstrating high repeatability of this method. The research findings indicated that the development of the multiplex Real-time PCR assay enables quicker, more accurate, and cost-effective detection of VZV, HSV, and EBV herpes family viruses in transplant patients, potentially leading to reduced treatment duration an associated complications.
Supplementary Information:
The online version contains supplementary material available at 10.1007/s12088-025-01487-8.
