title: Simultaneous Detection and Genotype Determination of HSV 1 and 2 by Real-time PCR Using Melting Curve Analysis and a Unique Pair of Primers.
abstract: Herpes simplex virus (HSV) is a human pathogen that causes different pathologic manifestations. Rapid and feasible detection and discrimination methods for HSV genotyping is a challenge in clinical laboratories, especially in children suffering from herpetic encephalitis. A quantitative real-time polymerase chain reaction (PCR)-based genotyping assay using SYBR Green I was established. We designed only 1 pair of primer for HSV 1 and 2, targeting thymidine kinase gene conserved region. HSV genotypes were determined by PCR using melting curve analysis with LightCycler. Different HSV genotypes were successfully detected in all clinical samples. The melting temperature for HSV 1 and 2 was 85.5±0.78°C and 89±0.53°C, respectively. These 2 genotypes were completely distinguished by means of the accurate melting assay. Importantly, detection was reliably performed within only 1 hour. The assay had no cross-reactivity across species, an excellent dynamic range from 10 to 10 copies per reaction, a good intra-assay and interassay reproducibility, and a detection limit of a single copy per reaction. Our homebrew designed and validated quantitative real-time PCR followed by a melting curve analysis provided a rapid and convenient screening test for differential identification of HSV genotypes 1 and 2. We recommend the large-scale application of this method for HSV 1 and 2 detection.
authors.affiliation: *Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran †Cellular and Molecular Biology Research Center ‡Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences §Day General Hospital Laboratory ¶Food and Drug Laboratory Research Center, Ministry of Health and Medical Education, Tehran ∥Department of Medical Biotechnology and Nanotechnology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
authors.forename: Mahdi
authors.initials: M
authors.lastname: Paryan
authors.forename: Samira
authors.initials: S
authors.lastname: Mohammadi-Yeganeh
authors.forename: Houri
authors.initials: H
authors.lastname: Rezvan
authors.forename: Vahid
authors.initials: V
authors.lastname: Kia
authors.forename: Ardalan
authors.initials: A
authors.lastname: Mansouri
authors.forename: Siamak
authors.initials: S
authors.lastname: Mirab Samiee
mesh_terms: D017931:DNA Primers; D005814:Genes, Viral; D006561:Herpes Simplex / Q000821:virology; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics*; D018258:Herpesvirus 2, Human / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D057230:Limit of Detection; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013937:Thymidine Kinase / Q000235:genetics
chemical_list: D017931:DNA Primers; D013937:Thymidine Kinase