Original Research Paper

HSV -1 & HSV-2 Detection by Optimized Real Time Triplex PCR Assay with Inbuilt Human Sample Quality Check

  Abstract :

Multiplex real-time (RT)-PCR assays have been widely used tools for detection of human herpesvirus (HSV) pathogens. Due to genome similarity among herpes group especially HSV-1 & HSV-2, most of the assay are cross-reactive or less sensitive. While the other assay available for detection of HSV-1 and HSV-2 virus doesnt provide any check over the quality of the sample. The present study was designed to develop a single-step triplex real-time polymerase chain reaction (PCR) assay for detection of HSV-1, HSV-2 separately along with sample quality check, using human -actin as a housekeeping gene.The primers and probes for the target genes of HSV-1, HSV-2, and -actin were designed and an RT triplex PCR assay was standardized modulating variables including annealing temperature, extension temperature, primers, probes, and other reagent concentrations. The assay was validated and sensitivity, a specificity of the assay was determined by various experiments. This novel assay was found to be sensitive, specific, and reproducible for the detection of HSV-1 and HSV-2 in patients sample. The technology was able to detect and quantify all genotypes of HSV-1 and HSV-2. The detection limit for different HSV viral genomes was found to be 100% for viral copies 100 copies/mL in a single-tube assay system. The present diagnostic assay can be routinely used in the diagnostic and prognostic assay of HSV-1 and HSV-2 in clinical samples; which is a major advantage in managing seriously/critically ill patients.

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