Rapid and simultaneous detection of 6 types of human herpes virus (herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes virus 6A/B, and human herpes virus 7) by multiplex PCR assay

Abstract

A multiplex PCR assay was developed that enabled the simultaneous detection of DNA from 6 types of human herpes virus, HSV-1/2, VZV, EBV, CMV, HHV-6A/B, and HHV-7, using appropriate primer sets and conventional PCR techniques and instruments, with PCR products for each type of virus designed to be easily distinguishable by size. Electropherograms obtained from conventional agarose gels showed that, for each type, the observed number of base pairs corresponded to the intended product and that bands were easily distinguishable from each other. A minimum of 20 copies of viral DNA in a reaction was sufficient to confirm the existence of each of the 6 types of human herpes virus. Comparison of the data obtained from this method and the data obtained from conventional TaqMan PCR using clinical specimens from various sources showed consistent results. The multiplex PCR method reported here for the detection and differentiation of human herpes viruses did not require special equipment or techniques such as hybridization analysis and sequencing analysis and, therefore, enabled us to easily and rapidly detect and identify the 6 types of human herpes virus using conventional methods.

Human herpesvirus 6-associated uveitis with optic neuritis diagnosed by multiplex PCR

“…A number of studies have suggested that the central nervous system is the site of persistent HHV-6 infection [3,[6][7][8][9]. However, only 3 cases of ocular inflammation-associated HHV-6 have been reported [10][11][12].…”

“…Briefly, DNA was extracted from specimens using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA). Six primer sets were used in the multiplex PCR assay [6]. The PCR primers (sense and antisense, respectively) were 5 0 TGT TGG CCT TCA TGA CCC TTG TGA AA 3 0 and 5 0 TAG CTC GAG AGC TTG ATC TTG TCG GTT 3 0 for herpes The results of the multiplex PCR showed genomic DNA for HHV-6 but not for HSV types 1 and 2, VZV, EBV, or CMV (Fig.…”

Salivary shedding of Epstein–Barr virus and cytomegalovirus in people infected or not by human immunodeficiency virus 1

“…Serology may sometimes be preferred rather than PCR [27]. However, the use of PCR in the present work is justifiable due to sensitivity of the test and the ability to amplify small amounts of the target sequence as showed in previous studies [29,32,[39][40][41][42][43][44][45][46]. In our study, precautions against PCR contamination were taken.…”

Minimum detection limit of an in-house nested-PCR assay for herpes simplex virus and varicella zoster virus

“…developed a multiplex-PCR assay for rapid and simultaneous detection of six types of human herpes viruses, including HSV and VZV. In that study, the PCR assay had a lower detection limit of approximately 20 viral copies per reaction (0.8 copies/µL) for each type of virus 15 . In this regard, there is a small variation of sensitivity in different studies, which can be explained by several factors, such as the sequence of primers chosen, enzyme activity, DNA extraction method and the detection method used.…”

“…In addition, for reactions with dilutions between 2.5 and 12 copies, 50% were positive by RT-PCR, and 12.5% were positive by nested-PCR 14 . Furthermore, Tanaka et al 15 . developed a multiplex-PCR assay for rapid and simultaneous detection of six types of human herpes viruses, including HSV and VZV.…”