Rapid detection of herpes simplex virus and varicella-zoster virus infections by real-time PCR

Abstract

The herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) and varicella-zoster virus (VZV) can cause life-threatening infections of the central nervous system and lead to severe infections in immunocompromised subjects and newborns. In these cases, rapid diagnosis is crucial. We developed three different real-time PCR assays based on TaqMan chemistry for the LightCycler instrument to detect HSV-1, HSV-2, and VZV. When the TaqMan assays were compared to our in-house nested PCR assays, the test systems had equal sensitivities of <or=10 plasmid copies per assay. When clinical samples were investigated by TaqMan PCR to detect HSV-1, HSV-2, and VZV DNA, 95, 100, and 96% of the samples determined to be positive by nested PCR, respectively, were positive by the real-time PCR assays. The specificities of all PCR assays were almost 100%. Furthermore, the TaqMan PCR assays could be performed within 2.5 h, whereas nested PCR results were available after 9 h. In addition to offering more rapid results, the TaqMan PCR assays appear to be less expensive than nested PCR assays due to less hands-on time. In summary, TaqMan PCR is an excellent alternative to conventional nested PCR assays for the rapid detection of HSV-1, HSV-2, and VZV in clinical samples.

FIG. 1.

Sensitivities of the PCR assays for detecting HSV-1, HSV-2, and VZV. (A to C) Results of real-time PCR. Plasmids were serially diluted from 10⁴ to 10¹ copies per assay. In the lower left parts of the graphs, results for the corresponding no-template controls are shown. Vertical (y) axis, fluorescence intensity; horizontal (x) axis, PCR cycle numbers. (D) Results of nested PCR to detect HSV-1, HSV-2, and VZV. Lane 1, molecular weight marker (MWM); lanes 2 to 4, 10³ to 10¹ copies of HSV-1 plasmid controls; lanes 5 and 6, no-template control; lane 7, MWM; lanes 8 to 10, 10³ to 10¹ copies of HSV-2 plasmid controls; lanes 11 and 12, no-template control; lane 13, MWM; lanes 14 to 16, 10³ to 10¹ copies of VZV plasmid controls; lanes 17 and 18, no-template control.