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LightCyler real-time PCR in combination with the melting curve analysis is a suitable method for diagnosis of HSV and VZV in specimens from skin lesions.
Detection of herpes simplex virus type 1, herpes simplex virus type 2
and varicella-zoster virus in skin lesions. Comparison of real-time PCR, nested PCR and virus isolation.
Schmutzhard J, Riedel HM, Wirgart BZ, Grillner L.
J Clin Virol 2004;29:120-26.
Summary:
Question
How do the results of a real-time PCR assay compare to the results of an in-house nested PCR assay and virus isolation for the detection of HSV-1, HSV-2, and VZV DNA in skin lesions?
Design
Real-time qualitative PCR assays performed on the LightCycler with SYBR Green detection were developed and evaluated and the results obtained on samples from dermal and genital lesions were compared to those of an in-house nested PCR assay and virus isolation for the detection of HSV-1, HSV-2, and VZV.
Participants
One hundred ten consecutive samples from dermal or genital lesions of patients with suspected mucocutaneous HSV infections and another 110 consecutive clinical samples from skin lesions of patients with suspected mucocutaneous VZV infections were tested.
Description of Tests and Diagnostic Standard
Swabs were collected in viral transport medium to which 2 mL EMEM containing 2% FBS were added. Virus isolation was performed by inoculating monolayers of green monkey kidney cells and human embryonic fibroblasts with 200 μl of specimen. The remainder of the sample was stored at –70oC until tested by PCR. The cultures were incubated and inspected for cytopathic effects during a period of 1 week for HSV and 3 weeks for VZV. Isolates were typed by using HSV-1 and HSV-2 monoclonal antibodies (HSV-1 and HSV-2 PathoDx, Diagnostic Products Corporation DCP Scandinavia) and VZV monoclonal antibody (VZV Merifluor clone:3B3, Orion Diagnostica).
DNA was extracted from 100 μl of each specimen using the QIAamp DNA Blood Mini Kit (Qiagen, VWR International). Every second sample processed was a negative water control. The in-house nested PCR assays for each virus were performed as two consecutive PCR reactions with two separate primer pairs. These assays were accredited and had been validated in earlier studies. Ten μl of the extracted DNA and 3 μl of the first round PCR product, respectively, were added to the two reactions. The PCR products were detected with gel electrophoresis. The detection limits of the nested PCR assays were determined using genomic DNA standards from the Swedish Institute for Infectious Disease Control. The real-time PCR assays were carried out on the LightCycler system (Roche Molecular Biochemicals) using SYBR Green I dye. Two primers from each of the 4 primers used in the nested PCR assays were used for the real-time reactions. PCR products were detected with melting curve analysis. The detection limits of the real-time PCR assays were determined using positive controls from the Sahlgrenska University Hospital, Gothenburg, Sweden.
Main Outcome Measures
The sensitivity, specificity, positive predictive values, and negative predictive values of the real-time PCRs as compared to the nested PCRs for the detection of HSV-1, HSV-2, and VZV were determined.
Main Results
No contamination was seen in the negative control samples. When the melting temperatures of the real-time PCR products were determined by using melting curve analysis for each viral assay, the differences in average melting temperatures between positive controls and positive samples were not statistically significant. A real-time PCR run, including the identification of the products by melting curve analysis was carried out within approximately 45 minutes. The results of the virus isolations, the nested PCRs, and the real-time PCRs for detection of HSV-1 and HSV-2 in 110 dermal and genital lesion swab samples and for detection of VZV in 110 skin swabs are shown in Table 1. The detection rate of VZV was increased more than three times by PCR compared to virus isolation. The effect of long transportation time on VZV culture was demonstrated by 12 samples that had been in transit for longer than 3 days. Eight were VZV positive by PCR while only 2 were positive by culture. The performances of virus isolation and the real-time PCR assays compared to the nested PCR assays are shown in Table 2.
Table 1. Results of virus isolation, nested PCR, and real-time PCR for detection of HSV-1, HSV-2, and VZV in skin lesion samples
Result by detection method Number (%) of samples with result by virus type
| Target |
Number of positive results by PCR assay |
| CMV |
EBV |
HSV-1/2 |
VZV |
| Herpes virus |
22 |
9 |
10 |
3 |
| IC |
106 |
109 |
109 |
111 |
Authors' Conclusions
The observation of IC-negative samples emphasizes the necessity of monitoring samples for adverse effects in PCR-based assays. The use of the constructed multiple IC streamlined the protocol and avoided the risk of handling errors because only one IC was required for all assays. Using a 1 mL sample volume with the automated DNA extraction procedure improved the sensitivity of the assays over those obtained with a manual procedure in a previous report.
DNA extraction and post-elution mixing on the MagNA Pure instrument proved markedly labor saving and provided excellent precision. The advantages of the assays described in this study include convenient and swift performance with reduced hands-on time, reduction of the risk of cross-contamination and pipetting errors, and the use of a single multiple IC DNA for monitoring sample adequacy.
Source of funding: None given
For correspondence:
Jörg Berg, Department of Laboratory Medicine (Zentral-Labor), Institute of Laboratory Medicine, General Hospital Linz, Allgemeines Krankenhaus, Krankenhausstrasse 9, A-4020 Linz, Austria. E-mail address: joerg.berg@akh.linz.at.
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