Literature review > Issue 8 > Review on Stocher et al. 

Rapid diagnosis of human herpes viruses is especially important for management of illnesses in specific patient populations including immunocompromised individuals and neonates. Real time PCR assays have become valuable tools for the rapid and accurate diagnosis of viral infections. These assays are more sensitive than viral culture, antigen detection, or serology for diagnosing acute infections.

This article by Stöcher et al. utilized a set of previously described real time PCR assays, performed on the LightCycler instrument using identical cycling parameters and reaction mixes but with viral specific primers and FRET probes, to detect EBV, CMV, HSV1, HSV2, and VZV. The development and validation of these assays was presented in an earlier paper [1]. The current article describes further modifications of the original assays to incorporate automated DNA extractions and PCR master mix set up. In addition, an internal control molecule used to monitor sample adequacy was synthesized with primer binding sites for all 4 herpes virus primer sets so that the same control could be used in a competitive PCR reaction for all 4 assays. The methods are well-described, proper controls were included, and the assays provided easily interpretable results.

The modifications described in this paper should improve the earlier assays. Compared to manual methods, automated DNA extraction and PCR reaction set up require less hands-on tech time and less technical skill, reduce the risk of specimen-to-specimen contamination (false positive results), and improve reproducibility. Although automated extraction instruments are expensive, their use can be justified to improve throughput in laboratories that perform large numbers of assays. However, when small numbers of specimens are being tested, manual methods often provide faster turn around times.

DNA amplification tests should include internal controls that are added to the specimens before DNA extraction to monitor for adequate DNA extraction and for the presence of enzyme-inhibiting factors that could lead to false negative results. For the assays described by Stöcher et al., the internal control was amplified using the same primers as the target DNA and differentiated from the target during amplification by hybridization with FRET probes containing a different fluorophore than the target probes and after amplification by its unique melting temperature. When used to test the plasma specimens, inclusion of the internal control prevented a false negative CMV result. However, exogenously-added controls such as the one used in this study do not identify specimens that were poorly collected or transported under conditions that might degrade the nucleic acids.

The authors stated that use of the new extraction method increased the sensitivity of the assays by about two-fold compared to the original PCR assays most likely because 5 times more DNA could be added per reaction. However, use of an internal control, especially one that competes for the same primers, can often decrease the sensitivity of a PCR assay. Samples containing low copy numbers of the viral target could give false negative results. Because the plasma specimens were tested by the modified herpes virus PCR assays only and were not also tested by the original assays, it is difficult to judge whether the modifications described in this paper resulted in more sensitive and specific herpes virus PCR assays when used to test clinical specimens.

This paper is one of many recent articles describing real time PCR assays for detection of human herpes viruses, including those employing the LightCycler instrument and either FRET probes and melting curve analysis [2,3,4] or SYBR Green and melting curve analysis [5,6,7,8] or those employing TaqMan chemistry [9,10,11,12]. Several of the assays also incorporated amplification controls [2,3,4,9,11] and/or automated specimen extraction [4,9,11]. Although real time PCR for herpes virus detection is rapid and accurate, the high cost of the instruments and the technical skill required to perform the assays will limit their use to settings with high test volume and substantial resources.

References [Issue and article number of SDI Literature Review summary and commentary]

1. Stöcher M, Leb V, Bozie M, Kessler HH, Halwachs-Baumann G, Landt O, Stekel H, Berg J. Parallel detection of five human herpes virus DNAs by a set of real-time polymerase chain reactions is a single run. J Clin Virol 2003;26:85-93.

2. Whiley DM, Syrmis MW, Mackay IM, Sloots TP. Preliminary comparison of three LightCycler PCR assays for the detection of herpes simplex virus in swab specimens. Eur J Clin Microbiol Infect Dis 2003;22:764-767. [Issue 7, article 10]

3. Whiley DM, Mackay IM, Syrmis MW, Witt MJ, Sloots TP. Detection and differentiation of herpes simplex virus types 1 and 2 by a duplex LightCycler PCR that incorporates an internal control PCR reaction. J Clin Virol 2004;30:32-38. [Issue 9, article 11]

4. Mengelle C, Sandres-Sauné K, Miédougé M, Mansuy J-M, Bouquies C, Izopet J. Use of two real-time polymerase chain reactions (PCRs) to detect herpes simplex type 1 and 2-DNA after automated extraction of nucleic acid. J Med Virol 2004;74:459-62. [Issue 11, article 10]

5. Scoular A, Gillespie G, Carman WF. Polymerase chain reaction for diagnosis of genital herpes in a genitourinary medicine clinic. Sex Transm Inf. 2002;78:21-25. [Issue 91, article 11]

6. Aldea C, Alvarez CP, Folgueira L, Delgado R, Otero JR. Rapid detection of herpes simplex virus DNA in genital ulcers by real-time PCR using SYBR Green I dye as the detection signal. J Clin Microbiol. 2002;40:1060-1062. [Issue 1, article 12]

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