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The use of automated DNA extraction and PCR mixture preparation and of an internal control increased the sensitivity of 4 herpes virus real-time PCR assays.
Automated detection of five human herpes virus DNAs by a set of LightCycler PCRs complemented with a single multiple internal control.
Stöcher M, Hölzl G, Stekel H, Berg J.
J Clin Virol 2004;29:171-78.
Summary:
Question
What is the performance of a set of 4 previously described real-time PCR assays for the detection of 5
human herpes viruses when the DNA extractions and PCR mixture preparations are automated and when
sample adequacy is monitored using a single internal control molecule for all the assays?
Design
A previously developed set of 4 real-time PCR assays, which were performed using the LightCycler and
fluorescence resonance energy transfer (FRET) hybridization probes for the detection of CMV, EBV, HSV-1,
HSV-2, and VZV DNA in plasma samples, were modified to incorporate automated viral DNA extraction
from a larger sample volume, automated pipetting of PCR mixtures, and monitoring for sample adequacy
by using a single control molecule (IC) that contained primer binding sites for each PCR of the set.
Participants
Purified genomic DNA from herpes virus reference standards was used to validate the PCR assays. One
hundred twelve consecutive clinical EDTA-plasma samples from HIV-infected individuals and bone marrow
or solid organ transplant patients were analyzed. Fourteen normal EDTA-plasma samples from individuals
who showed no clinical symptoms or laboratory findings of infectious diseases were spiked with IC DNA or
IC and herpes virus DNA and used as negative and positive controls, respectively.
Description of Tests and Diagnostic Standard
A single IC molecule was constructed that contained a fragment of the bacterial neomycin
phosphotransferase gene that was flanked on the 5’ and 3’ ends by each of the four forward and reverse
primer binding sequences, respectively, that were specific for each herpes virus type-specific PCR of the
set. The resulting 342 base pair construct provided a template for IC-specific products of 287, 287, 292,
and 285 base pairs for the CMV, EBV, HSV-1 and 2, and VZV primers, respectively. The IC DNA
concentration that did not negatively affect the detection limits of all 4 LightCycler PCRs was found at 500
copies/mL. Each herpes virus DNA was amplified in the presence of the IC, which was detected with a
pair of IC specific FRET hybridization probes. For each of the 4 PCRs, two mixtures were prepared
containing 1) primers, probes, MgCl2, and water, and 2) enzyme and buffer. The MagNA Pure LC
instrument (Roche Applied Science) and Total Nucleic Acid Isolation Kit-Large Volume were used for
automated DNA purification and for automated liquid handling for the preparation of the PCR mixtures.
The post-elution section of the instrument was programmed to combine PCR mixtures 1 and 2 with the
extracted DNA in the reaction capillaries. The post-elution program was run 4times for each of the 4
herpes virus specific PCRs. Nucleic acids were extracted from 1 mL of each plasma sample to which 500
copies of IC were added. The nucleic acids were eluted in 50 μl of buffer. Five μl were added to a PCR
reaction.
For each herpes virus type, normal plasmas were spiked with 500 copies/ml of IC DNA and from 5000 to 125 copies/mL of herpes DNA. Samples were tested in triplicate on two consecutive days. To determine reproducibility, the results of 10 replicate samples on one plate and one sample on 10 runs of the negative control (500 copies/mL of IC) were used to determine intra-assay and inter-assay coefficients of variation (CVs), respectively.
Main Outcome Measures
Herpes virus negative samples with real-time PCR crossing point (CP) values for the IC that were 1.5 greater than the CP for the negative control were considered inhibited. Assay detection limits were determined as the lowest herpes DNA concentration that was positive in three replicate samples tested on two consecutive days.
Main Results
The real-time PCR assay detection limits were 250 copies/mL of viral DNA for CMV, EBV, and HSV-1 and 2, and 500 copies/mL for VZV. The intra-assay CVs, determined from the CP values, were 1.2, 1.0, 2.1, and 1.9%, while the inter-assay CVs were 1.8, 1.4, 3.0, and 2.3% for CMV, EBV, HSV, and VZV assays, respectively. The results of the 112 plasma samples tested by each herpes virus assay are shown in the table. Two samples were positive for two viruses. Six samples were negative for the IC. After a 1:2 dilution of the plasma, DNA extraction and PCR were repeated. One sample was positive for CMV, the remaining 5 were herpes virus negative but IC positive. The hands-on time to analyze 14 plasma samples by all 4 assays was 1 hour. For 112 samples, all 4 assays were completed within 6 hours when 2 LightCycler and one MagNA Pure instruments were used.
Results of 4 real-time herpes virus PCR assays performed on 112 plasma samples
| 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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