Two-centre study comparing DNA preparation and PCR amplification protocols for herpes simplex virus detection in cerebrospinal fluids of patients with suspected herpes simplex encephalitis.

H H Hirsch; W Bossart

doi:10.1002/(sici)1096-9071(199901)57:1<31::aid-jmv5>3.0.co;2-j

Home/Archive/PubMed

PubMed · 9890419

Two-centre study comparing DNA preparation and PCR amplification protocols for herpes simplex virus detection in cerebrospinal fluids of patients with suspected herpes simplex encephalitis.

H H Hirsch·W Bossart

View original source ↗Export metadata ↓

Abstract

In a two-centre study, the routine DNA preparation and PCR amplification protocols were compared for herpes simplex virus (HSV) detection in cerebrospinal fluids (CSFs) of 43 patients with suspected herpes simplex encephalitis (HSE). The combined clinical, radiological and laboratory results indicated HSE in 6/43 (14%) patients. Discrepant PCR results between the two centres were obtained in 8 (18%) cases consisting of 5 false-positive and 3 false-negative results. Seven out of 8 (88%) discrepant results were associated with the method of CSF preparation using protease K digestion followed by heat inactivation. In contrast, CSF digestion with proteinase K followed by DNA purification on silica spin columns was better yielding discrepant PCR results in only 1 of 78 analyses (1.3%). The results point to the need for standardization and inter-laboratory quality control for routine clinical work.

Explore related subjects

DNA, Viral Encephalitis, Viral Endopeptidase K False Negative Reactions False Positive Reactions Herpes Simplex Hot Temperature Humans Polymerase Chain Reaction Reagent Kits, Diagnostic Simplexvirus Case Reports Comparative Study Journal Article Multicenter Study

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

H H Hirsch, W Bossart. 1999. Two-centre study comparing DNA preparation and PCR amplification protocols for herpes simplex virus detection in cerebrospinal fluids of patients with suspected herpes simplex encephalitis.. https://doi.org/10.1002/(sici)1096-9071(199901)57%3A1%3C31%3A%3Aaid-jmv5%3E3.0.co%3B2-j

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

Statistical methods for determining the accuracy of quantitative polymerase chain reaction-based tests.

Quantitative polymerase chain reaction (PCR)-based tests are used in several different scientific fields to determine levels of a target DNA sequence of interest (the target molecule). The accuracy of quantitative PCR-based tests can be assessed by using the assay to determine the number of copies of the target molecule in a sample with a known concentration of the target molecule. For example, a sample with a known concentration of a target DNA sequence is serially diluted into replicate aliquots and these are tested to determine if the observed quantity of the target is close to the expected quantity (given the concentration in the original sample and the dilution). Statistical methods that are conventionally used to assess the accuracy of these assays do not take into account the variability in the number of target molecules in each aliquot from the original sample. We develop methods that take into account this extra variation and which determine the accuracy of quantitative PCR-based tests in estimating the number of target molecules based on a set of assays of serial dilutions from an original sample with a known concentration of target molecules. These methods are applied to data from an experiment to test the accuracy of a real-time PCR assay at low HIV-1 DNA copy levels.

DNA, Viral↗

PubMed2007

The cost of replication fidelity in human immunodeficiency virus type 1.

Mutation rates should be governed by at least three evolutionary factors: the need for beneficial mutations, the benefit of minimizing the mutational load and the cost of replication fidelity. RNA viruses show high mutation rates compared with DNA micro-organisms, and recent findings suggest that the cost of fidelity might play a role in the evolution of increased mutation rates. Here, by analysing previously published data from HIV-1 reverse transcriptase in vitro assays, we show a trade-off between enzymatic accuracy and the maximum rate of polymerization, thus providing a biochemical basis for the fitness cost of fidelity in HIV-1. This trade-off seems to be related to inefficient extension of mispairs, which increases fidelity at the expense of the polymerization rate. Since in RNA viruses fast replication is critical for survival, this could impose a high cost of fidelity and favour the evolution of high mutation rates.

DNA, Viral↗

PubMed2007

OTK18, a zinc-finger protein, regulates human immunodeficiency virus type 1 long terminal repeat through two distinct regulatory regions.

It has previously been shown by our laboratory that OTK18, a human immunodeficiency virus (HIV)-inducible zinc-finger protein, reduces progeny-virion production in infected human macrophages. OTK18 antiviral activity is mediated through suppression of Tat-induced HIV-1 long terminal repeat (LTR) promoter activity. Through the use of LTR-scanning mutant vectors, the specific regions responsible for OTK18-mediated LTR suppression have been defined. Two different LTR regions were identified as potential OTK18-binding sites by an enhanced DNA-transcription factor ELISA system; the negative-regulatory element (NRE) at -255/-238 and the Ets-binding site (EBS) at -150/-139 in the LTR. In addition, deletion of the EBS in the LTR blocked OTK18-mediated LTR suppression. These data indicate that OTK18 suppresses LTR activity through two distinct regulatory elements. Spontaneous mutations in these regions might enable HIV-1 to escape from OTK18 antiretroviral activity in human macrophages.

DNA, Viral↗