New molecular virus detection methods and their clinical value in lower respiratory tract infections in children

Abstract

During the past decade, several new respiratory viruses and their subgroups have been discovered. All these new viruses, as well as previously known respiratory viruses, can be detected by sensitive PCR methods, which have become popular in the diagnostic workup of respiratory viral infections. Currently, respiratory viruses can be detected in up to 95% of children with lower respiratory tract illness. On the other hand, virus detection rates in asymptomatic children are also high (up to 68%), as are coinfection rates in symptomatic children (up to 43%) and justified concerns of causality have been raised. Imposing progress has been made in developing multiplex quantitative PCR assays; here, several primer sets are run within a single PCR mixture. These PCR assays give a better understanding of the dominant viral infection, of viral infections that may be incipient and of any waning infections than does a single-target PCR. Multiplex PCR assays are also gaining popularity due to their cost-effectiveness and short throughput time compared to multiple single-target PCRs. Our understanding of the indications of virus PCRs and our ability to interpret the results from a clinical point of view have improved. This paper reviews the progress in PCR assays and discusses their role in the diagnosis of lower respiratory tract infections in children.

Figure 1

Overview of the real-time qPCR protocol. 1. Before PCR, the genetic material needs to be purified from the sample in a sample-preparation room. 2. In a DNA-free reaction-preparation room, water, buffer, nucleotides, primers, probe and a heat-stabile polymerase enzyme are mixed and aliquoted in reaction tubes, which are taken to the sample room. 3. Purified sample DNA or RNA, standard dilutions, and controls are added to separate reaction tubes, which are then applied to the PCR instrument in a separate amplification room. 4. During PCR the carefully designed specific oligonucleotide primers (in red) are annealed to their complementary regions of the two heat-denatured single-stranded DNA molecules. Progeny strands are synthesized by extension of the primers along the full template strand by the action of the DNA polymerase. This is repeated in 30-45 temperature cycles causing an exponential amplification of the target DNA sequence. At each cycle, the target-specific fluorescent probe (in blue) is hybridized to the increasing amount of targets during which fluorescence is measured. 5. This increase in fluorescence is shown in real time on the screen. The fluorescence curves of the samples are analyzed by the computer and compared with those of the known standard, and the quantity of DNA is calculated. NPA, nasopharyngeal aspirate sample; NTP, deoxyribonucleoside triphosphates (dNTP: dATP, dGTP, dCTP, dTTP); qPCR, quantitative polymerase chain reaction.