Real-time reverse transcriptase PCR assay for detection of human metapneumoviruses from all known genetic lineages - PubMed (original) (raw)

Real-time reverse transcriptase PCR assay for detection of human metapneumoviruses from all known genetic lineages

Jeroen Maertzdorf et al. J Clin Microbiol. 2004 Mar.

Abstract

The discovery of human metapneumovirus and its implications for respiratory tract disease have emphasized the need for a sensitive, specific, and rapid assay to detect this virus in a clinical setting. It recently became clear that human metapneumovirus can be grouped into at least four genetic lineages. Previously described assays for the detection of human metapneumovirus were developed by using limited sequence information and failed to detect viruses from all four genetic lineages with comparable sensitivities. Here we describe the development and evaluation of a real-time reverse transcriptase PCR assay that detects human metapneumovirus from the four known genetic lineages with equal specificities and sensitivities.

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Figures

FIG. 1.

FIG. 1.

Hybridization blot showing detection of serial dilutions of viral RNAs of the prototype hMPV strains from genetic lineages A1 (NL/1/00), A2 (NL/17/00), B1 (NL/1/99), and B2 (NL/1/94) by using the conventional end-point RT-PCR assay targeting the hMPV L gene (15). Values above the lanes indicate the amounts of virus (in TCID50) from which RNA used in each reaction was obtained.

FIG. 2.

FIG. 2.

Entropy plots of oligonucleotide-annealing sites in hMPV sequences available from the GenBank database. The sequences recognized by the individual oligonucleotides were compared to all available hMPV sequences, and their heterogeneities are displayed as entropy values on the x axis. A higher entropy value indicates mismatches at a particular position in the oligonucleotide with a larger number of target sequences analyzed. The number of sequences upon which each plot was based is given in the upper right corner of each plot. Oligonucleotide positions are given in the 5′-3′ direction, with position 1 being the extreme 5′ nucleotide.

FIG. 3.

FIG. 3.

Alignment of sequences of primers and probes for the NL-N and ALT-N assays with the target sequences of the four prototype hMPV strains. Residue Y in the third position of the NL-N probe represents either a C or a T.

FIG. 4.

FIG. 4.

Real-Time RT-PCR amplification in the N (A) and L (B) gene assays of serially diluted RNAs of prototype hMPV strains. The values on the x axes indicate the amounts of RNA per reaction, ranging from undiluted (1e+0) to millionfold diluted (1e−6) RNAs. Ct values on the y axes indicate the first PCR cycle at which a positive signal was detected for each sample. Black circles and triangles represent the lineage A1 (NL/1/00) and A2 (NL/17/00) prototype virus strains, respectively. No amplification of viral RNA from lineage B viruses was detected in these experiments. Error bars indicate standard deviations for triplicate samples.

FIG. 5.

FIG. 5.

Real-time RT-PCR amplification of serially diluted RNAs from virus stocks (top panels) and RNA runoff transcripts from the nucleoprotein gene (bottom panels) of four prototype hMPV strains inthe NL-N assay (left panels) and the ALT-N assay (right panels). Black symbols are the same as those defined in the legend to Fig. 4. Grey circles and triangles represent the lineage B1 (NL/1/99) and B2 (NL/1/94) prototype virus strains, respectively. The values on the x axes in the top panels indicate the serial dilutions of RNA per reaction, ranging from 0-fold (1e−) to millionfold (1e−) dilutions. The values on the x axes in the bottom panels indicate the numbers of RNA molecules per reaction, ranging from 50 million (5e+7) to 50 (5e+1). Error bars indicate standard deviations. Ct, threshold cycle.

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