Molecular assays for detection of human metapneumovirus

Abstract

The recent description of the respiratory pathogen human metapneumovirus (hMPV) has highlighted a deficiency in current diagnostic techniques for viral agents associated with acute lower respiratory tract infections. We describe two novel approaches to the detection of viral RNA by use of reverse transcriptase PCR (RT-PCR). The PCR products were identified after capture onto a solid-phase medium by hybridization with a sequence-specific, biotinylated oligonucleotide probe. The assay was applied to the screening of 329 nasopharyngeal aspirates sampled from patients suffering from respiratory tract disease. These samples were negative for other common microbial causes of respiratory tract disease. We were able to detect hMPV sequences in 32 (9.7%) samples collected from Australian patients during 2001. To further reduce result turnaround times we designed a fluorogenic TaqMan oligoprobe and combined it with the existing primers for use on the LightCycler platform. The real-time RT-PCR proved to be highly reproducible and detected hMPV in an additional 6 out of 62 samples (9.6%) tested during the comparison of the two diagnostic approaches. We found the real-time RT-PCR to be the test of choice for future investigation of samples for hMPV due to its speed, reproducibility, specificity, and sensitivity.

FIG. 1.

Titration of 5-μl aliquots of hMPV amplicon and detection by ELAHA. The inset panel displays the first three corresponding bands on an agarose gel. There is a faint band visible up to a dilution of 1:2, whereas the ELAHA is positive to a dilution of 1:1,024. The horizontal line represents the ELAHA cutoff value of 0.2 OD units.

FIG. 2.

Comparison of assay sensitivity using symmetric (filled bars) (0.2 μM sense and antisense primers) and asymmetric (open bars) (0.2 μM sense and 1.0 μM antisense primers) RT-PCR-ELAHA. The cutoff for a positive ELAHA result is shown as a broken, horizontal line.

FIG. 3.

The effect of an asymmetric primer mix on the reaction efficiency of a strong hMPV-positive patient sample. Fluorescent melting-curve analysis (A) only produced peaks from the amplicon that was created in the presence of an excess of sense primer (0.5 μM MPV01.2-0.2 μM MPV02.2 or 1.0 μM MPV01.2-0.2 μM MPV02.2, indicated by a solid or broken line, respectively, with crosses), whereas amplification using an excess of antisense primer (0.2 μM MPV01.2-0.5 μM MPV02.2 or 0.2 μM MPV01.2-1.0 μM MPV02.2, indicated by a solid or broken line, respectively, without crosses) (B) produced a higher amplicon yield and extended the LP of amplification.

FIG. 4.

A weakly hMPV-positive patient sample was amplified by single-round (broken line) or dual-round (solid line) real-time RT-PCR. The second step of the reaction incorporated an asymmetric primer mix containing a fivefold excess of sense primer.

FIG. 5.

Using the same weakly positive patient sample that was used for Fig. 4, hMPV RNA was amplified by single-round RT-PCR using asymmetric primers with a fivefold excess of the sense primer, MPV01.2 (broken line), or of the antisense primer, MPV02.2 (solid line). The kinetic curves indicate that the latter primer mix is superior (A) and this is confirmed by the melt analysis which shows incomplete oligoprobe hydrolysis when using more of the sense primer (B).