Rare Mutation in Matrix Gene Invalidates RT-PCR Assay for Pandemic H1N1?

There are 8 genomic segments in the Influenza A genome. The matrix (M) gene is usually highly conserved, that is, mutations accumulate more slowly in it than the other genomic segments. This is presumably because mutations would interfere with the function of the two proteins it encodes. The relatively stable sequence of the M gene makes it an ideal target for RT-PCR assays used to detect the presence of influenza. This is because mutations can cause mismatches with PCR primers that could result in false negatives, ie, failure to detect a flu infection.

A recent publication by Zheng et al. in J. Clin. Microbiol. raises the possibility that a mutation in the pandemic H1N1 M gene has occurred which invalidates at least one RT-PCR assay used to test for H1N1 in patients. To summarise their findings, a nasopharyngeal sample taken from a 4 year old girl initially tested negative using the ProFlu+ assay (Gen-Probe). This assay relies on RT-PCR with primers specific to the M gene of influenza. Perhaps because the girl exhibited classic pandemic flu symptoms and was positive with a rapid flu test (Binax), further RT-PCR tests were done. These included RT-PCR tests for the NP gene and HA gene of pandemic H1N1. These tests were positive. A RT-PCR test to the M gene produced by another company, EraGen Biosciences, was also positive. Although the sequences of the primers used to detect the M gene by Gen-Probe and EraGen Biosciences are not provided, they are presumably different.

One possible interpretation of these results is that there is a mutation in the M gene that results in a mismatch with Gen-Probe primers but not the EraGen primers. The authors indicate that sequencing of the isolate that gave anomalous results may be underway but do not report them in this paper.

From the paper:

We believe that this is the first report of a sample that may indicate a mutation in the influenza A virus matrix gene. At this time, although it seems to be very rare, the true prevalence of this variant among all 2009 H1N1 viruses is unknown until more data are available. Because of the implication of misidentification with a single assay, this case underscores the need for cautious interpretation and additional testing when a negative RT-PCR result does not seem to fit clinical presentation.

This result highlights the need for rapid, ongoing and extensive sequencing of pandemic H1N1 isolates. As the virus mutates, RT-PCR assays are going to fail, at least some of the time. Without sequencing, it will be impossible to be certain that an assay that clinicians are relying on is still valid.


Zheng et al. (2009) Unique Finding of a 2009 H1N1 Influenza Virus Positive Clinical Sample Suggests Matrix Gene Sequence Variation. J. Clin. Microbiol.


2 thoughts on “Rare Mutation in Matrix Gene Invalidates RT-PCR Assay for Pandemic H1N1?

  1. Perhaps we should ask the CDC (or ask Congress to ask) why CDC do not publicly release all of the influenza sequence data that they have. They are particularly secretive about how they choose the vaccine strains. In fact even the M gene segment of a FluA strain the 2011 years vaccine was not published.

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