Underestimation of ammonia-oxidizing bacteria abundance by amplification bias in amoA-targeted qPCR

Arnaud Dechesne, Sanin Musovic, Alejandro Palomo, Vaibhav Diwan, Barth F. Smets

    Research output: Contribution to journalJournal articleResearchpeer-review

    386 Downloads (Pure)

    Abstract

    Molecular methods to investigate functional groups in microbial communities rely on the specificity and selectivity of the primer set towards the target. Here, using rapid sand filters for drinking water production as model environment, we investigated the consistency of two commonly used quantitative PCR methods to enumerate ammonia-oxidizing bacteria (AOB): one targeting the phylogenetic gene 16S rRNA and the other, the functional gene amoA. Cloning-sequencing with both primer sets on DNA from two waterworks revealed contrasting images of AOB diversity. The amoA-based approach preferentially recovered sequences belonging to Nitrosomonas Cluster 7 over Cluster 6A ones, while the 16S rRNA one yielded more diverse sequences belonging to three AOB clusters, but also a few non-AOB sequences, suggesting broader, but partly unspecific, primer coverage. This was confirmed by an in silico coverage analysis against sequences of AOB (both isolates and high-quality environmental sequences). The difference in primer coverage significantly impacted the estimation of AOB abundance at the waterworks with high Cluster 6A prevalence, with estimates up to 50-fold smaller for amoA than for 16S rRNA. In contrast, both approaches performed very similarly at waterworks with high Cluster 7 prevalence. Our results highlight that caution is warranted when comparing AOB abundances obtained using different qPCR primer sets.
    Original languageEnglish
    JournalMicrobial Biotechnology
    Volume9
    Issue number4
    Pages (from-to)519-524
    Number of pages6
    ISSN1751-7907
    DOIs
    Publication statusPublished - 2016

    Keywords

    • Applied Microbiology and Biotechnology
    • Biotechnology
    • Biochemistry
    • Bioengineering

    Fingerprint

    Dive into the research topics of 'Underestimation of ammonia-oxidizing bacteria abundance by amplification bias in amoA-targeted qPCR'. Together they form a unique fingerprint.

    Cite this