Underestimation of ammonia‐oxidizing bacteria abundance by amplification bias in amoA ‐targeted qPCR
| Autor: | Arnaud Dechesne, Alejandro Palomo, Barth F. Smets, Vaibhav Diwan, Sanin Musovic |
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| Rok vydání: | 2016 |
| Předmět: |
DNA
Bacterial 0301 basic medicine In silico 030106 microbiology Bioengineering Biology Real-Time Polymerase Chain Reaction DNA Ribosomal Applied Microbiology and Biotechnology Biochemistry Microbiology 03 medical and health sciences Microbial ecology Ammonia RNA Ribosomal 16S Environmental Microbiology False Negative Reactions Nitrosomonas DNA Primers Highlight Genetics Bacteria Phylogenetic tree Brief Report Sequence Analysis DNA biology.organism_classification 16S ribosomal RNA Bacterial Load 6. Clean water 030104 developmental biology Genes Bacterial Metagenomics Brief Reports Primer (molecular biology) Oxidation-Reduction Biotechnology |
| Zdroj: | Microbial Biotechnology Dechesne, A, Musovic, S, Palomo, A, Diwan, V & Smets, B F 2016, ' Underestimation of ammonia-oxidizing bacteria abundance by amplification bias in amoA-targeted qPCR ', Microbial Biotechnology, vol. 9, no. 4, pp. 519-524 . https://doi.org/10.1111/1751-7915.12366 |
| ISSN: | 1751-7915 |
| DOI: | 10.1111/1751-7915.12366 |
| Popis: | Summary 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. |
| Databáze: | OpenAIRE |
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