Development of droplet digital PCR assays to quantify genes involved in nitrification and denitrification, comparison with quantitative real-time PCR and validation of assays in vineyard soil
Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substa...
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| Published in | Canadian journal of microbiology Vol. 67; no. 2; pp. 174 - 187 |
|---|---|
| Main Authors | , , |
| Format | Journal Article |
| Language | English |
| Published |
Canada
NRC Research Press
01.02.2021
Canadian Science Publishing NRC Research Press |
| Subjects | |
| Online Access | Get full text |
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| Abstract | Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substances. Droplet digital PCR (ddPCR) is a technology for absolute, sensitive quantification of genes. This study optimized eight ddPCR assays to quantify total bacteria and archaea as well as the nitrification (bacterial and archaeal amoA) and denitrification (nirS, nirK, nosZI, nosZII) genes involved in the generation or reduction of the greenhouse gas nitrous oxide. Detection and quantification thresholds were compared with those of quantitative real-time PCR and were equal to, or improved, in ddPCR. To validate the assays using environmental samples, soil DNA was isolated from two vineyards in the Okanagan valley in British Columbia, Canada, over the 2017 growing season. Soil properties related to the observed gene abundances were determined. Total bacteria, nirK, and nosZII increased with time and the soil C/N ratio and NH 4 + -N concentration affected total archaea and archaeal amoA negatively. The results, compared with those of other studies, showed that ddPCR is a valid alternative to qPCR to quantify genes involved in nitrification or denitrification. |
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| AbstractList | Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substances. Droplet digital PCR (ddPCR) is a technology for absolute, sensitive quantification of genes. This study optimized eight ddPCR assays to quantify total bacteria and archaea as well as the nitrification (bacterial and archaeal amoA) and denitrification (nirS, nirK, nosZI, nosZII) genes involved in the generation or reduction of the greenhouse gas nitrous oxide. Detection and quantification thresholds were compared with those of quantitative real-time PCR and were equal to, or improved, in ddPCR. To validate the assays using environmental samples, soil DNA was isolated from two vineyards in the Okanagan valley in British Columbia, Canada, over the 2017 growing season. Soil properties related to the observed gene abundances were determined. Total bacteria, nirK, and nosZII increased with time and the soil C/N ratio and NH4+-N concentration affected total archaea and archaeal amoA negatively. The results, compared with those of other studies, showed that ddPCR is a valid alternative to qPCR to quantify genes involved in nitrification or denitrification.Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substances. Droplet digital PCR (ddPCR) is a technology for absolute, sensitive quantification of genes. This study optimized eight ddPCR assays to quantify total bacteria and archaea as well as the nitrification (bacterial and archaeal amoA) and denitrification (nirS, nirK, nosZI, nosZII) genes involved in the generation or reduction of the greenhouse gas nitrous oxide. Detection and quantification thresholds were compared with those of quantitative real-time PCR and were equal to, or improved, in ddPCR. To validate the assays using environmental samples, soil DNA was isolated from two vineyards in the Okanagan valley in British Columbia, Canada, over the 2017 growing season. Soil properties related to the observed gene abundances were determined. Total bacteria, nirK, and nosZII increased with time and the soil C/N ratio and NH4+-N concentration affected total archaea and archaeal amoA negatively. The results, compared with those of other studies, showed that ddPCR is a valid alternative to qPCR to quantify genes involved in nitrification or denitrification. Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substances. Droplet digital PCR (ddPCR) is a technology for absolute, sensitive quantification of genes. This study optimized eight ddPCR assays to quantify total bacteria and archaea as well as the nitrification (bacterial and archaeal amoA) and denitrification (nirS, nirK, nosZl, nosZll) genes involved in the generation or reduction of the greenhouse gas nitrous oxide. Detection and quantification thresholds were compared with those of quantitative real-time PCR and were equal to, or improved, in ddPCR. To validate the assays using environmental samples, soil DNA was isolated from two vineyards in the Okanagan valley in British Columbia, Canada, over the 2017 growing season. Soil properties related to the observed gene abundances were determined. Total bacteria, nirK, and nosZll increased with time and the soil C/N ratio and [NH.sub.4.sup.+]-N concentration affected total archaea and archaeal amoA negatively. The results, compared with those of other studies, showed that ddPCR is a valid alternative to qPCR to quantify genes involved in nitrification or denitrification. Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substances. Droplet digital PCR (ddPCR) is a technology for absolute, sensitive quantification of genes. This study optimized eight ddPCR assays to quantify total bacteria and archaea as well as the nitrification (bacterial and archaeal amoA) and denitrification (nirS, nirK, nosZI, nosZII) genes involved in the generation or reduction of the greenhouse gas nitrous oxide. Detection and quantification thresholds were compared with those of quantitative real-time PCR and were equal to, or improved, in ddPCR. To validate the assays using environmental samples, soil DNA was isolated from two vineyards in the Okanagan valley in British Columbia, Canada, over the 2017 growing season. Soil properties related to the observed gene abundances were determined. Total bacteria, nirK, and nosZII increased with time and the soil C/N ratio and NH 4 + -N concentration affected total archaea and archaeal amoA negatively. The results, compared with those of other studies, showed that ddPCR is a valid alternative to qPCR to quantify genes involved in nitrification or denitrification. Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substances. Droplet digital PCR (ddPCR) is a technology for absolute, sensitive quantification of genes. This study optimized eight ddPCR assays to quantify total bacteria and archaea as well as the nitrification (bacterial and archaeal ) and denitrification ( , , , ) genes involved in the generation or reduction of the greenhouse gas nitrous oxide. Detection and quantification thresholds were compared with those of quantitative real-time PCR and were equal to, or improved, in ddPCR. To validate the assays using environmental samples, soil DNA was isolated from two vineyards in the Okanagan valley in British Columbia, Canada, over the 2017 growing season. Soil properties related to the observed gene abundances were determined. Total bacteria, , and increased with time and the soil C/N ratio and NH -N concentration affected total archaea and archaeal negatively. The results, compared with those of other studies, showed that ddPCR is a valid alternative to qPCR to quantify genes involved in nitrification or denitrification. Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substances. Droplet digital PCR (ddPCR) is a technology for absolute, sensitive quantification of genes. This study optimized eight ddPCR assays to quantify total bacteria and archaea as well as the nitrification (bacterial and archaeal amoA) and denitrification (nirS, nirK, nosZI, nosZII) genes involved in the generation or reduction of the greenhouse gas nitrous oxide. Detection and quantification thresholds were compared with those of quantitative real-time PCR and were equal to, or improved, in ddPCR. To validate the assays using environmental samples, soil DNA was isolated from two vineyards in the Okanagan valley in British Columbia, Canada, over the 2017 growing season. Soil properties related to the observed gene abundances were determined. Total bacteria, nirK, and nosZII increased with time and the soil C/N ratio and NH₄⁺-N concentration affected total archaea and archaeal amoA negatively. The results, compared with those of other studies, showed that ddPCR is a valid alternative to qPCR to quantify genes involved in nitrification or denitrification. Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substances. Droplet digital PCR (ddPCR) is a technology for absolute, sensitive quantification of genes. This study optimized eight ddPCR assays to quantify total bacteria and archaea as well as the nitrification (bacterial and archaeal amoA) and denitrification (nirS, nirK, nosZI, nosZII) genes involved in the generation or reduction of the greenhouse gas nitrous oxide. Detection and quantification thresholds were compared with those of quantitative real-time PCR and were equal to, or improved, in ddPCR. To validate the assays using environmental samples, soil DNA was isolated from two vineyards in the Okanagan valley in British Columbia, Canada, over the 2017 growing season. Soil properties related to the observed gene abundances were determined. Total bacteria, nirK, and nosZII increased with time and the soil C/N ratio and NH4+-N concentration affected total archaea and archaeal amoA negatively. The results, compared with those of other studies, showed that ddPCR is a valid alternative to qPCR to quantify genes involved in nitrification or denitrification. Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substances. Droplet digital PCR (ddPCR) is a technology for absolute, sensitive quantification of genes. This study optimized eight ddPCR assays to quantify total bacteria and archaea as well as the nitrification (bacterial and archaeal amoA) and denitrification (nirS, nirK, nosZl, nosZll) genes involved in the generation or reduction of the greenhouse gas nitrous oxide. Detection and quantification thresholds were compared with those of quantitative real-time PCR and were equal to, or improved, in ddPCR. To validate the assays using environmental samples, soil DNA was isolated from two vineyards in the Okanagan valley in British Columbia, Canada, over the 2017 growing season. Soil properties related to the observed gene abundances were determined. Total bacteria, nirK, and nosZll increased with time and the soil C/N ratio and [NH.sub.4.sup.+]-N concentration affected total archaea and archaeal amoA negatively. The results, compared with those of other studies, showed that ddPCR is a valid alternative to qPCR to quantify genes involved in nitrification or denitrification. Key words: nitrification, denitrification, droplet digital PCR, vineyard, soil. La quantification des genes dans le sol est importante pour etablir un lien entre l'abondance des bacteries du sol et les cycles biogeochimiques. La PCR quantitative en temps reel est largement utilisee pour cette quantification, mais son utilisation avec des echantillons environnementaux est limitee par la faible efficacite des reactions ou par l'inhibition de la PCR par des substances du sol co-purifiees. La PCR numerique a gouttelettes (ddPCR, droplet digital PCR) est une technologie de quantification absolue et sensible des genes. Cette etude a optimise huit essais par ddPCR pour quantifier les bacteries et archees totales, les genes de nitrification (amoA bacterien et archeal) et de denitrification (nirS, nirK, nosZl, nosZll), impliques dans la generation ou la reduction de l'oxyde nitreux, un gaz a effet de serre. Les seuils de detection et de quantification ont ete compares a ceux de la PCR quantitative en temps reel et il s'est avere que ceux de la ddPCR etaient egaux ou superieurs. Pour valider les essais a l'aide d'echantillons environnementaux, l'ADN du sol a ete isole de deux vignobles de la vallee de l'Okanagan en Colombie-Britannique, au Canada, au cours de la saison de croissance 2017. Les proprietes du sol liees a l'abondance des genes observes ont ete determinees. Les bacteries totales, nirK et de nosZll augmentaient en fonction du temps, et le rapport C/N et la concentration de [NH.sub.4.sup.+]-N du sol affectaient negativement les archees totales et amoA archeal. Les resultats, compares a ceux d'autres etudes, ont montre que la ddPCR est une solution de remplacement valable a la qPCR pour quantifier les genes impliques dans la nitrification ou la denitrification. [Traduit par la Redaction] Mots-cles : nitrification, denitrification, PCR numerique a gouttelettes, vigne, sol. |
| Audience | Academic |
| Author | Nelson, Louise M. Larrabee, Melissa M. Voegel, Tanja M. |
| Author_xml | – sequence: 1 givenname: Tanja M. surname: Voegel fullname: Voegel, Tanja M. – sequence: 2 givenname: Melissa M. surname: Larrabee fullname: Larrabee, Melissa M. – sequence: 3 givenname: Louise M. surname: Nelson fullname: Nelson, Louise M. |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/32910858$$D View this record in MEDLINE/PubMed |
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| Keywords | PCR numérique à gouttelettes sol vineyard droplet digital PCR dénitrification nitrification soil vigne denitrification |
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| Snippet | Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for... |
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| SubjectTerms | Abundance Analysis Archaea Archaea - genetics Archaea - metabolism Assaying Bacteria Bacteria - genetics Bacteria - metabolism Biochemical assays Biogeochemical cycles British Columbia Canada Carbon/nitrogen ratio Comparative analysis Denitrification Denitrification - genetics Deoxyribonucleic acid DNA Droplets Expressed sequence tags Farms Genes Genes, Microbial Genetic aspects Greenhouse gases Growing season Measurement Nitrification Nitrification - genetics Nitrous oxide Polymerase chain reaction quantitative polymerase chain reaction Real time Real-Time Polymerase Chain Reaction - methods Soil - chemistry Soil bacteria Soil Microbiology Soil microorganisms Soil properties Soils Technology application vineyard soils Vineyards |
| Title | Development of droplet digital PCR assays to quantify genes involved in nitrification and denitrification, comparison with quantitative real-time PCR and validation of assays in vineyard soil |
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