ddPCR surpasses classical qPCR technology in quantitating bacteria and fungi in the environment.
droplet digital PCR
environmental microbiology
fungi to bacteria ratio
microbial quantification quantitative real-time PCR
Journal
Molecular ecology resources
ISSN: 1755-0998
Titre abrégé: Mol Ecol Resour
Pays: England
ID NLM: 101465604
Informations de publication
Date de publication:
Oct 2022
Oct 2022
Historique:
revised:
24
02
2022
received:
17
09
2021
accepted:
13
05
2022
pubmed:
20
5
2022
medline:
8
9
2022
entrez:
19
5
2022
Statut:
ppublish
Résumé
Quantitative real-time PCR (qPCR) has been widely used in quantifying bacterial and fungal populations in various ecosystems, as well as the fungi to bacteria ratio (F:B ratio). Recently, researchers have begun to apply droplet digital PCR (ddPCR) to this area; however, no study has systematically compared qPCR and ddPCR for quantitating both bacteria and fungi in environmental samples at the same time. Here, we designed probe-primer pair combinations targeting the 16S rRNA gene and internal transcribed spacer (ITS) for the detection of bacteria and fungi, respectively, and tested both SYBR Green and TaqMan approaches in qPCR and ddPCR methods for mock communities and in real environmental samples. In mock communities, the quantification results of ddPCR were significantly closer to expected values (p < .05), and had smaller coefficients of variations (p < .05) than qPCR, suggesting ddPCR was more accurate and repeatable. In environmental samples, ddPCR consistently quantified ITS and 16S rRNA gene concentrations in all four habitats without abnormal overestimation or underestimation, and the F:B ratio obtained by ddPCR was consistent with phospholipid fatty acid analysis. Our results indicated that ddPCR had better precision, repeatability, sensitivity, and stability in bacterial and fungal quantitation than qPCR. Although ddPCR has high cost, complicated processes and restricted detection range, it shows insensitivity to PCR inhibitors and the potential of quantifying long target fragments. We expect that ddPCR, which is complementary to qPCR, will contribute to microbial quantification in environmental monitoring and evaluation.
Identifiants
pubmed: 35587727
doi: 10.1111/1755-0998.13644
doi:
Substances chimiques
RNA, Ribosomal, 16S
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
2587-2598Subventions
Organisme : Key Projects of Sichuan Provincial Department of Science and Technology
ID : 2020YFSY0008
Organisme : Sichuan Provincial Department of Science and Technology
Organisme : National Natural Science Foundation of China
ID : 91851106
Organisme : National Natural Science Foundation of China
ID : U1906223
Organisme : National Key Research and Development Program
ID : 2019YFC1905001
Informations de copyright
© 2022 John Wiley & Sons Ltd.
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