Synthetic oligonucleotides as quantitative PCR standards for quantifying microbial genes.
C and N cycling
microbial and functional gene abundances
plasmid
qPCR methodology
qPCR standards
real-time quantitative PCR
synthetic DNA
Journal
Frontiers in microbiology
ISSN: 1664-302X
Titre abrégé: Front Microbiol
Pays: Switzerland
ID NLM: 101548977
Informations de publication
Date de publication:
2023
2023
Historique:
received:
17
08
2023
accepted:
09
10
2023
medline:
9
11
2023
pubmed:
9
11
2023
entrez:
9
11
2023
Statut:
epublish
Résumé
Real-time quantitative PCR (qPCR) has been widely used to quantify gene copy numbers in microbial ecology. Despite its simplicity and straightforwardness, establishing qPCR assays is often impeded by the tedious process of producing qPCR standards by cloning the target DNA into plasmids. Here, we designed double-stranded synthetic DNA fragments from consensus sequences as qPCR standards by aligning microbial gene sequences (10-20 sequences per gene). Efficiency of standards from synthetic DNA was compared with plasmid standards by qPCR assays for different phylogenetic marker and functional genes involved in carbon (C) and nitrogen (N) cycling, tested with DNA extracted from a broad range of soils. Results showed that qPCR standard curves using synthetic DNA performed equally well to those from plasmids for all the genes tested. Furthermore, gene copy numbers from DNA extracted from soils obtained by using synthetic standards or plasmid standards were comparable. Our approach therefore demonstrates that a synthetic DNA fragment as qPCR standard provides comparable sensitivity and reliability to a traditional plasmid standard, while being more time- and cost-efficient.
Identifiants
pubmed: 37942081
doi: 10.3389/fmicb.2023.1279041
pmc: PMC10627841
doi:
Types de publication
Journal Article
Langues
eng
Pagination
1279041Informations de copyright
Copyright © 2023 Han, Beck, Bürgmann, Frey, Stierli and Frossard.
Déclaration de conflit d'intérêts
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Références
FEMS Microbiol Ecol. 2004 Sep 1;49(3):401-17
pubmed: 19712290
Environ Microbiol. 2020 Aug;22(8):3446-3462
pubmed: 32510812
ISME J. 2012 Mar;6(3):680-91
pubmed: 22030674
Cytokine. 2007 Apr;38(1):1-7
pubmed: 17521914
Mem Inst Oswaldo Cruz. 2017 Mar;112(3):220-223
pubmed: 28225902
Appl Environ Microbiol. 2006 Aug;72(8):5181-9
pubmed: 16885263
Environ Microbiol Rep. 2019 Apr;11(2):173-184
pubmed: 30507072
J Microbiol Methods. 2019 Sep;164:105670
pubmed: 31325465
J Mol Diagn. 2021 May;23(5):521-531
pubmed: 33549859
Appl Environ Microbiol. 2003 Jun;69(6):3476-83
pubmed: 12788753
Microbiology (Reading). 2002 Nov;148(Pt 11):3521-3530
pubmed: 12427943
Sci Total Environ. 2022 Feb 10;807(Pt 1):150720
pubmed: 34610405
Appl Microbiol Biotechnol. 2014 Jul;98(13):6105-13
pubmed: 24831026
Clin Chem. 2009 Apr;55(4):611-22
pubmed: 19246619
Biomol Detect Quantif. 2015 Mar 11;3:9-16
pubmed: 27077029
Appl Microbiol Biotechnol. 2015 Aug;99(16):6855-63
pubmed: 26092754
Appl Environ Microbiol. 2008 Nov;74(21):6663-71
pubmed: 18776026
Environ Microbiol. 2014 Oct;16(10):3055-71
pubmed: 24118804
Water Res. 2021 Sep 15;203:117516
pubmed: 34412018
Nature. 2006 Aug 17;442(7104):806-9
pubmed: 16915287
Microbiologyopen. 2016 Aug;5(4):537-50
pubmed: 26987552
Sci Rep. 2016 Apr 19;6:24731
pubmed: 27090814
Adv Mar Biol. 2005;48:1-599
pubmed: 15797449
Nat Rev Microbiol. 2022 Jul;20(7):401-414
pubmed: 35132241
Environ Microbiol. 2008 Jun;10(6):1433-49
pubmed: 18279346
Appl Environ Microbiol. 2012 Jun;78(12):4481-9
pubmed: 22492459
FEMS Microbiol Lett. 1995 Oct 15;132(3):203-8
pubmed: 7590173
Oecologia. 1979 Jan;40(1):51-62
pubmed: 28309603
Microbiologyopen. 2020 Mar;9(3):e977
pubmed: 31927795
Bioinformatics. 2014 Aug 15;30(16):2310-6
pubmed: 24764462
J Microbiol Methods. 2014 Jul;102:1-7
pubmed: 24769406
Microbiol Rev. 1994 Dec;58(4):603-15
pubmed: 7854248
PLoS One. 2012;7(12):e51931
pubmed: 23284822
J Virol Methods. 2018 Jun;256:100-106
pubmed: 29514044
Noncoding RNA. 2017 Jun;3(2):
pubmed: 28730153
Appl Environ Microbiol. 1999 Apr;65(4):1652-7
pubmed: 10103263
J Appl Microbiol. 2020 Sep;129(3):768-774
pubmed: 32202037
FEMS Microbiol Ecol. 2003 May 1;44(1):3-15
pubmed: 19719646
Clin Chem Lab Med. 1998 May;36(5):255-69
pubmed: 9676381
Biotechniques. 2008 Apr;44(5):619-26
pubmed: 18474036
Appl Environ Microbiol. 1997 Dec;63(12):4704-12
pubmed: 9406389
J Immunol Methods. 2010 Mar 31;354(1-2):34-9
pubmed: 20109462
Appl Environ Microbiol. 2015 Nov;81(21):7570-81
pubmed: 26296731
Nucleic Acids Res. 1991 Jan 11;19(1):99-107
pubmed: 2011516
Front Microbiol. 2019 Apr 09;10:720
pubmed: 31024498
J Environ Qual. 2016 Mar;45(2):441-53
pubmed: 27065390
Nat Biotechnol. 2009 Dec;27(12):1091-4
pubmed: 20010582
PLoS One. 2011;6(12):e29101
pubmed: 22194997
Appl Environ Microbiol. 2015 Oct;81(20):7281-9
pubmed: 26253682
Mol Cell Proteomics. 2014 May;13(5):1352-8
pubmed: 24563536
FEMS Microbiol Ecol. 2009 Jan;67(1):6-20
pubmed: 19120456
Appl Environ Microbiol. 2005 Jul;71(7):4117-20
pubmed: 16000830
Res Microbiol. 2001 Jan-Feb;152(1):95-103
pubmed: 11281330
Biotechniques. 1999 Jan;26(1):112-22, 124-5
pubmed: 9894600
Forensic Sci Med Pathol. 2018 Mar;14(1):70-75
pubmed: 29353346
Appl Environ Microbiol. 2005 Sep;71(9):5362-70
pubmed: 16151126
Front Microbiol. 2016 Jun 14;7:903
pubmed: 27379043
Science. 2014 Nov 28;346(6213):1256688
pubmed: 25430773
ISME J. 2014 Jan;8(1):226-44
pubmed: 24030594
Nature. 2005 Feb 24;433(7028):861-4
pubmed: 15729341
Front Microbiol. 2012 Sep 26;3:348
pubmed: 23055998
Proc Natl Acad Sci U S A. 2005 Oct 11;102(41):14683-8
pubmed: 16186488
Appl Environ Microbiol. 2000 Nov;66(11):5066-72
pubmed: 11055964
J Microbiol Methods. 2002 Jul;50(2):155-64
pubmed: 11997166
Expert Rev Mol Diagn. 2005 Mar;5(2):209-19
pubmed: 15833050
Appl Environ Microbiol. 1983 Aug;46(2):491-8
pubmed: 16346372
Appl Environ Microbiol. 2011 Sep;77(17):6060-8
pubmed: 21742929
FEMS Microbiol Ecol. 2016 Mar;92(3):
pubmed: 26832204
PLoS One. 2010 Mar 05;5(3):e9545
pubmed: 20221433
Biotechniques. 2018 Apr;64(4):177-179
pubmed: 29661012