Specificities and Efficiencies of Primers Targeting Candidatus Phylum Saccharibacteria in Activated Sludge
Abstract
:1. Introduction
2. Results and Discussion
2.1. Assessment of Selected Saccharibacteria-Specific Primers
2.2. Determination of Optimal Annealing Temperatures
2.3. Evaluation of Primer Specificity by Cloning Analysis
2.4. Evaluation of Primer Specificity by qPCR
3. Materials and Methods
3.1. Primer Selection and Evaluation
3.2. Activated Sludge Samples
3.3. DNA Extraction and PCR Amplification
3.4. Cloning, Sequencing, and Phylogenetic Analysis
3.5. Real-Time Quantitative PCR
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Primer | Sequence (5′ to 3′) | Reference |
---|---|---|
TM7314F | GAGAGGATGATCAGCCAG | [10] |
TM7580F | AYTGGGCGTAAAGAGTTGC | [10] |
Sac1031F | AAGAGAACTGTGCCTTCGG | [17] |
400F | TATGAGTGAAGAATATGAC | [13] |
TM7-910R | GTCCCCGTCAATTCCTTTATG | [16] |
TM7-1177R | GACCTGACATCATCCCCTCCTTCC | [16] |
Sac1218R | GCGTAAGGGAAATACTGACC | [17] |
1110R | CAGTCCAAGTAGAAAAATAC | [13] |
Primer Set | Expected Fragment Length (bp) | DNA Band | Annealing Temperature (°C) | Number of Saccharibacteria Sequences a/Number of Total Clones Analyzed (Specificity) |
---|---|---|---|---|
TM7314F and TM7-910R | 596 | yes | 64 | 95/95 (100%) |
TM7314F and TM7-1177R | 863 | yes | 64 | 96/96 (100%) |
TM7580F and TM7-910R | 330 | yes | 66 | 94/94 (100%) |
TM7580F and TM7-1177R | 597 | yes | 63 | 94/94 (100%) |
Sac1031F and Sac1218R | 187 | no | Not determined | Not determined |
400F and 1100R | 700 | no | Not determined | Not determined |
Primer Set | Copy Number of Plasmid (Copies/ng-DNA) a | Measured Copy Number of Plasmid (Copies/ng-DNA) b | Copy Number of Saccharibacteria in Activated Sludge (Copies/g-MLSS) b |
---|---|---|---|
TM7314F and TM7-910R | 1.0 × 103 | 1.4 ± 0.1 × 103 | 6.8 ± 0.3 × 106 |
TM7314F and TM7-1177R | 9.6 × 103 | 1.4 ± 0.1 × 104 | 5.6 ± 1.1 × 106 |
TM7580F and TM7-910R | 8.9 × 103 | 1.8 ± 0.2 × 104 | 6.7 ± 1.1 × 107 |
TM7580F and TM7-1177R | 9.9 × 104 | 2.1 ± 0.3 × 105 | 3.5 ± 1.0 × 107 |
Primer | Sequence of Hydrogenophaga sp. (5′ to 3′) a |
---|---|
TM7314F | GAGAGGACGACCAGCCAC |
TM7580F | ACTGGGCGTAAAGCGTGCG |
TM7-910R | CTCAAAGGAATTGACGGGGAC |
TM7-1177R | GGAAGGTGGGGATGACGTCAAGTC |
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Takenaka, R.; Aoi, Y.; Ozaki, N.; Ohashi, A.; Kindaichi, T. Specificities and Efficiencies of Primers Targeting Candidatus Phylum Saccharibacteria in Activated Sludge. Materials 2018, 11, 1129. https://doi.org/10.3390/ma11071129
Takenaka R, Aoi Y, Ozaki N, Ohashi A, Kindaichi T. Specificities and Efficiencies of Primers Targeting Candidatus Phylum Saccharibacteria in Activated Sludge. Materials. 2018; 11(7):1129. https://doi.org/10.3390/ma11071129
Chicago/Turabian StyleTakenaka, Ryota, Yoshiteru Aoi, Noriatsu Ozaki, Akiyoshi Ohashi, and Tomonori Kindaichi. 2018. "Specificities and Efficiencies of Primers Targeting Candidatus Phylum Saccharibacteria in Activated Sludge" Materials 11, no. 7: 1129. https://doi.org/10.3390/ma11071129