Table 1.
Target genes, primers, thermal conditions, and reagent protocol used in the PCR assays for hemoplasmas based on the 16S rRNA, 23S rRNA, RNAse P, and dnaK genes.
| Target Gene | Primer Sequences | Thermal Conditions | Reagent Volumes and Concentrations | Fragment Size | Primers Reference |
|---|---|---|---|---|---|
| 16S rRNA | 1st round: 5′-AGAGTTTGATCCTGGCTCAG-3’ 5′-TACCTTGTTACGACTTAACT-3′ 2nd round: 5′-ATATTCCTACGGGAAGCAGC-3′ 5′-TACCTTGTTACGACTTAACT-3′ |
95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 10 min for both rounds. | 1st reaction: 2.5 μL from 10X Buffer, 0.75 μL from 50 mM MgCl2, 2 μL from 10 mM dNTP mix, 1 μL from each primer at 10 mM, 0.25 μL from 5 U/μL Taq polymerase, 12.5 μL from ultrapurified water and 5 μL from template DNA. 2nd reaction: Ultrapurified water (16.5 μL) and template DNA (1 μL) quantity changes. |
~1107 bp | Harasawa et al., 2014; Di Cataldo et al., 2020 |
| 23S rRNA | 5′-TGAGGGAAAGAGCCCAGAC-3′ 5′-GGACAGAATTTACCTGACAAGG-3′ |
94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 10 min. | 2.5 μL from 10X Buffer, 0.75 μL from 50 mM MgCl2, 2 μL from 10 mM dNTP mix, 1 μL from each primer at 10 mM, 0.25 μL from 5 U/μL Taq polymerase, 12.5 μL from ultrapurified water and 5 μL from template DNA. | ~800 bp | Mongruel et al., 2020 |
| RNAseP | 5′-GATKGTGYGAGYATATAA AAAATAAARCTCRAC-3′ 5′-GMGGRGTTTACCGCGTTTCAC-3′ |
95 °C for 2 min, followed by 50 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, extension at 72 °C for 30 s and final extension at 72 °C for 1 min. | 2.5 μL from 10X Buffer, 1.0 μL from 50 mM MgCl2, 2 μL from 10 mM dNTP mix, 1 μL from each primer at 10 mM, 0.25 μL from 5 U/μL Taq polymerase, 12.25 μL from ultrapurified water and 5 μL from template DNA. | ~164 bp | Maggi et al., 2013 |
| dnaK | 5′-GGGTGGAGATGATTGAGACCA-3’ 5′-AGCCACCCCTCCTAGAGTTT-3' |
95 °C for 5 min, followed by 45 cycles of denaturation at 95 °C for 20 s, annealing at 55.5 °C for 30 s, extension at 72 °C for 45 s and final extension at 72 °C for 7 min. | 2.25 μL from 10X Buffer, 1.0 μL from 50 mM MgCl2, 2 μL from 10 mM dNTP mix, 1 μL from each primer at 10 mM, 0.15 μL from 5 U/μL Taq polymerase, 12.6 μL from ultrapurified water and 5 μL from template DNA. | ~544 bp | Descloux et al., 2020 |