Table 1.
Primer pairs tested for the QPCR quantification of the honeybee microbiota major bacterial taxa.
| Primer pairs and sequences (5′→3′) | Targeted group and % coverage in SILVA database (in honeybee microbiota) | Amplicon size (bp) | Linearity (r2) Efficiency (E) |
validated DNA rangea and CQ range | Reference |
|---|---|---|---|---|---|
| Bacteria | |||||
| 341F CCTACGGGAGGCAGCAG * | Bacteria 81.7 | 169–195 | r2 = 0,999 | 50 pg to 50 ng | 11 |
| 534R ATTACCGCGGCTGCTGGCA * | E = 0,92 | CQ 10 to 22 | |||
| BAC338F ACTCCTACGGGAGGCAG * | Bacteria 77.4 | 443–469 | r2 = 0,999 | 50 pg to 10 ngb | 86 |
| BAC805R GACTACCAGGGTATCTAATCC * | E = 0,85 | CQ 13.5 to 23 | |||
| BACT1369F CGGTGAATACGTTCYCGG | Bacteria 25.4 | 133–146 | r2 = 0,999 | 50 pg to 50 ng | 77 |
| PROK1492R GGWTACCTTGTTACGACTT | E = 0,84 | CQ 11 to 23 | |||
| 926F AAACTCAAAKGAATTGACGG | Bacteria 78.4 | 172–179 | r2 = 0,999 | 50 pg to 50 ng | 6 |
| 1062R CTCACRRCACGAGCTGAC | E = 0,87 | CQ 11 to 22 | |||
| Bifidobacterium spp. | |||||
| Bifi-F2 TCGCGTCYGGTGTGAAAG * | Bifidobacteriaceae 89.3 | 243 | r2 = 0,999 | 50 pg to 50 ng | 70 |
| Bifi-R2 CCACATCCAGCRTCCAC * | Bifidobacterium spp. 89.0 (96.1, n=51) | E = 0,78 | CQ 14.5 to 25 | ||
| Bifi-F1 CAAGCGAGAGTGAGTGTACC | Bifidobacterium spp. 2.5 (88.7, n=53) | 165 | r2 = 0,999 | 50 pg to 50 ng | 53 |
| Bifi-R1 GCCGATCCACCGTTAAGC | E = 0,90 | CQ 14.5 to 25.5 | |||
| Act920F3 TACGGCCGCAAGGCTA | Actinobacteria 86.7 | 299–307 | r2 = 0,999 | not validated | 6 |
| Act1200R TCRTCCCCACCTTCCTCCG | Bifidobacteriaceae 84.8 | E = 0,67 | |||
| Bifidobacterium spp. 86.5 (94.2, n=52) | |||||
| Lactobacillus spp. | |||||
| Lact-F1 TAACGCATTAAGCACTCC * | Lactobacillus 3 (28.6, n=1,705) | 270 | r2 = 0,998 | 50 pg to 50 ng | 53 |
| Lact-R1 GCTGGCAACTAATAATAAGG * | L. kunkeei (0, n=875) | E = 0,89 | CQ 13.5 to 24.5 | ||
| Firm-4 (0, n=88) | |||||
| Firm-5 (97.2, n=424) | |||||
| Lact-F2 AGCAGTAGGGAATCTTCCA * | Lactobacillus 87.2 (95.7, n=1,690) | 341 | r2 = 0,997 | 50 pg to 10 ngb | 70 |
| Lact-R2 CACCGCTACACATGGAG * | L. kunkeei (96.8, n=875) | E = 1,08 | CQ 14 to 23 | ||
| Firm-4 (97.7, n=88) | |||||
| Firm-5 (91.7, n=424) | |||||
| 928F-Firm TGAAACTYAAAGGAATTGACG | Firmicutes 73.7 | 149–157 | r2 = 0,998 | 50 pg to 10 ngb | 6 |
| 1040FirmR ACCATGCACCACCTGTC | Lactobacillales 73.3 | E = 0,83 | CQ 14.5 to 23.5 | ||
| Lactobacillus (43.6) (69.1, n=1,705) | |||||
| L. kunkeei (97.7-8, n=875) | |||||
| Firm-4 (97.7, n=88) | |||||
| Firm-5 (0.5, n=424) | |||||
| Mollicutes 39.8 | |||||
| Alphaproteobacteria | |||||
| a682F CDAGTGTAGAGGTGAAATT * | Alphaproteobacteria 63.6 | 245–250 | r2 = 0,998 | 50 pg to 50 ng | 6 |
| 908aR CCCCGTCAATTCCTTTGAGTT * | Bartonella 25.4 (89.6, n=77) | E = 0,91 | CQ 14.5 to 25 | ||
| Acetobacteraceae 0.8 (10.0, n=201) | |||||
| Gammaproteobacteria | |||||
| 1080γF TCGTCAGCTCGTGTYGTGA * | Gammaproteobacteria 61.2 | 146–160 | r2 = 0,999 | 50 pg to 50 ng | 6 |
| γ1202R CGTAAGGGCCATGATG * | Gilliamella 96.9 (97.1, n=516) | E = 0,90 | CQ 14.5 to 25 | ||
| Snodgrasella 0 (0, n=342) | |||||
| Enterobacteriales 94.0 | |||||
| Snodgrasella alvi | |||||
| Neiss-F AAGCGGTGGATGATGTGG * | Neisseriaceae 79.4 | 194–199 | r2 = 0,999 | 50 pg to 50 ng | 53 |
| Neiss-R TGATGGCAACTAATGACAAGG * | Snodgrassella 86.4 (95.1, n=344) | E = 0,90 | CQ 13.5 to 23.5 | ||
| Snodgrassella alvi 100 (95.6, n=226) | |||||
| Beta-1009-qtF CTTAGAGATAGGAGAGTG * | Snodgrasella 77.3 (93.0, n=344) | 127–128 | r2 = 0,999 | 50 pg to 50 ng | 60 |
| Beta-1115-qtR AATGATGGCAACTAATGACAA * | Snodgrassella alvi 76.9 (94.2, n=226) | E = 0,87 | CQ 15 to 24 | ||
| Snod-F GGAATTTCTTAGAGATAGGAAAGTG | Snodgrasella 11.4 (0, n=344) | 136 | r2 = 0,999 | 50 pg to 50 ng | 85 |
| Snod-R TTAATGATGGCAACTAATGACAA | Snodgrassella alvi 23.1 (0, n=226) | E = 0,86 | CQ 15 to 26 | ||
| Gilliamella apicola | |||||
| G1-459-qtF GTATCTAATAGGTGCATCAATT * | Gilliamella 23.0 (71.6, n=232) | 210 | r2 = 0,999 | 50 pg to 50 ng | 60 |
| G1-648-qtR TCCTCTACAATACTCTAGTT * | Gilliamella apícola 22.9 (80.7, n=109) | E = 0,91 | CQ 14.5 to 25.5 | ||
| Gill-F CCTTTGTTGCCATCGATTAGG | Gilliamella 28.0 (1.7, n=355) | 249 | r2 = 0,999 | 50 pg to 50 ng | 85 |
| Gill-R GACATTCTGATTCACGATTACTAGC | Gilliamella apícola 37.5 (0.9, n=233) | E = 0,83 | CQ 16.5 to 28 | ||
| Past-F TTGTTGCCAGCGATTAGG | Gilliamella apícola 4.2 (2.1, n=233) | 243 | r2 = 0,998 | 200 pg to 50 ngc | 53 |
| Past-R ATTCTGATTCACGATTACTAGC | Frischella perrara 100 (94.9, n=59) | E = 0,75 | CQ 18 to 27.5 | ||
| Bacteroidetes | |||||
| 798cfbF CRAACAGGATTAGATACCCT | Bacteroidia 86.2 (100, n=44) | 202–208 | r2 = 0,998 | 50 pg to 50 ng | 6 |
| cfb967R GGTAAGGTTCCTCGCGTAT | Spirochaetaceae 42.8 | E = 0,90 | CQ 21.5 to 33 d | ||
a
total DNA from the honeybee gut
b
not quantitative for higher DNA concentrations
c
not quantitative for lower DNA concentrations
d
high CQ precluded any analyses
Selected pairs are indicated by an *.