Table 1.
Primers used in the first step (18SMP and 16SMP) and second step (PriMP and HypMP) multiplex PCR assays including assay names, primer targets, primer sequences (forward followed by reverse primers), the expected product size, concentration of each primer (Conc.) in the PCR, the gene of the primers are based on, and the detection limits expressed as DNA template copies per reaction.
| Assay name | Targeted taxa | Primer names and sequences (5′-3′) | Product size (bp) | Conc. (µM) | Gene | Detection limit (templates) |
|---|---|---|---|---|---|---|
| 18SMP | Parasitoid-group | ParG-S452: AACYAGARTTCCRACCAGAGATGG | 196 ± 4 (193, 203) | 1 | 18S | / |
| ParG-A435: CATGGTAGGCRYAGAACCTACCA | ||||||
| Rhopalosiphum padi | Rho-pad-S440: TAATAATATAAAATTAAACCAAATTCCATTA | 136 | 0.2 | COI | 750 | |
| Rho-pad-A442: TGATGTATTTAAATTACGATCAGTAAGAAG | ||||||
| Sitobion avenae | Sit-ave-S433: TCATCACTTAGAATTCTTATTCGTCTT | 259 | 0.08 | COI | 750 | |
| Sit-ave-A437: AGAAACTACAGATTATTATTATTAATGATGGT | ||||||
| Metopolophium dirhodum | Met-dir-S436: CCTTTATCAAATAACATTGCACATAAC | 105 | 0.22 | COI | 750 | |
| Met-dir-A440: AATAAAGTTAATTGCTCCTAAAATTGAG | ||||||
| Hamiltonella defensa | Ham-def-S441: TTTGGAGGTTGCGGTCTTG | 233 | 0.02 | 16S | 150 | |
| Ham-def-A443: CCATGCAGCACCTGTCTCAC | ||||||
| Regiella insecticola | Reg-ins-S443: AAGTTGCCTTCGGGAGCC | 426 | 0.022 | 16S | 150 | |
| Reg-ins-A446: AAGCTACCTACTTCTTTTGCCG | ||||||
| Pea aphid X-type symbiont (PAXS) | PAXS-S447: ACTGTGGCTTGCGGAGCA | 604 | 0.038 | 16S | 150 | |
| PAXS-A450: TAAGCTACCTACTTCTTTTGCAAAA | ||||||
| 16SMP | Aphidiinae | AphG-S458: WATAATYTTAAGTCWAATCTGCC | 213 ± 2 (211, 216) | 1 | 16S | 15 |
| ParG-A462: AARTTCTAWAGGGTCTTMTCGTCT | ||||||
| Aphelinus-Hyperparasitoid | HypG-S460: TAAYTGTACWAAGGTAGCATAATCAWTT | 155 ± 2 (152, 158) | 0.8 | 16S | 15 | |
| ParG-A462: AARTTCTAWAGGGTCTTMTCGTCT | ||||||
| Dendrocerus spp. | DenG-S461: CTAAGGTAGCATAATAATTAGTTTATTAATTGT | 317/318 | 0.2 | 16S | 15 | |
| DenG-A461: GCTGTTATCCCTAAAGTAATTTAATCA | ||||||
| R. padi | Rho-pad-S437: CCTTAGAATCTTAATTCGAYTAGAACTAAGT | 478 | 0.14 | COI | 7500 | |
| Rho-pad-A458: TGTAATTAAAATTGATCAAGGGAATAAT | ||||||
| S. avenae | Sit-ave-S433/Sit-ave-A437 | 259 | 0.08 | COI | 7500 | |
| M. dirhodum | Met-dir-S436/Met-dir-A440 | 105 | 0.12 | COI | 7500 | |
| H. defensa | Ham-def-S441/Ham-def-A443 | 233 | 0.02 | 16S | 750 | |
| R. insecticola | Reg-ins-S443/Reg-ins-A446 | 426 | 0.022 | 16S | 750 | |
| PAXS | PAXS-S447/PAXS-A450 | 604 | 0.038 | 16S | 750 | |
| PriMP | Aphidius ervi | Aph-erv-S817: TCATGCTTTTGTAATAATTTTYTTTATG | 324 | 0.4 | COI | 75 |
| Aph-erv-A816: ATAGTACTAATAAAATTAATAGCTCCYATRATAGAG | ||||||
| Aphidius avenae | Aph-pic-S820: CGAATAGAATTAAGARTTACTGGTACTTTT | 605 | 1 | COI | 75 | |
| Aph-pic-A820: CCAAAAAATCAAAATAAATGTTGATAC | ||||||
| Aphidius uzbekistanicus | Aph-uzb-S821: TTTATTTTTGGTATATGATCTGGAGTC | 290 | 0.2 | COI | 75 | |
| Aph-uzb-A821: CCTCTAATTAATAATAAAATTAAAGATGGAAC | ||||||
| Ephedrus plagiator | Eph-pla-S823: TTAGGTCATAGAGGAATATCAGTTGATA | 134 | 0.4 | COI | 75 | |
| Eph-pla-A824: TGATCYTTAACTATTCCTAAAGAATTTATTG | ||||||
| Praon gallicum | Pra-gal-S824: GATACCTGGTAGATTAATTGGAAGG | 482 | 0.4 | COI | 75 | |
| Pra-gal-A826: CTAAAACTGGTAAAGATAACAATAAMAATAA | ||||||
| Praon volucre | Pra-vol-S828: TGGAATATGAGCAGGAATAGTAGGRT | 371 | 0.2 | COI | 75 | |
| Pra-vol-A829: CTAAATCTACAGARATTCCYCTATGTCTAG | ||||||
| Aphidius spp. | AphG-S818: GAGAAGACCCTTTAGAATTTTATAWTAAWT | 180/181 | 2.4 | 16S | 150 | |
| AphG-A818: CCCTAAGGTAATTTATTTTAAWATWCTAAAAA | ||||||
| HypMP | Aphelinus abdominalis | Aphe-abd-S802: TGGTTTTTTGATTATAATTTAAAATCTG | 368 | 0.8 | 16S | 1500 |
| Aphe-abd-A802: TTATTTCAAAATAATTTGGATAAAATAAATA | ||||||
| Alloxysta victrix | All-vic-S801: AACAAATTTTTTTATTYTAAATAATACTTATAAG | 101 | 0.4 | 16S | 750 | |
| All-vic-A801: TTAYGCTGTTATCCCTAAGGTAACTAG | ||||||
| Asaphes suspensus | Asa-sus-S808: AAAGACGAGAAGACCCTATAGAATTTA | 138 | 1.8 | 16S | 15000 | |
| Asa-sus-A806: RAWTRATGTTAATTTATTAAAAAAATTTATTAAT | ||||||
| Asaphes vulgaris | Asa-vul-S809: ATTATTAAAAGACGAGAAGACCCTG | 173 | 1 | 16S | 1500 | |
| Asa-vul-A807: TCATAATTTATGGATAAAAAATTTATTAATATATA | ||||||
| Coruna clavata | Cor-cla-S811: ACAAAGGTAGCATAATCAATTGTCTTA | 255 | 0.8 | 16S | 15000 | |
| Cor-cla-A809: TATAAATTTATTTAAAAAATTTATTAAATTTTTAC | ||||||
| Dendrocerus carpenteri | Den-car-S814: TTATAGGATCAATTAACTTTTTRTCWACTCTAC | 205 | 0.1 | COI | 150 | |
| Den-car-A812: CCTCCWCCRCATGGGTCAAAG | ||||||
| Phaenoglyphis villosa | Pha-vil-S816: TCTTAATAATATAAGATACTGATTATTATTACCAG | 306 | 0.2 | COI | 150 | |
| Pha-vil-A815: GGTAAAGATAATAATAATAAAATTGTTGTTAGG |