Table 1.
Sequences of new hydrazine synthase primers targeting the hzsA subunit of anammox bacteriaa
| Primerb | Position (bp) | Sequence |
|---|---|---|
| Forward | ||
| hzsA_370F | 370–389 | 5′-CACAAGAADGGYGGYGGDTG-3′ |
| hzsA_382F | 382–400 | 5′-GGYGGDTGYCAGATATGGG-3′ |
| hzsA_526F | 526–543 | 5′-TAYTTTGAAGGDGACTGG-3 |
| hzsA_757F | 757–773 | 5′-AGTTCNAAYTWTGATCC-3′ |
| hzsA_757F Scalindua | 757–773 | 5′-AGTTCNAAYTWTGACCC-3′ |
| hzsA_862F | 862–878 | 5′-GTDGAYAACTGGGAYGG-3′ |
| hzsA_1318F | 1318–1338 | 5′-TATCAGCCRTTTGAYCAGGTG-3′ |
| hzsA_1597F | 1597–1615 | 5′-WTYGGKTATCARTATGTAG-3′ |
| hzsA_1600F | 1600–1617 | 5′-GGKTATCARTATGTAGAG-3′ |
| hzsA_1600F Scalindua | 1600–1620 | 5′-GGKTATCARTATGTAGAAG-3′ |
| Reverse | ||
| hzsA_887R | 868–887 | 5′-GGATAHGCRCCRTCCCAGTT-3′ |
| hzsA_1353R | 1333–1353 | 5′-ATAHCCTTCMACHTTCACCTG-3′ |
| hzsA_1829R | 1813–1829 | 5′-TCATACCACCARTTGTA-3′ |
| hzsA_1829R Scalindua | 1813–1829 | 5′-CTGAACCACCARTTGTA-3′ |
| hzsA_1857R | 1838–1857 | 5′-AAABGGYGAATCATARTGGC-3′ |
| hzsA_2210R | 2191–2210 | 5′-GGRTTDACATAYTTWCCRCC-3 |
| hzsA_2390R | 2371–2390 | 5′-ATRTTRTCCCAYTGYGCHCC-3′ |
The positions indicated refer to the “Ca. Kuenenia stuttgartiensis” hzsA gene (kuste2861).
Several primer set were tested, but generally the primer set hzsA_526F/hzsA_1857R performed best for most of the samples. In addition, for the Ooijpolder freshwater ditches, a nested PCR with the primer set hzsA_382F/hzsA_2390R followed by the primer set hzsA_526F/hzsA_1857R or a direct PCR with the primer set hzsA_526F/hzsA_1829R was used. For the RT-PCR and qPCR, primer set hzsA_1597F/hzsA1857R is recommended. PCR products of the expected size have been obtained for Northeast Greenland and from the Barents Sea samples, using the primer combination hzsA_526F/hzsA_1829R. The primers with “Scalindua” added to their names were designed specifically to target marine “Ca. Scalindua” species.