Positive samples for N. mikurensis in the qPCR assay were further validated with a nested PCR assay (Figure 1 ). The nPCR included primers targeting the 16S rRNA gene of N. mikurensis, to amplify a 1259-bp-long amplicon (Table 1 ). The 25 μL reactions of the first round of amplification consisted of 12.5 μL Supreme NZYTech Taq 2× Green Master Mix (NZYTech), 0.5 μL of each of the primers Neo_16S_95_F/Neo_16S_1393_R (10 μM, Table 1 ), and 9 μL RNAse free water.
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