PCR amplification of a 570-bp fragment from the strA gene of S. griseus subsp. griseus from agricultural streptomycin formulation batch 12 (Table 1) spiked with different amounts of S. griseus subsp. griseus DNA. PCR was performed directly on a suspension of the formulation (A to C) or after DNA extraction using the BioSprint96 DNA plant kit (a to c). Sample pretreatments were as follows: none (A and a), 50 μg nontarget (P. agglomerans) DNA g⁻¹ formulation added (B and b), 40 mM sodium metaphosphate added to formulation suspension (C and c), and S. griseus subsp. griseus DNA only (formulation absent) (D). The amounts of spiked S. griseus subsp. griseus DNA g⁻¹ formulation in the different lanes are as follows: 30 μg (lanes 1), 5 μg (lanes 2), 1 μg (lanes 3), 0.2 μg (lanes 4), 0.04 μg (lanes 5), and 0 μg (formulation only) (lanes F). (Bottom right panel) Effect of pretreatment with different concentrations of sodium metaphosphate (0 to 160 mM) on DNA extraction and strA amplification efficiency on a sample of formulation batch 12 containing 1 μg of spiked S. griseus subsp. griseus DNA g⁻¹ formulation. The optimal concentration of sodium metaphosphate is about 40 mM. F lanes are negative controls and contain formulation with no spiked S. griseus subsp. griseus DNA and 40 mM sodium metaphosphate. Lanes M, size markers (base pair ladder from Sigma). Positive bands in the gels are shown in white boxes.

Detection limit of 16S rRNA genes (top band in gels) and strA (bottom band in gels) of S. griseus subsp. griseus in different agricultural streptomycin formulation mixtures after sample pretreatment with 40 mM sodium metaphosphate and DNA extraction using the BioSprint96 DNA plant kit. PCR of 16S rRNA genes and PCR of strA were performed as separate reactions, and then the products were combined before loading on the gel. Formulation mixtures consisted in equal parts of different batches of the same formulation manufactured by the same producer, with the exception of mixture D (Table 1), where different formulations with similar textures were used. Spiked S. griseus subsp. griseus DNA was added at the following rates (g⁻¹ formulation): 5 μg (lanes 2), 1 μg (lanes 3), 200 ng (lanes 4), 40 ng (lanes 5), 8 ng (lanes 6), 1.6 ng (lanes 7), 0.3 ng (lanes 8), and 0 ng (formulation only) (lanes F). (Bottom right panel) Effect of pretreatment with different concentrations of sodium metaphosphate (0 to 160 mM) on DNA extraction and 16S rRNA gene amplification on a sample of formulation batch 12 amended with 1 μg of spiked S. griseus subsp. griseus DNA g⁻¹ formulation. The optimal concentration of sodium metaphosphate was about 40 mM. F lanes are negative controls and contain formulation with no spiked S. griseus subsp. griseus DNA and 40 mM sodium metaphosphate. Lanes M, size markers (base pair ladder from Sigma). The observable detection limits on the gels are indicated by the white boxes.