From the analysis of exudates released by Methylosinus trichosporium OB3b and isolated following the one-step purification protocol published by Choi et al. [21], however, we found that the elemental composition (mainly C, N, and S) diverges largely from the theoretical composition of methanobactin according to the structure proposed by Behling et al. [16].
Here, we report an improved method yielding highly pure, copper-free methanobactin extending the published one-step resin extraction method with a subsequent purification step with HPLC using a reversed-phase C18 column.
The CoV screening was performed by amplifying a 179-nt stretch of the CoV RdRp gene using our previously published one-step pan-CoV RT-PCR method [24].
To demonstrate the superior sensitivity of the two-step approach, the lyssavirus qRT-PCR was compared with a previously published one-step lyssavirus qRT-PCR [ 33].
AKAV was detected in one bovine brain sample (1/123) by both the one-step mRT-PCR and published RT-qPCR [ 14] (Table 1).
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Genome equivalents in serum and homogenized tissues were determined using a previously published TaqMan one-step RT-qPCR assay targeting the genomic fragment encoding the nonstructural protein 551.
The reverse transcriptase (RT) and protease (PR -coding regions were amPR -coding separegionsactions using gene-specific primers and one-step RT-PCR techniques previously published [ 20].
Other strains were constructed by PCR-mediated one-step gene replacement using a published set of tagging cassettes and standard PEG/lithium acetate transformation (Longtine et al. 1998).
The E. coli K12 BW25113 Δ acrAB and E. coli NST74 Δ acrAB strains were constructed using the one-step inactivation of chromosomal genes using published methods (Datsenko et al., 2000).
When compared to previously published RT-qPCRs, the one-step mRT-qPCR provided comparable level of sensitivity and specificity.
