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The method of DNA isolation from root nodules used in this study yielded PCR-amplifiable DNA for both bacterial and plant PCR target sequences in all cases.
For each PCR assay, a plasmid was produced that contains the respective PCR target sequence.
PCR target sequences were then confirmed by Mayo Clinic's Advanced Genomic Technology Center DNA Sequencing Core.
These results facilitated the design of a short pCS20 probe and a large PCR target that improved the sensitivity of the E. ruminantium detection assay.
To amplify the regions of interest we used the HaloPlex PCR target enrichment system (Agilent Technologies).
Using the Clustal Multiple Alignment Algorithm, we identified a highly conserved 245-bp PCR target on the above seven tetracycline resistance genes (Table 1).
For each PCR, target single-stranded DNA primed by the SWPOP primer in the exclusive one low-stringency cycle is converted into double-stranded form in the following high-stringency cycle due to the presence of a perfect annealing site for the specific primer.
Threshold values used for PCR analysis were set within the linear range of PCR target amplification.
This leads to inaccuracies when counting PCR target copy numbers as whole genome equivalents.
PCR target regions, based on specific deletions, were used to distinguish between M. tuberculosis and M. bovis.
PCR target products were used as reaction standards at concentrations ranging from 1×102 to 1×108 molecules per µL.
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