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Each assay was tested using as many positive mutation controls as available to us.
The sensitivity of the assay was tested using 0.5 40 ng pristine template DNA in the PCR reaction.
The assay was tested using blood from infected rats and was found to be sensitive, detecting less than one genomic equivalent of T. evansi.
This TaqMan real-time assay was tested using four different reagent kits and was shown to be robust and stable, with no significant differences in sensitivity between kits.
Specificity of the assay was tested using purified DNA from 67 species and subspecies of Fusarium as well as 50 species comprising 22 genera of other filamentous fungi and yeasts.
The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains.
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In addition, the NGS SNP assay and three CE-based assays (two STR and one InDel assay) were tested using a dilution series consisting of 0.5 ng, 0.1 ng, and 0.05 ng non-degraded DNA.
Differences between treatments (concentration of nanoparticles) in antimicrobial assay were tested using one-way ANOVA (GraphPad Prism, version 5, La Jolla, CA, USA) followed by Tukey's honestly significant difference (HSD) test, for differences that were significant at 5% probability.
Next, extracts that showed significant cytotoxicity to Panc-1 cells (> 80% cell death at a testing concentration of 100 μg/mL) in the PC biosensor assay were tested using a colorimetric MTT assay on two additional pancreatic cell lines (Mia-Paca2, and Capan-1).
Specificity of the two assays was tested using plasmid DNA for each event.
The diagnostic sensitivity of both assays was tested using samples from ELISA positive animals from 6 seropositive flocks.
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