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Multiplex results obtained with one of the multiplex polymerase mix linked extremely well with the uniplex reference.
Through this approach, the results represented with out of 31 blood samples 1 15 shows the positive with E. coli and remaining 14 only 11 were correlated with multiplex results of Vibrio cholera, remaining the urine samples all are positive with 17 samples correlate with the Salmonella typhi.
The multiplex assays were repeated, without variation, up to four times for each sample, and real-time quantitative PCR reported copy number consistent with the ability to repeat the multiplex results.
Finally, our Multiplex results also indicate that the level of TNF-α, a pro-inflammatory cytokine, was lower for wounds treated with CD34+ cells and ECs, or only CD34+ cells encapsulated in fibrin gel.
It is perhaps possible that this discrepancy is caused by the much larger size of our multiplex assay (44 amplicons in contrast to the 10 of the original study), plus differences in the way the multiplex results were assessed (gel visualization of PCR products in the original, but capillary electrophoresis SNP typing in this study).
However, abundant proteins present in bodily fluids, such as serum, may affect multiplex results.
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96 SNPs (divided evenly across the multiplexes) resulted in successful assays with >97% call rates and were used for future analyses (Additional file 7: Table S3).
The accuracy of this new molecular tool was tested and validated by comparing multiplex PCR results with species identification based on sequences at mitochondrial and nuclear markers.
For a given isolate, integration of PCR and multiplex pyrosequencing results allows the determination of the ESBL-producing status (available at https://ucl-irec-ctma.shinyapps.io/Pyrosequencing-TEM-SHV/).
Of particular interest was validating multiplex PCR results by incorporating a probe extension step.
The control region haplotypes (also multiplexed and re-amplified from the same DNA aliquot) are 100% consistent with the two Andaman specific haplgroups assigned by the multiplex SBE results.
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