Fig 1.
Overview of IS6110-5’3’FP original protocol and its simplified highly performing version.
(A). The original IS6110-5’3’FP protocol as described in Thabet et al. (2014) [11]. (B). The new protocol of IS6110-5’3’FP. In this new protocol version, aside from using the frequently cutting BstUI enzyme instead of HincII, there is no need for plasmid library amplification in E. coli, a modification that considerably shortens the method turnaround. Moreover, amplification in E. coli could result in the loss of some IS6110-containing plasmid most likely because of clone instability. Therefore, omission of this step increases the sensitivity of the method.
Fig 2.
Box plot showing the sizes of IS6110 polymorphic amplicons generated by IS6110-5’3’FP using the 11-banded laboratory reference strain genomic DNA digested either by BstUI or HincII.
The IS6110-5’3’FP products were fractionated without being diluted on an ABI PRISM 3100 capillary DNA sequencer (Applied Biosystems Inc., CA, USA). The boxes show the 25% to 75% interquartile range.
Fig 3.
IS6110-5’3’FP chromatograms of the 11-banded laboratory reference strain using the original (HincII-based) and the optimized version developed herein (BstUI-based).
x-axis = fragments size in base pairs (bp); y-axis = fluorescence intensity in relative fluorescence units (RFU).
Table 1.
Number of IS6110-5’3’FP-generated peaks relative to IS6110 copies as determined from IS6110 RFLP profiles.
Fig 4.
Assessment of the discriminatory power of IS6110-5’3’FP and 24-loci MIRU-VNTR.
M. tuberculosis strain collections belonging to Haarlem (A), LAM (B), and Beijing (C) genotypes were used.
Table 2.
Comparison of the discriminatory power of IS6110-5’3’FP and 24-loci MIRU-VNTR.
Table 3.
Comparative cost estimates (USD).