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. 2017 Dec 11;11(12):e0006077. doi: 10.1371/journal.pntd.0006077

Fig 4. Agarose-MAMA is capable of genotyping Y. pestis directly from complex clinical samples if pathogen targets are at sufficient levels.

Fig 4

(A) Agarose-MAMA (Mad-43) gel showing the PCR products for two template controls at the expected size for each respective ancestral and derived allele state (#1 and #2, respectively). Three complex clinical samples (#3-#5) also yielded PCR products with the size expected of an ancestral genotype. Two other complex clinical samples (#6 & #7) showed no PCR products but displayed a banding pattern consistent with NTC negative controls. (B) To assess the relative quantity of the Y. pestis target in the five clinical samples (#3, #4, #5, #6, #7), we generated amplification plots of these clinical samples on a TaqMan 3a assay used to target Y. pestis chromosomal DNA. Three clinical samples (#3-#5) showed amplified at a mid-range cycle-time (Ct) value consistent with high copy numbers of template DNA [21]. These same samples showed a robust signal of a PCR product on the MAMA gel. The two samples (#6 & #7) that failed on the MAMA gel showed a high Ct value and failed amplification, respectively, when tested by real-time PCR. The pairing of TaqMan 3a assay with MAMA gel results on the same templates provided insight to the genotyping capability of MAMA tools on complex clinical samples with low-level target template.