Conjugated critical reagent characterization for ligand-binding assays: using MALDI-TOF-MS as an orthogonal tool to assess assay performance

Accelerating antimicrobial susceptibility testing (AST) is a priority in the development of novel microbiological methods. The MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA) has recently been described as a rapid phenotypic AST method. In this proof-of-principle study, we expanded this method to simultaneously test 24 antimicrobials. An Enterobacterales panel was designed and evaluated using 24 clinical isolates. Either one or two (only for antimicrobials with the EUCAST “I” category) breakpoint concentrations were tested. Microdroplets containing bacterial suspensions with antimicrobials and growth controls were incubated directly on the spots of a disposable MALDI target inside a humidity chamber for 6, 8 or 18 h. Broth microdilution was used as the standard method. After 6 and 8 h of incubation, the testing was valid (i.e., growth control was successfully detected) for all isolates and the overall categorical agreement was 92.0% and 92.7%, respectively. Although the overall assay performance applying short incubation times is promising, the lower performance with some antimicrobials and when using the standard incubation time of 18 h indicates the need for thorough standardization of assay conditions. While using “homebrew” utensils and provisional evaluation algorithms here, technical solutions such as dedicated incubation chambers, tools for broth removal and improved software analyses are needed.

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MALDI-TOF MS Genotyping of Polymorphisms Related to 1-Carbon Metabolism Using Common and Mass-Modified Terminators

Clinical Chemistry ◽

10.1373/clinchem.2008.115378 ◽

2009 ◽

Vol 55 (1) ◽

pp. 139-149 ◽

Cited By ~ 8

Author(s):

Klaus Meyer ◽

Åse Fredriksen ◽

Per Magne Ueland

Keyword(s):

Carbon Metabolism ◽

Accurate Determination ◽

Average Error ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Sufficient Power ◽

Assay Performance ◽

Allele Specific ◽

Tof Ms

Abstract Background: Large cohort studies may provide sufficient power to disentangle the role of polymorphisms related to 1-carbon metabolism and chronic diseases, but they require fast, accurate, high-throughput genotyping techniques. MALDI-TOF mass spectrometry has been adapted to rapid fine mapping using various approaches for allele discrimination. We developed a genotyping method based on MALDI-TOF MS and compared assay performance for formats based on standard and mass-modified terminators. Methods: The assay includes 20 polymorphisms of 14 genes involved in 1-carbon metabolism (BHMT 742G>A, CBS 844ins68 and 699C>T, CTH 1364G>T, DHFR del19, NOS3 –786T>C and 894G>T, FOLR1 1314G>A, MTHFD1 –105T>C and 1958G>A, MTHFR 677C>T and 1298A>C, MTR 2756A>G, MTRR 66A>G and 524C>T, SLC19A1 80G>A, SHMT1 1420C>T, TCN2 67A>G and 776C>G, and TYMS 1494del6). Results: Missing calls were observed for 4.7% of the DNA samples, attributed to failed liquid sample handling. Highly accurate genotyping was obtained by mass-modified as well as standard ddNTPs, with an average error rate of ≤0.1% by analysis of sample duplicates. A semiquantitative approach enabled unambiguous identification of the CBS 844ins68. Cluster plots of the relative allele intensities showed allele-specific bias according to type of minisequencing terminator and revealed a potential structural variation in the BHMT gene. Conclusions: MALDI-TOF MS–based genotyping using either standard or mass-modified terminators allows the accurate determination of single nucleotides as well as structural genetic variants. This was demonstrated with 20 polymorphisms involved in 1-carbon metabolism.

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