Multiplex Microsphere PCR (mmPCR) Allows Simultaneous Gram Typing, Detection of Fungal DNA, and Antibiotic Resistance Genes

Browne, Daniel J., Liang, Fang, Gartlan, Kate H., Harris, Patrick N.A., Hill, Geoffrey R., Corrie, Simon R., and Markey, Kate A. (2022) Multiplex Microsphere PCR (mmPCR) Allows Simultaneous Gram Typing, Detection of Fungal DNA, and Antibiotic Resistance Genes. Laboratory Medicine, 53 (5). pp. 459-464.

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Objective: To show the high analytical specificity of our multiplex microsphere polymerase chain reaction (mmPCR) method, which offers the simultaneous detection of both general (eg, Gram type) and specific (eg, Pseudomonas species) clinically relevant genetic targets in a single modular multiplex reaction.

Materials and Methods: Isolated gDNA of 16S/rRNA Sanger-sequenced and Basic Local Alignment Tool–identified bacterial and fungal isolates were selectively amplified in a custom 10-plex Luminex MagPlex-TAG microsphere-based mmPCR assay. The signal/noise ratio for each reaction was calculated from flow cytometry standard data collected on a BD LSR Fortessa II flow cytometer. Data were normalized to the no-template negative control and the signal maximum. The analytical specificity of the assay was compared to single-plex SYBR chemistry quantitative PCR.

Results: Both general and specific primer sets were functional in the 10-plex mmPCR. The general Gram typing and pan-fungal primers correctly identified all bacterial and fungal isolates, respectively. The species-specific and antibiotic resistance–specific primers correctly identified the species- and resistance-carrying isolates, respectively. Low-level cross-reactive signals were present in some reactions with high signal/noise primer ratios.

Conclusion: We found that mmPCR can simultaneously detect specific and general clinically relevant genetic targets in multiplex. These results serve as a proof-of-concept advance that highlights the potential of high multiplex mmPCR diagnostics in clinical practice. Further development of specimen-specific DNA extraction techniques is required for sensitivity testing.

Item ID: 74255
Item Type: Article (Research - C1)
ISSN: 1943-7730
Keywords: multiplex PCR, flow cytometry, MagPlex-TAG microspheres, antibiotic resistance testing, Gram typing, fungi, analytical specificity
Copyright Information: © The Author(s) 2022. Published by Oxford University Press on behalf of American Society for Clinical Pathology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Date Deposited: 18 May 2022 09:02
FoR Codes: 32 BIOMEDICAL AND CLINICAL SCIENCES > 3202 Clinical sciences > 320220 Pathology (excl. oral pathology) @ 100%
SEO Codes: 20 HEALTH > 2001 Clinical health > 200101 Diagnosis of human diseases and conditions @ 100%
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