PMID- 35460243
OWN - NLM
STAT- MEDLINE
DCOM- 20220907
LR  - 20221110
IS  - 1943-7730 (Electronic)
IS  - 0007-5027 (Print)
IS  - 0007-5027 (Linking)
VI  - 53
IP  - 5
DP  - 2022 Sep 1
TI  - Multiplex Microsphere PCR (mmPCR) Allows Simultaneous Gram Typing, Detection of 
      Fungal DNA, and Antibiotic Resistance Genes.
PG  - 459-464
LID - 10.1093/labmed/lmac023 [doi]
AB  - 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.
CI  - (c) The Author(s) 2022. Published by Oxford University Press on behalf of American 
      Society for Clinical Pathology.
FAU - Browne, Daniel J
AU  - Browne DJ
AUID- ORCID: 0000-0003-1840-8904
AD  - Division of Immunology, QIMR Berghofer Medical Research Institute, Brisbane, 
      Australia.
AD  - Centre for Molecular Therapeutics, Australian Institute of Tropical Health and 
      Medicine, James Cook University, Cairns,Australia.
FAU - Liang, Fang
AU  - Liang F
AD  - Division of Immunology, QIMR Berghofer Medical Research Institute, Brisbane, 
      Australia.
FAU - Gartlan, Kate H
AU  - Gartlan KH
AD  - Division of Immunology, QIMR Berghofer Medical Research Institute, Brisbane, 
      Australia.
AD  - School of Medicine, University of Queensland, Brisbane, Australia.
FAU - Harris, Patrick N A
AU  - Harris PNA
AUID- ORCID: 0000-0002-2895-0345
AD  - Faculty of Medicine, UQ Centre for Clinical Research, University of Queensland, 
      Royal Brisbane and Women's Hospital, Brisbane, Australia.
FAU - Hill, Geoffrey R
AU  - Hill GR
AD  - Division of Immunology, QIMR Berghofer Medical Research Institute, Brisbane, 
      Australia.
AD  - Division of Hematopoietic Transplantation, Fred Hutchinson Cancer Research 
      Center, Seattle, WA, USA.
FAU - Corrie, Simon R
AU  - Corrie SR
AD  - Department of Chemical Engineering, ARC Centre of Excellence in Convergent 
      Bio-Nano Science and Technology, Monash and QLD Nodes, Monash University, 
      Clayton, Australia.
FAU - Markey, Kate A
AU  - Markey KA
AD  - Division of Immunology, QIMR Berghofer Medical Research Institute, Brisbane, 
      Australia.
AD  - School of Medicine, University of Queensland, Brisbane, Australia.
AD  - Division of Hematopoietic Transplantation, Fred Hutchinson Cancer Research 
      Center, Seattle, WA, USA.
AD  - Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
LA  - eng
GR  - P30 CA008748/CA/NCI NIH HHS/United States
GR  - Prestige Research Training Program/
GR  - Queensland Health Junior Clinical Research Fellowship/
PT  - Journal Article
PL  - England
TA  - Lab Med
JT  - Laboratory medicine
JID - 0250641
RN  - 0 (Anti-Bacterial Agents)
RN  - 0 (DNA Primers)
RN  - 0 (DNA, Fungal)
SB  - IM
MH  - *Anti-Bacterial Agents
MH  - DNA Primers/genetics
MH  - DNA, Fungal/genetics
MH  - Drug Resistance, Microbial
MH  - Humans
MH  - Microspheres
MH  - *Multiplex Polymerase Chain Reaction/methods
MH  - Sensitivity and Specificity
PMC - PMC9435484
OTO - NOTNLM
OT  - Gram typing
OT  - MagPlex-TAG microspheres
OT  - analytical specificity
OT  - antibiotic resistance testing
OT  - flow cytometry
OT  - fungi
OT  - multiplex PCR
EDAT- 2022/04/24 06:00
MHDA- 2022/09/08 06:00
PMCR- 2022/04/23
CRDT- 2022/04/23 08:34
PHST- 2022/04/24 06:00 [pubmed]
PHST- 2022/09/08 06:00 [medline]
PHST- 2022/04/23 08:34 [entrez]
PHST- 2022/04/23 00:00 [pmc-release]
AID - 6572827 [pii]
AID - lmac023 [pii]
AID - 10.1093/labmed/lmac023 [doi]
PST - ppublish
SO  - Lab Med. 2022 Sep 1;53(5):459-464. doi: 10.1093/labmed/lmac023.