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09:03
Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus
Published on: September 5, 2013
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Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer
Yu-Chieh Liao1, Han-Chieh Wu2, Ci-Hong Liou2
1Institute of Population Health Sciences, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan.
Frontiers in Microbiology
|April 15, 2022
Summary
Oxford Nanopore Technologies
Area of Science:
- Microbiology
- Genomics
- Molecular Biology
Background:
- Molecular typing is crucial for laboratory and clinical applications.
- Next-generation sequencing offers high-throughput molecular analysis but faces accessibility barriers due to high capital costs.
- Oxford Nanopore Technologies' MinION device provides real-time analysis, rapid library preparation, and low cost, but its raw accuracy is a limitation.
Purpose of the Study:
- To develop a concise multilocus sequence typing protocol for *Staphylococcus aureus* using the MinION device.
- To evaluate the impact of carryover DNA on MinION flow cell performance through successive runs.
- To demonstrate the effectiveness of the portable MinION sequencer for accurate and routine molecular typing.
Main Methods:
- Developed a multilocus sequence typing protocol for *Staphylococcus aureus*.
- Utilized multiplex polymerase chain reaction and the Rapid Barcoding Kit for sample barcoding.
- Employed the Oxford Nanopore Technologies MinION device for sequencing.
- Investigated MinION flow cell reusability by washing and performing successive sequencing runs to assess carryover DNA effects.
Main Results:
- Successfully performed accurate molecular typing of 467 *Staphylococcus aureus* samples.
- Sequenced a total of 3,269 kilobases of genetic material across 5 MinION runs.
- Demonstrated that the MinION flow cell can be reused effectively, maintaining accuracy.
- Confirmed the MinION device's capability for accurate typing despite potential carryover DNA.
Conclusions:
- The developed protocol enables accurate and rapid molecular typing of *Staphylococcus aureus*.
- The portable MinION sequencer is effective for routine molecular typing applications.
- MinION flow cell reusability is feasible, contributing to cost-effectiveness and reducing waste.
- The study validates the MinION as a valuable tool for accessible, high-throughput molecular typing in diverse settings.
Keywords:
MinIONmolecular typingmultilocus sequence typingmultiplex polymerase chain reactionnanopore sequencingMore Related Videos
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