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Updated: May 5, 2026
Optimized PCR-based Detection of Mycoplasma
Published on: June 20, 2011
High-throughput rapid amplicon sequencing for multilocus sequence typing of Mycoplasma ovipneumoniae from archived
Isaac Framst1, Rebecca M Wolking2, Justin Schonfeld3
1Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada.
Introduction:
Spillover events of Mycoplasma ovipneumoniae have devastating effects on the wild sheep populations. Multilocus sequence typing (MLST) is used to monitor spillover events and the spread of M. ovipneumoniae between the sheep populations. Most studies involving the typing of M. ovipneumoniae have used Sanger sequencing. However, this technology is time-consuming, expensive, and is not well suited to efficient batch sample processing.
Methods:
Our study aimed to develop and validate an MLST workflow for typing of M. ovipneumoniae using Nanopore Rapid Barcoding sequencing and multiplex polymerase chain reaction (PCR). We compare the workflow with Nanopore Native Barcoding library preparation and Illumina MiSeq amplicon protocols to determine the most accurate and cost-effective method for sequencing multiplex amplicons. A multiplex PCR was optimized for four housekeeping genes of M. ovipneumoniae using archived DNA samples (N = 68) from nasal swabs.
Results:
Sequences recovered from Nanopore Rapid Barcoding correctly identified all MLST types with the shortest total workflow time and lowest cost per sample when compared with Nanopore Native Barcoding and Illumina MiSeq methods.
Discussion:
Our proposed workflow is a convenient and effective method for strain typing of M. ovipneumoniae and can be applied to other bacterial MLST schemes. The workflow is suitable for diagnostic settings, where reduced hands-on time, cost, and multiplexing capabilities are important.
Insights
A new Nanopore Rapid Barcoding workflow offers a faster and cheaper method for multilocus sequence typing (MLST) of Mycoplasma ovipneumoniae. This approach aids in monitoring disease spread in wild sheep populations.
Area of Science:
- Veterinary Microbiology
- Genomics
- Molecular Diagnostics
Background:
- Mycoplasma ovipneumoniae causes significant losses in wild sheep populations.
- Multilocus sequence typing (MLST) is crucial for tracking M. ovipneumoniae spread.
- Traditional Sanger sequencing for MLST is slow and costly.
Purpose of the Study:
- To develop and validate a Nanopore Rapid Barcoding sequencing workflow for M. ovipneumoniae MLST.
- To compare this workflow against Nanopore Native Barcoding and Illumina MiSeq methods.
- To identify the most accurate and cost-effective sequencing approach.
Main Methods:
- Optimized a multiplex PCR for four M. ovipneumoniae housekeeping genes.
- Utilized archived nasal swab DNA samples (N=68).
- Compared Nanopore Rapid Barcoding with Nanopore Native Barcoding and Illumina MiSeq.
Main Results:
- Nanopore Rapid Barcoding correctly identified all MLST types.
- This method demonstrated the shortest overall workflow time.
- It also offered the lowest cost per sample.
Conclusions:
- The proposed workflow is an effective and convenient tool for M. ovipneumoniae strain typing.
- This method is suitable for diagnostic settings prioritizing speed, cost, and multiplexing.
- The workflow can be adapted for other bacterial MLST schemes.
