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Design and validation of Dolosigranulum pigrum specific PCR primers using the bacterial core genome
Maliha Aziz1, Amber Palmer1, Søren Iversen2
1Antibiotic Resistance Action Center, Department of Environmental and Occupational Health, Milken Institute School of Public Health, George Washington University, 800 22nd Street NW, Washington, DC, 20052, USA.
Abstract:
Dolosigranulum pigrum-a lactic acid bacterium that is increasingly recognized as an important member of the nasal microbiome. Currently, there are limited rapid and low-cost options for confirming D. pigrum isolates and detecting D. pigrum in clinical specimens. Here we describe the design and validation of a novel PCR assay targeting D. pigrum that is both sensitive and specific. We designed a PCR assay targeting murJ, a single-copy core species gene identified through the analysis of 21 D. pigrum whole genome sequences. The assay achieved 100% sensitivity and 100% specificity against D. pigrum and diverse bacterial isolates and an overall 91.1% sensitivity and 100% specificity using nasal swabs, detecting D. pigrum at a threshold of 1.0 × 104 D. pigrum 16S rRNA gene copies per swab. This assay adds a reliable and rapid D. pigrum detection tool to the microbiome researcher toolkit investigating the role of generalist and specialist bacteria in the nasal environment.
Insights
A new PCR assay effectively detects Dolosigranulum pigrum, a key nasal bacterium. This rapid and specific tool aids researchers in understanding the nasal microbiome and D. pigrum
Area of Science:
- Microbiology
- Genomics
- Molecular Biology
Background:
- Dolosigranulum pigrum is an increasingly recognized component of the human nasal microbiome.
- Current methods for identifying D. pigrum in clinical samples are limited in speed and cost-effectiveness.
Purpose of the Study:
- To design and validate a novel, sensitive, and specific PCR assay for the detection of D. pigrum.
- To provide a reliable tool for microbiome research focused on the nasal environment.
Main Methods:
- A PCR assay was designed targeting the single-copy murJ gene, identified from 21 D. pigrum whole genome sequences.
- The assay's sensitivity and specificity were validated against various bacterial isolates and clinical nasal swab samples.
Main Results:
- The assay demonstrated 100% sensitivity and specificity against pure cultures of D. pigrum and other bacteria.
- Using nasal swabs, the assay achieved 91.1% sensitivity and 100% specificity, detecting D. pigrum at a threshold of 1.0 × 10^4 16S rRNA gene copies per swab.
Conclusions:
- The developed PCR assay offers a reliable and rapid method for D. pigrum detection.
- This tool enhances the capacity of researchers to investigate the role of D. pigrum in the nasal microbiome.
