Nested PCR to optimize rpoB metabarcoding for low-concentration and host-associated bacterial DNA

Area of Science:

• Microbiology
• Genetics
• Bioinformatics

Background:

• Housekeeping genes are effective taxonomic markers for bacterial microbiota in metabarcoding.
• The rpoB gene offers high accuracy and species-level resolution but faces amplification challenges with degenerate primers.
• Host-associated microbiota often involves low bacterial DNA concentrations and eukaryotic DNA matrices.

Purpose of the Study:

• To optimize the rpoB gene amplification for improved characterization of bacterial microbiota.
• To address limitations of degenerate rpoB primers in low-input and complex samples.
• To enhance the accuracy and efficiency of host-associated microbiota analysis.

Main Methods:

• Developed and evaluated a two-step (nested) PCR approach for rpoB gene amplification.
• Utilized in silico analysis to assess primer universality.
• Tested the nested PCR method on commercial mock samples and field-collected insect samples.

Main Results:

• The nested PCR approach significantly increased amplification efficiency for dilute bacterial DNA samples.
• No bias in bacterial composition was observed in mock samples compared to single-step PCR.
• Nested PCR outperformed single-step PCR in amplifying bacterial DNA from insect oral secretions and larvae.

Conclusions:

• The nested rpoB PCR method provides a robust strategy for characterizing host-associated bacterial microbiota.
• This optimized method enhances amplification efficiency and accuracy, especially for challenging samples.
• The improved technique offers a reliable solution for microbiome research.