Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

DNA Isolation01:24

DNA Isolation

40.2K
DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
40.2K

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Panorama: A robust pangenome-based method for predicting and comparing biological systems across species.

PLoS computational biology·2026
Same author

16S rRNA sequence dataset for the identification of the French hoverflies (Diptera, Syrphidae).

Biodiversity data journal·2026
Same author

Argonaute protein and small RNA expression patterns in Spodoptera frugiperda (Lepidoptera, Noctuidae).

Gene·2026
Same author

Retrotransposed gene copies persist under relaxed selection in wild Spodoptera frugiperda.

Molecular biology and evolution·2026
Same author

Priority effects drive fungal and nematode emergence from insect larvae.

FEMS microbiology ecology·2026
Same author

The fecal microbiota of lactating Holstein cows: A meta-analysis highlighting key microbial profiles and methodological challenges.

Journal of dairy science·2026

Related Experiment Video

Updated: Sep 11, 2025

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
11:22

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

Published on: October 15, 2019

29.4K

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

Laëtitia Leclerc1, Gabryelle Agoutin2, Thierry Brévault3

  • 1DGIMI, INRAE, Univ Montpellier, Montpellier, France.

Microbiology Spectrum
|August 12, 2025
PubMed
Summary
This summary is machine-generated.

We optimized the rpoB gene sequencing method for bacterial microbiota analysis. A nested PCR approach enhances amplification efficiency for low-concentration or host-associated bacterial DNA, improving accuracy in metabarcoding studies.

Keywords:
Spodoptera frugiperdahost-associated microbiotainsectmetabarcodingnested PCRrpoB

More Related Videos

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization
12:37

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

Published on: April 14, 2016

39.0K
Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR
11:37

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Published on: June 11, 2016

17.9K

Related Experiment Videos

Last Updated: Sep 11, 2025

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
11:22

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

Published on: October 15, 2019

29.4K
Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization
12:37

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

Published on: April 14, 2016

39.0K
Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR
11:37

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Published on: June 11, 2016

17.9K

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.