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

You might also read

Related Articles

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

Sort by
Same author

A multi-site laboratory evaluation of the MEDSCAN application for automated POC-CCA interpretation.

Frontiers in parasitology·2026
Same author

Achievement of 15-Minute Adaptive PCR Benchmark with 1370 nm Laser Heating.

Biosensors·2025
Same author

L-DNA-Based Melt Analysis Enables Within-Sample Validation of PCR Products.

Analytical chemistry·2024
Same author

Implementing L-DNA analogs as mirrors of PCR reactant hybridization state: theoretical and practical guidelines for PCR cycle control.

Analytical methods : advancing methods and applications·2024
Same author

Correction to "Low-Resource Nucleic Acid Extraction Method Enabled by High-Gradient Magnetic Separation".

ACS applied materials & interfaces·2023
Same author

A safer framework to evaluate characterization technologies of exhaled biologic materials using electrospun nanofibers.

Nanoscale·2023

Related Experiment Video

Updated: Sep 19, 2025

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray
07:35

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Published on: April 25, 2014

12.9K

Single-Sample Melt-Based Screening for Rifampicin Susceptibility in the Emerging Mutation Hotspot at rpoB Codon 491.

Nicole A Malofsky1, Swayashreyee B Dhungel1, Megan E Pask1

  • 1Department of Biomedical Engineering, Vanderbilt University, Nashville, Tennessee 37235, United States.

ACS Infectious Diseases
|June 17, 2025
PubMed
Summary
This summary is machine-generated.

A new assay detects rifampicin resistance mutations in Mycobacterium tuberculosis by analyzing rpoB codon 491 variants. This method improves upon existing tests by accurately identifying all three major mutations, aiding in better treatment and control of tuberculosis.

Keywords:
L-DNAemerging hotspotmelt analysismutation screeningrifampicin resistancetuberculosis

More Related Videos

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test
08:10

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Published on: April 9, 2012

81.1K
Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria
10:27

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Published on: November 10, 2015

11.8K

Related Experiment Videos

Last Updated: Sep 19, 2025

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray
07:35

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Published on: April 25, 2014

12.9K
Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test
08:10

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Published on: April 9, 2012

81.1K
Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria
10:27

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Published on: November 10, 2015

11.8K

Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • Mutations at rpoB codon 491 in Mycobacterium tuberculosis are linked to rifampicin resistance.
  • Current diagnostic tests fail to detect all relevant codon 491 mutations, leading to untreated resistant infections.
  • Existing real-time PCR assays may miss critical variants, hindering effective tuberculosis treatment.

Purpose of the Study:

  • To develop a novel single-sample screening method for detecting all three sequence-identified rpoB codon 491 variants (I491F, I491N, I491M).
  • To address the limitations of current genotypic tests in identifying rifampicin resistance in Mycobacterium tuberculosis.
  • To provide a highly sensitive and specific diagnostic tool for rpoB mutations associated with drug resistance.

Main Methods:

  • Development of a single-sample screening method using asymmetric PCR followed by melt analysis.
  • Incorporation of a melt probe matching the susceptible sequence and double-stranded L-DNA for single-sample classification.
  • Evaluation of the assay on synthetic rpoB wild-type and variant sequences using two calibrated PCR instruments.

Main Results:

  • The developed assay achieved 100% sensitivity and 100% specificity across both PCR instruments.
  • The new method accurately classified all three variants (I491F/N/M), unlike the André assay which misclassified I491N and I491M.
  • Single-sample classification based on within-sample T_m differences effectively identified rifampicin susceptibility.

Conclusions:

  • The developed asymmetric PCR and melt analysis assay is a robust tool for detecting rpoB codon 491 mutations in Mycobacterium tuberculosis.
  • This assay overcomes the limitations of existing genotypic tests, enabling accurate identification of rifampicin resistance.
  • The assay design is adaptable for detecting other clinically significant mutation hotspots, offering broad applicability in molecular diagnostics.