Detection of rpoB Gene Mutations Using Helicase-Dependent Amplification

2013 ◽  
pp. 89-98 ◽  
Author(s):  
Wanyuan Ao ◽  
Robert Jenison
Keyword(s):  
Gene Mutations ◽  
Rpob Gene

scholarly journals Biological microchips for detection of multiple drug resistant M. tuberculosis strains isolated in Kyrgyz Republic

2008 ◽  
pp. 64-66
Author(s):  
J. T. Isakova ◽  
Z. K. Goncharova ◽  
A. A. Aldashev
Keyword(s):  
Drug Resistance ◽  
Pulmonary Tuberculosis ◽  
Gene Mutations ◽  
Rpob Gene ◽  
Multiple Drug ◽  
Drug Resistant ◽  
Kyrgyz Republic ◽  
Newly Diagnosed ◽  
Single Drug ◽  
Multiple Drug Resistant

The aim of the study was to estimate spread of primary and secondary multiple drug resistant Mycobacterium tuberculosis (MBT) and to characterize rpoB, katG, inhA, and ahpC gene mutations of rifampicin (RIF) and isoniazid (INH) resistant MBT strains isolated from tuberculosis patients in Kyrgyz. We obtained 493 specimens from patients with pulmonary tuberculosis which were diagnosed based on clinical, X-ray, and bacteriological examination. Among them, newly diagnosed pulmonary tuberculosis was in 445 patients (90.2 %), and 48 of the patients (9.8 %) have already been treated for tuberculosis. Mutations of rpoB, KatG, inhA, and ahpC genes associated with RIF and INH resistance were detected by biological chip test. Sensitive MBT strains were detected in 47 % and resistant strains were in 53 % of the newly diagnosed patients. Single-drug resistance to RIF only was detected in 3 % of cases; resistance to INH was found in 20 %, resistance to both the drugs was detected in 30 % of the patients. In pre-treated patients single-drug resistance to RIF was defined in 4 % of cases, resistance to INH was in 8 %, resistance to both the drugs was estimated in 75 % of the patients. Therefore, we suppose that there is a high prevalence of multi-drug resistant MBT in Kyrgyz Republic: 30 % among newly diagnosed patients and 75 % among pre-treated patients. The main cause of RIF-resistance of MBT is Ser531→Leu mutation of rpoB gene, and the main cause of INHresistance is Ser315→Thr mutation of katG gene.

scholarly journals Resistance profiles and rpoB gene mutations of Mycobacterium tuberculosis isolates in Taiwan

2013 ◽  
Vol 46 (4) ◽  
pp. 266-270 ◽  
Author(s):  
Yun-Ho Lin ◽  
Chun-Hsi Tai ◽  
Chia-Ru Li ◽  
Chin-Fu Lin ◽  
Zhi-Yuan Shi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Gene Mutations ◽  
Rpob Gene

scholarly journals Does Xpert MTB/RIF Assay Give Rifampicin Resistance Results Without Identified Mutation? Review of Cases from Addis Ababa, Ethiopia.

2019 ◽  
Author(s):  
Ayinalem Alemu ◽  
Mengistu Tadesse ◽  
Getachew Seid ◽  
Helina Mollalign ◽  
Kirubel Eshetu ◽  
...  
Keyword(s):  
Molecular Beacon ◽  
Addis Ababa ◽  
Gene Mutations ◽  
Rpob Gene ◽  
Rifampicin Resistance ◽  
Reference Laboratory ◽  
Probe Type ◽  
Dna Amount ◽  
National Tuberculosis ◽  
E Region

Abstract Background: Xpert MTB/RIF Assay is currently used in Ethiopia for the rapid diagnosis of Mycobacterium tuberculosis (MTB) and mutations that confer Rifampicin resistance. Rifampicin resistance is determined based on any mutation in the 81 bp of rpoB gene using five overlapping probes represented as Probe A (codons 507–511), Probe B (codons 512–518), Probe C (codons 518–523), Probe D (codons 523–529) and Probe E (codons 529–533). In this review, we assessed the frequency of missed probe types for Rifampicin Resistance results. Methods: Data were reviewed from specimens received and tested using Xpert® MTB/RIF Assay at Ethiopian National Tuberculosis Reference Laboratory, in Addis Ababa from 15 July 2016 to 31 December 2018 retrospectively. All archived data were reviewed carefully to describe missed probe types and quantity of DNA in the sample. Results: A total of 100 specimens were reported as MTB Detected Rifampicin Resistance Detected by Xpert MTB/RIF assay. More than half (55%) of these results were reported from male patients. The median age was 28.0 years (5 months to 88 years). Majorities (62%) of the cases were detected from sputum. Among the total of 38 extra pulmonary samples, lymph node aspirates were accounted 50% (19/38). The most common mutations (81.0%) were found in Probe E region followed by Probe D (10.0%), and Probe B (3.0%). Mutations in Probe A and Probe C regions were not observed. However six (6.0%) Rifampicin resistance cases were found without any missed probe type. The delta Ct max is ≥4.3. No specimen yielded Rifampicin resistance associated with more than one probe failure or mutation combinations. Conclusion: Mutations associated with Probe E (codons 529–533) region were identified as the commonest rpoB gene mutations. The Rifampicin resistance results found without any identified missing probe needs a further study. Lower DNA amount was observed in extrapulmonary specimens compared with sputum. Keywords: Rifampicin-resistance, Molecular Beacon, DNA probes, Xpert MTB/RIF Assay

scholarly journals Does Xpert MTB/RIF Assay Give Rifampicin Resistance Results Without Identified Mutation? Review of Cases from Addis Ababa, Ethiopia.

2020 ◽  
Author(s):  
Ayinalem Alemu ◽  
Mengistu Tadesse ◽  
Getachew Seid ◽  
Helina Mollalign ◽  
Kirubel Eshetu ◽  
...  
Keyword(s):  
Molecular Beacon ◽  
Addis Ababa ◽  
Gene Mutations ◽  
Rpob Gene ◽  
Rifampicin Resistance ◽  
Reference Laboratory ◽  
Probe Type ◽  
Dna Amount ◽  
National Tuberculosis ◽  
E Region

Abstract Background: Xpert® MTB/RIF assay is currently used in Ethiopia for the rapid diagnosis of Mycobacterium tuberculosis (MTB) and mutations that confer Rifampicin resistance. Rifampicin resistance is determined based on any mutation in the 81 bp of rpoB gene using five overlapping probes represented as Probe A (codons 507–511), Probe B (codons 512–518), Probe C (codons 518–523), Probe D (codons 523–529) and Probe E (codons 529–533). In this review, we assessed the frequency of missed probe types for Rifampicin Resistance results. Methods: Data were reviewed from specimens received and tested using Xpert® MTB/RIF assay at Ethiopian National Tuberculosis Reference Laboratory, in Addis Ababa from 15 July 2016 to 31 December 2018 retrospectively. All archived data were reviewed carefully to describe missed probe types and the quantity of DNA in the sample. Results: A total of 100 specimens were reported as MTB Detected Rifampicin Resistance Detected by Xpert® MTB/RIF assay. More than half (55%) of these results were reported from male patients. The median age was 28.0 years (5 months to 88 years). Majorities (62%) of the cases were detected from sputum. Among the total of 38 extrapulmonary samples, lymph node aspirates were accounted for 50% (19/38). The most common mutations (81.0%) were found in the Probe E region followed by Probe D (10.0%), and Probe B (3.0%). Mutations in Probe A and Probe C regions were not observed. However, six (6.0%) Rifampicin resistance cases were found without any missed probe type. The delta Ct max is ≥4.3. No specimen yielded Rifampicin resistance associated with more than one probe failure or mutation combinations. Conclusion: Mutations associated with Probe E (codons 529–533) region were identified as the commonest rpoB gene mutations. The Rifampicin resistance results found without any identified missing probe needs further study. The lower DNA amount was observed in extrapulmonary specimens compared with sputum. Keywords: Rifampicin-resistance, Molecular Beacon, DNA probes, Xpert® MTB/RIF Assay

Prevalence and Distribution of Multidrug-Resistant Mutations in Mycobacterium tuberculosis in Tanzania

2019 ◽  
Vol 1 (1) ◽  
pp. 15-22
Author(s):  
John C. Mgogwe ◽  
Hadija H. Semvua ◽  
Oliva Safari ◽  
Gibson E. Kapanda ◽  
Balthazar M. Nyombi ◽  
...  
Keyword(s):  
Gene Mutations ◽  
Rpob Gene ◽  
Multidrug Resistant ◽  
Sputum Smear ◽  
Sensitivity Testing ◽  
Cross Sectional ◽  
Important Approach ◽  
Mdr Tb ◽  
Frequent Mutations ◽  
Lowenstein Jensen

Background: Molecular identification of mutations resulting in multidrug-resistant tuberculosis (MDR-TB) is an important approach for improving understanding of MDR-TB epidemiology and planning for appropriate interventions. We aimed to estimate the prevalence and distribution of mutations causing MDR-TB as well as determine the gene distribution among patients previously treated for TB. Methods: This was a cross-sectional, hospital-based study conducted from April 2017 to October 2018 at Kibong’oto Infectious Diseases Hospital (KIDH). KIDH is the national MDR-TB referral hospital. Participants were patients presumptively diagnosed with MDR-TB and referred to KIDH from district and regional hospitals across Tanzania. Sputum samples were collected and analysed using the Xpert MTB/RIF assay, direct sputum smear fluorescence microscopy, culture on Lowenstein-Jensen medium, and line probe assay using the GenoType MTBDRplus VER 2.0 system. Demographic information and mutation frequencies were reported as counts and percentages and analysed using descriptive statistics. Results: A total of 208 (69.3%) participants had rpoB gene mutations conferring resistance to only rifampicin; 92 (30.7%) had rpoB, katG, and inhA mutations conferring resistance to rifampicin and isoniazid; 78 (26%) had rpoB and katG mutations conferring resistance to rifampicin and isoniazid; and 14 (4.7%) had rpoB and inhA mutations conferring resistance to rifampicin and isoniazid. Conclusion: The mutation prevalences identified in this study indicate the most frequent mutations were the S531L mutation of the rpoB gene, the S315T1 mutation of the katG gene, and the S315T mutation in the promoter region of the inhA gene. To control the emergence and spread of MDR-TB, drug sensitivity testing must be carried for GeneXpert-confirmed TB patients prior to initiating second-line anti-TB regimens.

scholarly journals Automatic Identification of Individual rpoB Gene Mutations Responsible for Rifampin Resistance in Mycobacterium tuberculosis by Use of Melting Temperature Signatures Generated by the Xpert MTB/RIF Ultra Assay

2019 ◽  
Vol 58 (1) ◽  
Author(s):  
Yuan Cao ◽  
Heta Parmar ◽  
Ann Marie Simmons ◽  
Devika Kale ◽  
Kristy Tong ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Melting Temperature ◽  
Gene Mutations ◽  
Test Sample ◽  
Rpob Gene ◽  
Automatic Identification ◽  
Content Type ◽  
Automated Algorithm ◽  
Wide Range ◽  
Rifampin Resistance

ABSTRACT Molecular surveillance of rifampin-resistant Mycobacterium tuberculosis can help to monitor the transmission of the disease. The Xpert MTB/RIF Ultra assay detects mutations in the rifampin resistance-determining region (RRDR) of the rpoB gene by the use of melting temperature (Tm) information from 4 rpoB probes which can fall in one of the 9 different assay-specified Tm windows. The large amount of Tm data generated by the assay offers the possibility of an RRDR genotyping approach more accessible than whole-genome sequencing. In this study, we developed an automated algorithm to specifically identify a wide range of mutations in the rpoB RRDR by utilizing the pattern of the Tm of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific “Tm signature” reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the Tm distances between the test sample and the reference Tm values. Validated using a set of clinical isolates, the algorithm correctly identified RRDR genotypes of 93% samples with a wide range of rpoB single and double mutations. Our analytical approach showed a great potential for fast RRDR mutation identification and may also be used as a stand-alone method for ruling out relapse or transmission between patients. The algorithm can be further modified and optimized for higher accuracy as more Ultra data become available.

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