A novel approach to automated genotyping of Mycobacterium tuberculosis using a panel of 15 MIRU VNTRs

Matsumoto, Tomoshige; Koshii, Yuriko; Sakane, Kazu; Murakawa, Tomomi; Hirayama, Yukio; Yoshida, Hisako; Kurokawa, Masashi; Tamura, Yoshitaka; Nagai, Takayuki; Kawase, Ichiro

doi:10.1016/j.mimet.2013.03.022

Cited by 15 publications

(10 citation statements)

References 6 publications

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“…The concordance reached 99.9% for the size range 100 to 900 bp, which corresponds to 98% of the processed data. These results are in line with previous observations (16) and demonstrate the capability of the automated method for MIRU-VNTR typing. However, the optimization of the automated sizing algorithm remains to be carried out to obtain full results for longer amplicons.…”

Section: Discussionsupporting

confidence: 92%

High-Throughput Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Genotyping for Mycobacterium tuberculosis Epidemiological Studies

et al. 2015

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dThe emergence of drug-resistant forms of tuberculosis (TB) represents a major public health concern. Understanding the transmission routes of the disease is a key factor for its control and for the implementation of efficient interventions. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) marker typing is a well-described method for lineage identification and transmission tracking. However, the conventional manual genotyping technique is cumbersome and time-consuming and entails many risks for errors, thus hindering its implementation and dissemination. We describe here a new approach using the QIAxcel system, an automated high-throughput capillary electrophoresis system that also carries out allele calling. This automated method was assessed on 1,824 amplicons from 82 TB isolates and tested with sets of markers of 15 or 24 loci. Overall allele-calling concordance between the methods from 140 to 1,317 bp was 98.9%. DNA concentrations and repeatability and reproducibility performances showed no biases in allele calling. Furthermore, turnaround time using this automated system was reduced by 81% compared to the conventional manual agarose gel method. In sum, this new automated method facilitates MIRU-VNTR genotyping and provides reliable results. Therefore, it is well suited for field genotyping. The implementation of this method will help to achieve accurate and cost-effective epidemiological studies, especially in countries with a high prevalence of TB, where the high number of strains complicates the surveillance of circulating lineages and requires efficient interventions to be carried out in an urgent manner.T uberculosis (TB) remains a major public health concern worldwide. The widespread emergence of multidrug resistant (MDR-TB) strains and extensively and extremely drug-resistant (XDR-TB and XXDR-TB) strains has hampered the management of treatment of the disease and the control of TB outbreaks (1). Many epidemiological questions remain unresolved, particularly in regard to the risk factors associated with the transmission of the bacilli to the host and to the prevalence of strain reactivation versus exogenous reinfection. Various genotype lineages of Mycobacterium tuberculosis have been described that can be associated with drug resistance, a higher rate of transmission, a higher rate of progression to disease, virulence, and vaccine escape (2, 3). The emergence of whole-genome sequencing (WGS) technologies can provide a complete genomic picture of lineage characterization and drug-resistant mutations. However, these technologies are presently not affordable for most laboratories, considering the costs and specialized skills required for data analysis. Thus, methods on the bench still have their value, as they have a faster turnaround time and can handle a higher batch capacity at a lower cost. One of these in particular, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) marker typing, is commonly used for effective genotyping. There ...

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Section: Discussionsupporting

confidence: 92%

High-Throughput Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Genotyping for Mycobacterium tuberculosis Epidemiological Studies

et al. 2015

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“…It would increase accuracy to 99.63% in Albania and 99.19% in Tunisia with a very high overall rate of concordance of 99.41%. These results are in line with other studies performed so far on MIRU-VNTR typing of MTBC with the QIAxcel system [14,15].…”

Section: Discussionsupporting

confidence: 93%

Accuracy of the QIAxcel Automated System for MIRU-VNTR Genotyping of Mycobacterium tuberculosis in Two Limited Resource Settings

Tafaj

Ghariani

Trovato

et al. 2020

JCM

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Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing of Mycobacterium tuberculosis complex (MTBC) isolates, based on 24 loci, is still widely used as the standard for routine molecular surveillance of tuberculosis (TB). QIAxcel system is proposed as an affordable tool that could replace conventional gel electrophoresis and provide high concordance with the reference methods regarding MIRU-VNTR typing of MTBC. We aimed to evaluate the QIAxcel accuracy for allele calling of MIRU-VNTR loci in two regional reference laboratories. A total of 173 DNA were used for the study. Results obtained with QIAxcel were compared to the reference results obtained with an ABI 3730 DNA analyzer. In Albania, the overall agreement with the reference method was 97.92%. A complete agreement result was obtained for 17 loci. In Tunisia, the overall agreement with the reference method was 98.95%. A complete agreement result was obtained for 17 loci. Overall agreement in both centers was 98.43%. In our opinion, use of QIAxcel technology has the potential to be reliable, given an optimized algorithm. Inaccuracies in sizing of long fragments should be solved, especially regarding locus 4052.

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“…However, genotyping M. tuberculosis using traditional methods is laborious. Thus, Matsumoto et al [39] presented a new approach that used the QIAxcel CE system for VNTR analysis, in which allelic ladders representing the loci of 15 mycobacterial interspersed repetitive units (MIRU)-VNTR were used to define the peaks and the samples were continuously analyzed in a 96-well plate. A high agreement (99.2%) was found between the results of Hitachi i-chips and that of the QIAxcel CE system.…”

Section: Detecting and Diagnosing Bacteriamentioning

confidence: 99%

Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease

Lian

Zhao

2016

Clinical Chemistry and Laboratory Medicine (CCLM)

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Rapid transmission, high morbidity, and mortality are the features of human infectious diseases caused by microorganisms, such as bacteria, fungi, and viruses. These diseases may lead within a short period of time to great personal and property losses, especially in regions where sanitation is poor. Thus, rapid diagnoses are vital for the prevention and therapeutic intervention of human infectious diseases. Several conventional methods are often used to diagnose infectious diseases, e.g. methods based on cultures or morphology, or biochemical tests based on metabonomics. Although traditional methods are considered gold standards and are used most frequently, they are laborious, time consuming, and tedious and cannot meet the demand for rapid diagnoses. Disease diagnosis using capillary electrophoresis methods has the advantages of high efficiency, high throughput, and high speed, and coupled with the different nucleic acid detection strategies overcomes the drawbacks of traditional identification methods, precluding many types of false positive and negative results. Therefore, this review focuses on the application of capillary electrophoresis based on nucleic detection to the diagnosis of human infectious diseases, and offers an introduction to the limitations, advantages, and future developments of this approach.

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A novel approach to automated genotyping of Mycobacterium tuberculosis using a panel of 15 MIRU VNTRs

Cited by 15 publications

References 6 publications

High-Throughput Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Genotyping for Mycobacterium tuberculosis Epidemiological Studies

High-Throughput Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Genotyping for Mycobacterium tuberculosis Epidemiological Studies

Accuracy of the QIAxcel Automated System for MIRU-VNTR Genotyping of Mycobacterium tuberculosis in Two Limited Resource Settings

Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease

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