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 Table of Contents  
Year : 2022  |  Volume : 11  |  Issue : 1  |  Page : 23-29

Validation and comparative analysis of kogene mycobacterial interspersed repetitive unit-variable number of tandem repeat typing kit and its application on clinically isolated mycobacterium tuberculosis samples from national tuberculosis hospital, Republic of Korea

Clinical Research Center, Masan National Tuberculosis Hospital, Changwon, South Korea

Date of Submission16-Jan-2022
Date of Decision26-Jan-2022
Date of Acceptance26-Feb-2022
Date of Web Publication12-Mar-2022

Correspondence Address:
Sungweon Ryoo
Masan National Tuberculosis Hospital, Gapo-ro 215, MasanHappo-gu, Changwon-si, Gyeongsangnamdo 51755
South Korea
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmy.ijmy_8_22

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Background: Tuberculosis (TB) remains a serious public health burden in Korea. Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) is preferred for epidemiological TB investigation. Until recently, the difficulty lies in epidemiological TB investigation due to the absence of commercialized MIRU-VNTR in Korea. Here, we have evaluated the newly designed MIRU-VNTR kit by Kogenebiotech, Korea. Materials and Methods: A total of 200 samples, where 100 are Mycobacrerium tuberculosis (M. tuberculosis), and the other 100 are non-M. tuberculosis, were used. Initially, the Kogenebiotech MIRU-VNTR typing kit (KoMIRU) was compared with Multilocus Variable Number Tandem Repeat Genotyping of M. tuberculosis typing kit (MVNTR) by Philip Supply for validation purpose. Then, Limit of Detection for DNA copies was optimized. Finally, KoMIRU and Genoscreen MIRU-VNTR typing kit (GeMIRU) were tested and comparatively analyzed for its specificity and sensitivity. Results: The study showed that the KoMIRU has slightly higher discriminatory power over MVNTR, 100% versus 97.5%. In comparative analysis, the KoMIRU has shown comparable capability as GeMIRU, showing 100% for sensitivity and specificity with a 95% CI value of 96.38 to 100.00%. Also, no discrepancies were observed on discriminated lineage strains between KoMIRU and GeMIRU. Out of 100, 84 were identified as Beijing strains, and remains were identified as NEW-1 (n = 8), Uganda (n = 6), East African Indian (EAI) (n = 6), Turkey (n = 2), and Haarlem (n = 1). Conclusion: In this study, KoMIRU has shown a comparable capability to GeMIRU. Furthermore, previous researches had suggested an association between lineage strains and drug resistance; hence, the implementation of KoMIRU can help in TB control and prevention.

Keywords: Mycobacterium tuberculosis lineage, Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat, tuberculosis, tuberculosis genotyping

How to cite this article:
Jung J, Kang T, Hwang Y, Ryoo S. Validation and comparative analysis of kogene mycobacterial interspersed repetitive unit-variable number of tandem repeat typing kit and its application on clinically isolated mycobacterium tuberculosis samples from national tuberculosis hospital, Republic of Korea. Int J Mycobacteriol 2022;11:23-9

How to cite this URL:
Jung J, Kang T, Hwang Y, Ryoo S. Validation and comparative analysis of kogene mycobacterial interspersed repetitive unit-variable number of tandem repeat typing kit and its application on clinically isolated mycobacterium tuberculosis samples from national tuberculosis hospital, Republic of Korea. Int J Mycobacteriol [serial online] 2022 [cited 2022 Dec 7];11:23-9. Available from: https://www.ijmyco.org/text.asp?2022/11/1/23/339521

  Introduction Top

Despite readily available treatment and antitubercular drugs, tuberculosis (TB) remains a serious public health burden in a global setting.[1],[2],[3],[4] To overcome against prevalent TB, identifying strains of TB for its transmission dynamics is inevitable.[5]

The Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing is the most promising typing and there is high demand for optimized and user-friendly rapid MIRU-VNTR typing to enable active TB case finding and establish national epidemiological TB surveillance.[6]

In this study, given that the need for optimized MIRU-VNTR typing is high, we have evaluated novel MIRU-VNTR typing. Here, we have validated and compared it with other MIRU-VNTR typing kit.

  Methods Top


This study was commenced on June 1, 2019, and was exempted from Institutional Review Board approval for experiment involving human subjects as no direct/invasive experiments involving human subjects required. Hence, consent for human/patient is not required for this study. The clinical Mycobacterium tuberculosis samples were procured from the TB and infectious disease biobank of Masan National Tuberculosis Hospital, South Korea which collects clinical TB samples on patient's consent.

DNA samples

We used a total number of 200 DNA samples in this study. Of 200 samples, 100 DNA samples provided are isolated from clinical M. tuberculosis deposited in TB and infectious disease biobank of Masan National Tuberculosis Hospital, South Korea, from 2019 to 2020.[7] The other 100 DNA samples provided by ATCC, Korean Collection for Type Culture, Korean Veterinary Culture Collection, National Culture Collection for Pathogens, Korea Bank for Pathogenic Viruses, Vircell, and Promega, from 2019 to 2020 and are non-M. tuberculosis. These include bacteria, viruses, and human [Supplemental Table S1]. Also, for control use purposes, M. tuberculosis H37Rv (ATCC 25618D-2) was used throughout the experiments. The MIRU-VNTR testing was carried out using these DNA samples above and our MIRU-VNTR typing kit.

Validation of Kogenebiotech Mycobacterial Interspersed Repetitive Unit

A validation analysis was carried out to determine whether designed primers have differential typing capability. In this work, KoMIRU and Multilocus Variable Number Tandem Repeat Genotyping of M. tuberculosis typing kit (MVNTR) by Philip Supply were carried out on the first 40 DNA samples of clinical M. tuberculosis according to the technical manual.[8] We made some modifications. Polymerase chain reaction (PCR) amplification has performed this work with SimpliAmp Thermal Cycler (ThermoFisher Scientific, USA) and, instead of Qiagen Hotstart Taq Polymerase kit (Qiagen, Germany), PowerAmp 2X Premix (Kogenebiotech, Korea) was used. ABI 3500 Genetic Analyzer was used (Applied Biosystems, USA) instead of ABI 3100 genetic analyzer (Applied Biosystems, USA).

The designed primers of KoMIRU and MVNTR were tabulated for comparison [Supplemental Table S2]. Then, results were interpreted using the MIRU-VNTRplus database (URL: https://www.miru-vntrplus.org/MIRU/index. faces) to determine the strain lineage of M. tuberculosis samples.

Optimization of limit of detection

Upon successful validation of KoMIRU, we performed optimization of KoMIRU. Multiplex PCR or KoMIRU was carried out to optimize the limit of detection (LoD). The PCR mixture was composed of 10 ㎕ of PowerAmp 2X Premix (Kogenebiotech, Korea), 5 ㎕ of each Primer Mixture from 1 to 6, and 5 ㎕ of the DNA template. Each Primer mixture contains four different target loci for amplification [Supplemental Table S2]. In this testing, M. tuberculosis H37Rv (ATCC 25618D-2) was used with additional amounts of DNA copies varies from 5 to 50,000 with 5 intervals, 5, 50, 500, 5,000, and 50,000 copies per ㎕, to determine LoD. The DNA of M. tuberculosis H37Rv was initially measured for its concentration by using NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, USA). We calculated the copy number based on measured DNA concentration and genome size (Full genome size: 4,411,532 bp). We used PCR-grade nuclease-free water for negative control.

Amplification was done using the SimpliAmp Thermal Cycler (ThermoFisher Scientific, USA) with the following conditions: 5 min at 95°C for initial denaturation, 35 cycles of 30 s at 95°C, another 30 s at 63°C, and 90 s at 72°C for annealing and 5 min at 72°C for the final extension. We performed experiments triplicate.

The amplicons were then proceeded to fragment assay. The reaction mixture was prepared by using 9 ㎕ of Formamide HiDi for DNA denaturation, 0.5 ㎕ of 1500 LIZ, and 1 ㎕ of amplicon or allelic ladder. Those prepared reaction mixtures were dispensed to plates and centrifuged for few seconds. Then each plate was placed in a thermal cycler with the following conditions: 5 min at 95°C and 3 min at 4°C. Once the reaction was done, the plates were equipped in ABI 3500 Genetic Analyzer (Applied Biosystems, USA) for analysis. The operation of the genetic analyzer was set and conducted according to the manufacturer's manual.[9] Then, results were interpreted using the MIRU-VNTRplus database to determine the strain lineage of M. tuberculosis samples.

Evaluation of interpersonal reproducibility

The evaluation of reproducibility was tested using KoMIRU. The same protocol which was used for the optimization of LoD was used with some modification. These modifications made are (1) three DNA concentrations were used: 50, 100, 200 copies per ㎕ where each labeled as ×1 LoD, ×2 LoD, and ×4 LoD, respectively; (2) two technicians with similar research capability experimented once per day for 10 straight days. Based on the result, peaks of MIRU-VNTR markers were analyzed, and from it, coefficient of variance (CV) was calculated. If CV lesser than 5% is observed, it is considered as successfully reproduced. We did experiments duplicate.

Comparative analysis with commercially available Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat typing kit

The comparative analysis was conducted using KoMIRU and GeMIRU.

For the KoMIRU, the same protocol which was used for the optimization of LoD was used with some modification. These modifications made are: 1) DNA concentration of samples used were adjusted to 50 copies per ㎕; 2) all 200 samples, 100 clinical M. tuberculosis, and 100 non-M. tuberculosis samples were used and M. tuberculosis H37Rv was not used during this evaluation.

For the GeMIRU, the protocol used in this experiment was referred by the manufacturer's manual and related article.[10],[11]

The results from both KoMIRU and GeMIRU were analyzed and compared for similarity in silico using the MIRU-VNTRplus database to determine strain lineage of M. tuberculosis samples [Table 1]. We did experiments triplicate.
Table 1: Comparative analysis on detection rate of target locus

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  Results Top

Validation of Kogenebiotech Mycobacterial Interspersed Repetitive Unit

KoMIRU and MVNTR were conducted on 40 clinical M. tuberculosis samples for comparison and validation purposes. The result of both typings, in general, shows a high rate of amplification on each target locus. However, the MVNTR showed 97.5% of amplification on 3 target locus, Mtub 04, ETRB, and QUB 26 [Table 1]. Hence, the KoMIRU has shown a slightly higher amplification rate than that of MVNTR, indicating higher discriminatory power.

Determination of optimum DNA concentration

Upon successful validation compared with MVNTR, the five different concentrations, from 5 to 50,000 copies per ㎕, were directed to the test to determine optimum DNA concentration. Of 5 different concentrations tested, the result turned out that all DNA concentrations were amplified except the lowest concentration, 5 copies per ㎕ [Table 2]. Based on the result, DNA concentration of 50 copies per ㎕ is determined as optimized minimum DNA concentration required for amplification. Hence, DNA concentration of 50 copies per ㎕ was set forward as standard and used for subsequent experiments and analyses.
Table 2: Result of mycobacterial interspersed repetitive unit-variable number tandem repeat locus amplification on different DNA concentration

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Evaluation of interpersonal reproducibility

An interpersonal reproducibility test was carried out to test and evaluate the stability of the KoMIRU typing and human-derived influence affected by performing technician, interpersonal reproducibility test was carried out. Two technicians with comparable research capability to each other were selected and conducted KoMIRU typing using 3 different concentrations, ×1 LoD, ×2 LoD, and ×4 LoD, once per day for 10 days and made typing duplicate. We made an analysis based on peaks observed from 24 target locus of the MIRU-VNTR. The raw data of peaks, mean value, SD and CV, presented as a percentile, were derived and analyzed.

Typically, CV value lesser than 5% is considered as no significant difference. In the result, the actual CV values observed mainly were 0.01% [Supplemental Table S3]. A primer amplifying certain target loci, Mtub 30, showed the CV value of 0.12 on × 1 LoD but is considered ignorable as it is lower than the threshold, 5%. Thus, the interpersonal reproducibility evaluation confirmed that KoMIRU is highly reproducible and less likely to be affected by human-derived factors.

Comparative analysis of Kogenebiotech Mycobacterial Interspersed Repetitive Unit and Genoscreen Mycobacterial Interspersed Repetitive Unit

Comparative analysis was carried out to compare KoMIRU to that of GeMIRU to determine sensitivity and specificity.

Initially, both KoMIRU and GeMIRU typing were conducted on samples (n = 200) where 100 are clinical M. tuberculosis samples, and the other 100 samples are non-M. tuberculosis samples. Also, the strains of M. tuberculosis were determined and classified into different categories according to the MIRU-VNTRplus database.

Of 200 samples typed using 2 MIRU-VNTR kits, 100 samples of clinical M. tuberculosis samples were confirmed as M. tuberculosis while those non-M. tuberculosis samples were not amplified, showing 100% for both sensitivity and specificity with 95% CI value of 96.38-100.00%, indicating proposed typing is precise and reliable [Table 3]. On result interpretation, both MIRU-VNTR typing kits were correctly discriminated M. tuberculosis and non-M. tuberculosis.
Table 3: Sensitivity and specificity of Kogenebiotech mycobacterial interspersed repetitive unit targeting 24 mycobacterial interspersed repetitive unit-variable number tandem repeat loci for Mycobacterium tuberculosis differentiation

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Those typed M. tuberculosis samples were classified according to their specific strains. Of 100 samples, a large proportion of samples were determined as Beijing strains, 84 out of 100 (84%), while the remaining were found to be non-Beijing strains such as NEW-1 (n = 8), Uganda (n = 6), EAI (n = 6), Turkey (n = 2), and Haarlem (n = 1) [Figure 1] and [Table 4]. Furthermore, it is noted that no discrepancies were observed on those typed lineage strains between KoMIRU and GeMIRU, showing equal discriminatory power. Interestingly, those non-Beijing strains, except EAI, are geographically distant lineage strains, Uganda, Turkey, Haarlem, and relatively unique strain, NEW-1.
Figure 1: Radial genetic tree based on Kogenebiotech Mycobacterial Interspersed Repetitive Unit data of 100 isolates of Mycobacterium tuberculosis. A radial genetic tree was generated using Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat Plus database. 100 isolates of Mycobacterium tuberculosis were identified into 6 lineages, a represents Beijing strain; b represents Turkey strain; c represents Haarlem strain; d represents Uganda strain; e represents NEW-1 strain; and f represents EAI strain

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Table 4: Result of comparative analysis of Kogenebiotech mycobacterial interspersed repetitive unit and genoscreen mycobacterial interspersed repetitive unit

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  Discussion Top

Recent investigations have revealed that particular strains of M. tuberculosis are strongly associated with hypervirulence and certain drug resistance patterns. Several studies have investigated the prevalence of each strain at certain regions yet, more studies should be done to understand TB transmission dynamics.[12],[13],[14],[15],[16] Therefore, rapid identification of strains significantly contributes to TB epidemiology and clinical treatment for countries, especially where TB is prevalent.[17],[18],[19],[20] In terms of the epidemiological aspect, genotyping on TB isolates performs a significant role in analyzing TB transmission from local contact tracing to comparisons of TB prevalence landscape at a global level. In addition, recent studies revealed that genotyping, especially MIRU-VNTR, is suitable for determining re-infections and relapses of TB.[21]

For GeMIRU, its accuracy and utility in actual real-world setting had been globally proved. Thus, it was implemented as standardized a genotyping tool in the number of countries.[22],[23] The KoMIRU, a MIRU-VNTR typing kit that utilizes the same principle of GeMIRU but with unique primer designs, also determines strains of M. tuberculosis by amplifying 24-locus. In this study, the validation and comparative analysis of KoMIRU has shown the expected comparable result as GeMIRU, 100% of concordance rate. Also, in comparison with MVNTR, the KoMIRU has shown slightly higher sensitivity.

Those clinical 100 M. tuberculosis used in this study were procured from the biobank of MNTH, a hospital dedicated to infectious disease in Korea. Based on analysis on clinical M. tuberculosis samples, the study identified 6 different strain types of M. tuberculosis. These are Beijing, NEW-1, Uganda, EAI, Turkey, and Haarlem.

Recent related studies have reported the prevalence of Beijing strain in Korea, but other strains were not reported nor reported as non-Beijing strain.[24],[25] Our study firstly presents diverse strains of M. tuberculosis that are prevalent in Korea. Understanding strains and their molecular and clinical feature is highly significant. For instance, the Beijing, Haarlem, and other minor strains are known for its association with hypervirulence and drug resistance, such as rifampicin and ofloxacin resistance for Beijing strain and MDR for Haarlem, as well as TB outbreaks.[26],[27],[28],[29],[30],[31],[32],[33],[34],[35],[36] Also, in the case of NEW-1, although only a few studies are available to make rationale, M. tuberculosis with this strain is presumed to have a higher mutation rate. Thus, a strong association with global dissemination of MDR has been suggested.[32],[37],[38],[39],[40],[41],[42],[43] For EAI strain, this was reported for a low positive rate via Tuberculin skin test, and it is less likely to cause severe TB due to reduced transmissibility.[44] In the case of the Uganda strain and Turkey strain, the Uganda strain is primarily predominant in regions of Africa, while for the Turkey strain, more active investigations are required to understand its characteristics.[45],[46]

The effectiveness of preventive TB intervention using GeMIRU has been evaluated and allows GeMIRU as the global standard. Our study found that KoMIRU has the equal discriminatory capability as GeMIRU and is higher than that of MVNTR [Supplementary Table 1], [Supplementary Table 2], [Supplementary Table 3]. The application of KoMIRU is expected to contribute to investigations on TB transmission in Korea. Also, the KoMIRU allows rapid determination of TB strains; hence, based on the association of lineage strains and drug resistance, this could help in customizing TB treatment regimens during the initial TB treatment phase.

Limitation of study

The limitation of this study lies in the use of relatively small number of clinical M. tuberculosis samples (n = 100). In addition, it is noted that those clinical M. tuberculosis samples were collected from those visited and/or hospitalized patients at Masan National Tuberculosis Hospital, South Korea, whom consented for their clinical samples to be collected at the TB and infectious disease biobank of Masan National Tuberculosis Hospital, South Korea. Thus, the MIRU-VNTR results do not display any close or approximate prevalence of TB strains at national level. However, for evaluation purpose, the study proves sufficiently that the KoMIRU has high sensitivity and specificity in statistical context.

Author contributions

All authors were involved in setting of research concept and design. J. J. and T. K. conducted literature review/search. J. J. performed experiments and acquisition of data. T. K. was in charge of analysis of data, manuscript writing and editing. Finally, all authors were contributed to the manuscript review. All authors agreed to submit this article.

Ethical clearance

Public Institutional Review Board Designated by the Ministry of Health and Welfare approved this study (P01-201908-33-001).

Financial support and sponsorship

This research has been financially supported by the Tuberculosis Clinical Research Program (4600-4631-304) of the Clinical Research Center, Masan National Tuberculosis Hospital.

Conflicts of interest

There are no conflicts of interest.

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Bazira J, Asiimwe BB, Joloba ML, Bwanga F, Matee MI. Mycobacterium tuberculosis spoligotypes and drug susceptibility pattern of isolates from tuberculosis patients in South-Western Uganda. BMC Infect Dis 2011;11:81.  Back to cited text no. 46


  [Figure 1]

  [Table 1], [Table 2], [Table 3], [Table 4]


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