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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 11  |  Issue : 3  |  Page : 268-272

The Utility of a real-time polymerase chain reaction kit for differentiating between Mycobacterium tuberculosis and the Beijing familythe


Clinical Research Center, Masan National Tuberculosis Hospital, Gyeongsangnam-do, South Korea

Date of Submission03-Jun-2022
Date of Decision21-Jul-2022
Date of Acceptance08-Aug-2022
Date of Web Publication12-Sep-2022

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


DOI: 10.4103/ijmy.ijmy_99_22

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  Abstract 


Background: Tuberculosis (TB) is a severe public health challenge in Korea. Of all Mycobacterium tuberculosis (M. tb) strains, the Beijing genotype strain reportedly correlates with hypervirulence and drug resistance. Hence, an early identification of the Beijing genotype strain of M. tb plays a significant role in initial TB treatment. Kogenebiotech® (KoRT-polymerase chain reaction [PCR]) has developed a real-time PCR 17 18 kit to determine the Beijing genotype strain classified as M. tb. To determine the feasibility of the commercially produced KoRT-PCR kit in identifying the M. tb strain. Methods: We used 100 clinical isolates of M. tb and 100 non-M. tb samples for the assessment. We evaluated the overall concordance between the KoRT-PCR kit and the mycobacterial interspersed repetitive unite variable number tandem repeat typing kit (GenoScreen, Lille, France). Moreover, we measured the detection limits based on the chromosomal DNA copies for the KoRT-PCR kit. In addition, we determined the reproducibility among individual technicians using the KoRT-PCR. Results: The KoRT-PCR kit successfully discriminated all M. tb (confidence interval [CI]: 96.38%–100.00% for both sensitivity and specificity) and Beijing genotype strain (CI: 95.70%–100.00% for sensitivity and 96.87%–100.00% for specificity). We confirmed no significant deviation in the reproducibility between the technicians. Conclusions: The KoRT-PCR kit displayed sufficient capability of discriminating the Beijing genotype strain, which enabled the rapid identification of the Beijing genotype strain from the M. tb clinical isolates.

Keywords: Beijing strain, Mycobacterium tuberculosis diagnostics, substrain identification, tuberculosis


How to cite this article:
Kang T, Lee DG, Jung J, Ryoo S. The Utility of a real-time polymerase chain reaction kit for differentiating between Mycobacterium tuberculosis and the Beijing familythe . Int J Mycobacteriol 2022;11:268-72

How to cite this URL:
Kang T, Lee DG, Jung J, Ryoo S. The Utility of a real-time polymerase chain reaction kit for differentiating between Mycobacterium tuberculosis and the Beijing familythe . Int J Mycobacteriol [serial online] 2022 [cited 2022 Nov 26];11:268-72. Available from: https://www.ijmyco.org/text.asp?2022/11/3/268/355935




  Introduction Top


Tuberculosis (TB) is a significant global health issue owing to rapid airborne transmission and high mortality.[1],[2] Mycobacterium tuberculosis (M. tb) with the Beijing genotype strain has recently emerged globally and caused outbreaks.[3],[4] The Beijing strain of M. tb is significant and could hasten the disease progression.[5] Moreover, this genotype is prone to severe clinical manifestation owing to hypervirulence and multidrug resistance.[6],[7],[8],[9],[10],[11] Researchers have frequently identified the Beijing strain among extensively drug-resistant TB strains.[12],[13],[14] TB, which predominantly occurs in East Asia, including Korea, occupies the genotype of the Beijing (East Asian) family.

Approximately 88.1% of the genotypes isolated from Korean patients with multidrug-resistant TB belong to the Beijing family.[15] The proportion of foreign patients with TB in their domestic counterparts has increased in Korea, and the majority of these foreign patients are from high TB burden countries.[16] This necessitates distinguishing the genotype of M. tb introduced from abroad and understanding the substantial role of the transmission route and the prevalence of the Beijing strain on National TB Control Programs. Researchers have developed several methods for distinguishing these strains. The most preferred strategies to recognize the Beijing genotype strains are IS6110 DNA fingerprinting and spoligotyping.[17],[18],[19] However, these methods are labor intensive; therefore, investigations on the Beijing strains of M. tb display slow progress. Hillemann et al. reported on a rapid real-time polymerase chain reaction (RT-PCR)-based method using the TaqMan probe.[20] They successfully applied PCR targeting IS6110 to identify the Beijing and non-Beijing strains.

Furthermore, Nagai et al. developed a multiplex loop-mediated isothermal amplification assay using a Beijing-specific single-nucleotide polymorphism on Rv0679c for detecting the Beijing lineage strains of M. tb.[21] However, the majority of reports are limited to experimental results for research purposes in the laboratory. There is a continuous need for M. tb and Beijing family diagnostic kits produced as standardized procedures and certificates by the licensing agency. In this study, we aimed to evaluate the KoRT-PCR kit composed of a primer and probe set capable of rapidly and accurately detecting M. tb and that of the Beijing strain concurrently. Currently, the accurate and simple RT-PCR-based diagnostic kit enables the rapid and appropriate detection of dnaA-IS6110, a specific target gene for M. tb of the Beijing strain, with a detection limit of 50 copies/uL of chromosomal DNA. The continuous monitoring of the pathogenesis and transmission of M. tb can contribute to managing patients with TB from neighboring countries and establishing an effective initial TB treatment program.


  Methods Top


DNA sample procurement

In this study, we procured 200 chromosomal DNAs from bacteria samples. Of these 200 DNA samples, 100 M. tb isolates were obtained from the biobank of the Masan National TB Hospital (MNTH), Korea, whereas the remaining 100 non-M. tb isolates were procured from biobanks and repositories, including the American Type Culture Collection (Virginia, USA), the Korean Collection for Type Culture (Jeongeup, Korea), Korea Veterinary Culture Collection (Gimcheon, Korea), the National Culture Collection for Pathogens (Cheongju, Korea), the Korea Bank for Pathogenic Viruses (Seoul, Korea), Vircell (Granada, Spain), and Promega (Wisconsin, USA) [Supplemental Table S1].[22] This study used the bacterial DNA similar to that in a previous publication.[23]



We used M. tb H37Rv (ATCC 25618D-2) for an internal control.

The identification of M. tb and Beijing genotype strain

The 100 M. tb DNA isolates were extracted as described previously.[24] These clinically isolated M. tb samples were identified using the mycobacterial interspersed repetitive unite variable number tandem repeat (MIRU-VNTR) typing kit (GenoScreen, Lille, France) according to the manufacturer's protocol.

Briefly, the 24 loci of M. tb DNA were amplified via multiplex PCR and discriminated through capillary electrophoresis using 3500 Series Genetic Analyzers (Applied Biosystems, Massachusetts, USA).[25] We analyzed and interpreted the obtained data using the MIRU-VNTRplus database (URL: https://www.miru-vntrplus.org/MIRU/index.faces) to determine the M. tb strains.[26],[27],[28]

Designing and composing the KoRT-polymerase chain reaction kit

We sorted the sequences after searching for the M. tb and Beijing family gene sequences at the National Center for Biological Information (https://www.ncbi.nlm.nih.gov/). We obtained a database of 130 sequences for M. tb, including 30 Beijing families.

The obtained nucleotide sequences were analyzed by a multialignment method, in which several nucleotide sequences were bundled and aligned. Following the multialignment, we secured a conserved region comprising a nucleotide sequence without any mutations. We prepared the primer and probe sets by designing the nucleotide sequences within the conserved region [Supplemental Table S2].



This kit comprises a ×2 RT-PCR master mix, primer/probe mix, and positive control. A total of 15 μL PCR master mixes, 10 μL ×2 RT-PCR master mixes, 5 μL primer/probe mix, and 5 μL DNA templates were added to the prepared PCR master mix. The probe/primer mix comprises two fluorescence channels, namely the fluorescein amidites (FAM) fluorescence and victoria (VIC) fluorescence for detecting the M. tb and Beijing strain, respectively.

Limit of detection optimization in DNA concentration

We measured the limit of detection (LOD) of the KoRT-PCR kit using five different DNA concentrations of M. tb H37Rv (ATCC 25618D-2). We prepared quantified M. tb-specific IS6110 and Beijing family M. tb-specific dnaA-IS6110 DNA of each target gene by serial dilution with nuclease-free water (Applied Biosystems™, USA) to generate 200 copies/μL, 100 copies/μL, 50 copies/μL, 25 copies/μL, and 12.5 copies/μL. We performed multiple RT-PCR (ABI7500; Applied Biosystems 7500 RT-PCR Instrument System, Applied Biosystems, USA), and the results were analyzed using a detection software (ABI Version 2.3, USA) with the following conditions: one cycle of 2 min at 50°C for the initial preheating and 10 min at 95°C for denaturation, followed by 35 cycles of 15 s at 95°C for annealing and 1 min at 60°C for the final extension. We performed the experiment in triplicate.

Sensitivity and specificity analysis

We determined the analytical specificity of the KoRT-PCR kit through real-time multiplex PCR using a primer and probe set for the simultaneous detection of the M. tb and Beijing family M. tb with two channels, namely dnaA-IS6110 and IS6110 for the Beijing M. tb and M. tb target gene, respectively. Multiple RT-PCR were performed under similar LOD optimization method conditions.

Evaluating interpersonal reproducibility

Duplicate aliquots of each DNA were created for each technician with three DNA concentrations of 50/μL, 100/μL, and 200/μL copies using the identical protocol to assess the reproducibility.

We calculated the cycle threshold (Ct), standard deviation (SD), and the coefficient of variance (CV) based on the RT-PCR results. A CV value <5% was considered reproducible.


  Results Top


The identification of M. tb strain

We assessed the 100 clinical M. tb samples using a MIRU-VNTR typing kit to determine their specific strain. Of these samples, 84 samples were identified as Beijing strains. The remaining samples were identified as non-Beijing strains, including eight, six, two, and one isolate of NEW-1, Uganda and EAI, Turkey, and Haarlem, respectively. Intriguingly, the non-Beijing strains were initially isolated from geographically distant regions, except the EAI strain.

Limit of detection optimization in DNA concentration

We determined the DNA concentration to optimize the concentration for KoRT-PCR. This experiment selected five different DNA concentrations, namely 12.5 copies, 25 copies, 50 copies, 100 copies, and 200 copies per μL. Of five different DNA concentrations, those equal to or above 50 copies per μL displayed a 100% amplification rate, whereas concentrations of 12.5 copies and 25 copies per μL displayed an amplification rate of 66.7% and 0%, respectively. Thus, we selected 50 copies per μL DNA concentration for the subsequent experiment and analysis as this concentration was optimized.

Sensitivity and specificity analysis

Upon conducting KoRT-PCR on 200 samples, 100 clinical M. tb samples were amplified for VIC fluorescence. Of these samples, only 84 Beijing strains were amplified for FAM fluorescence. The non-M. tb samples were amplified neither for VIC nor FAM fluorescence. We calculated the sensitivity and specificity [Table 1]. For M. tb discrimination, the KoRT-PCR kit displayed 100% sensitivity and specificity (95% confidence interval [CI]: 96.38%–100%). Simultaneously, both sensitivity and specificity for M. tb with Beijing strain discrimination was 100% (CI: 95.70%–100% and 96.87%–100%). In other words, the KoRT-PCR kit displayed good discriminatory power for the M. tb and Beijing strain.
Table 1: Sensitivity and specificity of the KoRT-polymerase chain reaction kit for differentiating between Mycobacterium tuberculosis and Mycobacterium tuberculosis with Beijing strain

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Evaluating interpersonal reproducibility

We performed the interpersonal reproducibility test to evaluate and determine any deviation in reproducibility between the performing technicians. In this experiment, we selected two technicians with comparable research capabilities. They performed KoRT-PCR using three different DNA concentrations, namely 1X LOD, 2X LOD, and 4X LOD, once per day for 10 days. We conducted an analysis based on the Ct value, SD, and CV.

In general, a CV value <5% was considered nonsignificant. We obtained CV values of 0.73%, 0.64%, and 0.73% for 1X LOD, 2X LOD, and 4X LOD, respectively [Table 2]. Thus, the KoRT-PCR kit was less likely to be influenced by the DNA concentration or human-derived factors.
Table 2: Results of evaluating interpersonal reproducibility using the KoRT-polymerase chain reaction kit

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  Discussion Top


Each strain of M. tb has a unique feature, and specific strains are strongly associated with particular patterns of drug resistance and hypervirulence.[8],[29],[30] Despite the significant role of strain identification, it is not easily conducted in epidemiological and medical fields. This is because M. tb strain identification requires a complicated protocol combined with in silico analysis and takes prolonged time for data interpretation. Previously, IS6110-restriction fragment length polymorphism (IS6110-RFLP) was the available option for strain identification; however, it was unsuitable for Beijing strain-specific typing. Moreover, the IS6110-RFLP requires an average of 44 days until the result is released for interpretation.[31] Considering our failure to perform rapid strain identification, the consideration of the strain feature of M. tb during initial treatment remains challenging.[32] The KoRT-PCR kit was developed following the high demand for Beijing strain-specific diagnostic tools.

The Beijing strain of M. tb is a specific type of genotypic lineage strain prevalent worldwide, particularly in the Eastern Asian region.[33],[34] In Korea, approximately 82.4% of the total patients with TB among foreigners had reported the Beijing strain.[35] The significance this strain is not only its high prevalence but also its clinical manifestation. The Beijing strain exhibits a more significant correlation with antitubercular drugs, such as rifampicin, ofloxacin, and multidrug resistance.[36],[37] In addition, this strain and specific drug resistance can act as a critical risk factor for treatment failure because of increased spreading capability via an intervening febrile and other immunological responses.[38],[39],[40],[41],[42]

For this study, we procured 100 clinical M. tb isolates from MNTH biobank. MNTH is dedicated to infectious diseases, particularly TB. It is an appropriate institution for using and evaluating the Beijing strain identification kit.[22]

We recommend developing a convenient and accurate multiplex PCR system to discriminate different M. tb genotypes. In addition, well-characterized clinical information and background could help us anticipate the goal, thus enabling the optimization of personalized medical treatment.


  Conclusion Top


In summary, M. tb and Beijing strain-specific RT-PCR were handled and evaluated for this study. The KoRT-PCR kit displayed sufficient discriminative capability for the M. tb with Beijing strain. Using the KoRT-PCR kit in a practical setting for clinical and epidemiological purposes will likely enable the rapid identification of the Beijing strain and contribute to TB control and treatment.[41],[43],[44],[45],[46] Moreover, it could contribute to understanding the dynamics of the TB transmission landscape in Korea, which will eventually provide greater insights into the mechanism of M. tb dissemination.

Ethical statement

Public Institutional Review Board designated by the Ministry of Health and Welfare approved this study (Approval No. P01-201908-33-001).

Financial support and sponsorship

This work was supported by the Tuberculosis Clinical Research Program (Grant number: 4600-4631-304) of the Clinical Research Center, Masan National Tuberculosis Hospital, and was funded by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (grant No. 2018R1A5A2021242).

Conflicts of interest

There are no conflicts of interest.



 
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