Dielectrophoresis Concentration Method for Increased Sensitivity of the Loop-Mediated Isothermal Amplification Test for the Mycobacterium tuberculosis Complex

Research Article

J Bacteriol Mycol. 2020; 7(8): 1157.

Dielectrophoresis Concentration Method for Increased Sensitivity of the Loop-Mediated Isothermal Amplification Test for the Mycobacterium tuberculosis Complex

Takeda K1,2,3*, Chikamatsu K1, Aono A1, Igarashi Y1, Morishige Y1, Murase Y1, Takaki A1, Yamada H1 and Mitarai S1,2

1Department of Mycobacterium Reference and Research, The Research Institute of Tuberculosis, Japan

2Department of Basic Mycobacteriology, Graduate School of Biomedical Science, Nagasaki University, Japan

3Center for Pulmonary Diseases, National Hospital Organization Tokyo National Hospital, Japan

*Corresponding author: Keita Takeda, Center for Pulmonary Diseases, National Hospital Organization Tokyo National Hospital, 3-1-1 Takeoka, Kiyose, Tokyo 204-8585, Japan

Received: October 23, 2020; Accepted: November 13, 2020 Published: November 20, 2020


Background: An effective concentration method is required to detect Mycobacterium tuberculosis (MTB) in paucibacillary specimens. Dielectrophoresis (DEP), a phenomenon in which a force is exerted on a dielectric particle subjected to a non-uniform electric field, is useful for concentrating bacterial specimens.

Objectives: To investigate whether the procedure using the DEP method increases nucleic acid amplification test (NAAT) sensitivity.

Methods: First, the capture rates were examined for multiple electrode settings by calculating the bacterial load before and after DEP using quantitative real-time PCR. These results were used to determine the optimal electrode setting. Second, conventional loop-mediated isothermal amplification (LAMP) was performed using 30 μL DNA purified using a PURE DNA extraction kit from 60 μL Mycobacterium bovis bacille de Calmette et Guérin culture fluid. The minimally-diluted specimen (theoretical concentration, 103 cfu/mL), which showed 10 consecutive negative results by LAMP, was subjected to DEP. One milliliter of the specimen was concentrated to 50 μL in the DEP chip, and LAMP was performed in 30 μL of the specimens without DNA purification.

Results: At 73.2-84.9%, the capture rate was the highest in the setting with 100 kHz frequency. LAMP with DEP was performed using conventional LAMPnegative specimens, and eight of ten tests (80%) were positive. The sensitivity was higher than that of the conventional LAMP method (p = 0.0007).

Conclusion: The DEP method has the potential to increase NAAT sensitivity for the MTB complex in paucibacillary specimens.

Keywords: Mycobacterium Tuberculosis Complex; Dielectrophoresis; Concentration; Nucleic Acid Amplification Test


TB: Tuberculosis; MTB: Mycobacterium Tuberculosis; DEP: Dielectrophoresis; NAAT: Nucleic Acid Amplification Test; BCG: Bacille de Calmette et Guérin; LAMP: Loop-Mediated Isothermal Amplification


Tuberculosis (TB) remains a serious global health concern, with 1.5 million reported deaths in 2019 [1]. Accurate and rapid diagnosis is important to control TB. Bacterial culture and Nucleic Acid Amplification Tests (NAAT) are the definitive diagnostic tools for active Mycobacterium Tuberculosis (MTB) [2]. Liquid culture shows the highest sensitivity among MTB laboratory examinations; however, it takes several weeks to detect MTB. Thus, NAAT is essential for rapid diagnosis, but unfortunately, it has a lower sensitivity than that of bacterial liquid culture. In reality, approximately 20-50% of pulmonary tuberculosis cases globally are diagnosed clinically without bacteriological confirmation [1]. Low bacillary burden and insufficient MTB recovery from specimens are among the reasons why NAAT is not as sensitive as liquid culture [3,4].

To increase the diagnostic sensitivity of active MTB, many new molecular methods have been developed. For example, Xpert MTB/ RIF Ultra (Cepheid, Sunnyvale, CA, USA), which amplifies multiple target genes, has a higher sensitivity than that of Xpert MTB/RIF [5]. Other molecular methods have also been developed [6]. However, to date, none of the molecular methods has a higher sensitivity than that of liquid culture [5,7].

Another strategy for improving the sensitivity of bacterial confirmation tests is to modify specimen processing. Centrifugation is generally performed to concentrate and recover MTB. However, centrifugation is not an efficient technique to collect bacteria, particularly in low bacillary specimens [4,8], necessitating the development of other concentration methods.

The Dielectrophoresis (DEP) method is a good strategy for concentrating target cells [9,10], thereby increasing the sensitivity of the bacterial confirmation test. DEP is a phenomenon in which a force is exerted on a dielectric particle subjected to a non-uniform electric field. The DEP method can be efficacious for target cell discrimination and isolation [9-13]. For mycobacteria, M. bovis bacille de Calmette et Guérin (BCG) and M. smegmatis have been captured using DEP methods [14,15].

In this study, the optimal DEP electrode setting for M. bovis BCG was examined by calculating the capture rate using quantitative realtime PCR. It was also verified whether specimen processing using the DEP method increases the sensitivity of NAAT using Loop-Mediated Isothermal Amplification (LAMP) in a paucibacillary M. bovis BCG specimen that showed negative results using the conventional LAMP method.

Materials and Methods

Specimen Preparation

M. bovis BCG was cultured at 37°C in MycoBroth (Kyokuto Pharmaceuticals, Tokyo, Japan) until the optical density at 530 nm (OD530) of the culture medium reached 0.10. The MycoBroth culture fluid was centrifuged, and the cell pellets were resuspended twice in DEP buffer. The DEP buffer consisted of 8.5% (w/v) sucrose, 0.3% (w/v) dextrose [11], and 1.0% Tween 20, and the buffer was deionized using SMNUPB ion-exchange resin (Mitsubishi Chemical, Tokyo, Japan). The final conductivity of the DEP buffer was 10 μS/cm.

Dielectrophoresis Device Fabrication

The function generator was the prototype of the ConseeMo a instrument (AFI, Kyoto, Japan). The ConseeMo a is a compact device that can concentrate bacteria in a specimen using a microfluidic chip. The dedicated X-23 chip (AFI, Kyoto, Japan; Figure 1) has a 50 μL capacity, and was used after loading with bovine serum albumin (BSA; 2 mg/mL; Thermo Fisher Scientific, Basingstoke, UK) for 1 h to minimize cell adhesion to the DEP chip.