Development and Clinical Validation of Multiple Cross Displacement Amplification Combined With Nanoparticles-Based Biosensor for Detection of Mycobacterium tuberculosis: Preliminary Results

Abstract

Tuberculosis is still a major threat to global public health. Here, a novel diagnosis assay, termed as multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor (MCDA-LFB), was developed to simultaneously detect IS6110 and IS1081 of Mycobacterium tuberculosis (MTB) in DNA extracted from reference strain H37Rv and clinical samples. The amplification can be finished within 30 min at a fixed temperature (67°C), thus the whole procedure, including rapid template preparation (15 min), isothermal reaction (30 min) and result reporting (2 min), can be completed within 50 min. The limit of detection of multiplex MCDA assay was 10 fg per reaction. By using the multiplex MCDA protocol, cross-reaction with non-mycobacteria and non-tuberculous mycobacteria (NTM) strains was not observed. Among clinically diagnosed TB patients, the sensitivity of liquid culture, Xpert MTB/RIF and multiplex MCDA assay was 42.0% (50/119), 49.6% (59/119), and 88.2% (105/119), respectively. Among culture positive samples, the sensitivity of Xpert MTB/RIF and multiplex MCDA assay was 86.0% (43/50) and 98.0% (49/50), respectively. Among culture negative samples, the sensitivity of Xpert MTB/RIF and multiplex MCDA assay was 23.2% (16/69) and 81.2% (56/69), respectively. The specificity was 100% (60/60) for Xpert MTB/RIF and 98.3% (59/60) for multiplex MCDA. Therefore, the multiplex MCDA assay for MTB detection is rapid, sensitive and easy to use and may be a promising test for early diagnosis of TB.

Keywords: MCDA-LFB; Mycobacterium tuberculosis; lateral flow biosensor; limit of detection; multiple cross displacement amplification.

FIGURE 1

Schematic view of sequences and location of MCDA primers. The nucleotide sequences of the sense strand of IS6110 (A) and IS1081 (B) are shown. The locations of primers are marked with different colors. Right and left arrows indicate the sense and complementary sequences.

FIGURE 2

Optimization of amplification temperature for MCDA primer sets. The MCDA assays for MTB detection were monitored by real-time measurement of turbidity. The threshold value was 0.1, thus turbidity of >0.1 was regarded to be positive. Eight kinetic graphs (1–8) were obtained at different temperatures (61 –68°C at 1°C intervals), with 1 ng H37Rv template per reaction for IS6110 primer set (A) and IS1081 primer set (B), respectively.

FIGURE 3

Sensitivity of MCDA assay using serially diluted genomic template of H37Rv. (A) IS6110 MCDA assay; (B) IS1081 MCDA assay; (C) Multiplex MCDA assay for both IS6110 and IS1081. Biosensors (Aa, Ba, Ca)/tubes (Ab, Bb, Cb) 1–9 represented the DNA templates of H37Rv 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg per reaction and blank control (DW).

FIGURE 4

Optimized reaction time for multiplex MCDA assay. Four different duration times (A, 10 min; B, 20 min; C, 30 min; D, 40 min) were determined at optimal amplification temperature (67°C). Biosensors 1, 2, 3, 4, 5, and 6 represent genomic DNA template of H37Rv 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, 100 ag. Biosensors 7 and 8 represent negative control (genomic DNA from M. abscessus 1 ng) and blank control (DW).

FIGURE 5

Specificity of multiplex MCDA assay using DNA templates from different bacteria. Biosensor 1 represents reference strain H37Rv. Biosensors 2–7 represent cultured MTB strains from clinical patients. Biosensor 8 represents BCG strain. Biosensors 9–29 represent 21 different non-mycobacteria bacteria (details shown in Table 2). Biosensors 30–38 represent nine different NTM strains (details shown in Table 2). Biosensor 39 represents blank control (DW).