Development and clinical evaluation of a real-time multiple cross displacement amplification assay for rapid and sensitive detection of Mycobacterium tuberculosis

Abstract

Molecular techniques of nucleic acid testing recommended by the World Health Organization (WHO) for the Mycobacterium tuberculosis (MTB) detection were considered to have the potential access to the accurate tuberculosis (TB) notifications. In this study, a new method, which coupled real-time (rt) fluorescence technique with multiple cross displacement amplification (MCDA), was developed for the rapid, sensitive and specific detection of MTB (termed MTB-rt-MCDA). According to the principle of the rt-MCDA test, a set of ten primers were designed for the MCDA reaction, of which one was engineered with a restrictive endonuclease recognition site, a fluorophore and a quencher for achieving the real-time fluorescence detection. MTB-rt-MCDA test was conducted under the optimized conditions (67 °C, 40 min) on the real-time fluorescence platform. The MTB-rt-MCDA assay accurately identified the MTB strains with no cross reaction with other bacteria. The lowest detectable genomic DNA concentration of the MTB-rt-MCDA assay was 25 fg/μl. We employed the genomic DNA templates extracted from sputum of clinical cases for validating the practical applicability of this assay, and the detection power of the MTB-rt-MCDA assay was comparable to that of the Xpert method and MCDA-based biosensor detection and superior to smear microscope method. The complete process of the MTB-rt-MCDA assay, including rapid extraction of DNA and rt-MCDA test, takes less than 1 h. In conclusion, the presented MTB-rt-MCDA assay provided an effective and simple option for the rapid screening of MTB infection.

Keywords: Lateral flow biosensor; Multiple cross displacement amplification; Mycobacterium tuberculosis; Rapid diagnosis; Real-time.

Fig. 1

Schematic illustration of the principle of the MTB-rt-MCDA assay. A, Location and sequences of the primers used in this study targeting the sdaA gene of MTB. Right and left arrows show sense and complementary sequences, respectively. The colored text indicates the position of primers, including two displacement primers (F1 and F2), two cross primers (CP1 and CP2), and six amplification primers (C1, C2, D1, D2, R1 and R2). B, Schematic diagram of the MCDA reactions with the modified primers. Primer CP1 extended with an endonuclease recognition site at the 5′ end was modified with a fluorophore (FAM, F) at the 5′ end and a quencher (BHQ1, Q) in the middle. After amplification, double stranded target amplicons with the restriction endonuclease recognition site were generated. After recognized and cleaved by restriction endonuclease, the fluorescence signal was released. C, The entire process of the MTB-rt-MCDA detection system. The whole process, including rapid DNA extraction, MCDA reaction and endonuclease cleavage, and real-time fluorescence detection, could be completed within 1 h.

Fig. 2

Confirmation of the MTB-rt-MCDA assay. For primer verification, MTB-MCDA assay was carried out at 65 °C for 40 min and the results were reported using real-time turbidimeter (A), VDR (B), and LFB (C). The MTB-rt-MCDA assay was performed at 65 °C for 40 min using real-time fluorescence detector monitoring the results (D). DNA templates extracted from MTB strains and Mycoplasma pneumoniae were used as positive and negative controls, respectively, and the DW as blank control. DW, distilled water; TL, testing line; CL, control line.

Fig. 3

Temperature optimization for MTB-rt-MCDA assay. The MTB-MCDA reactions were conducted at temperatures ranging from 64 to 69 °C and the kinetic curves at different temperatures (A∼F) were acquired from real-time tubidimeter.

Fig. 4

Sensitivity confirmation of the MTB-rt-MCDA assay. Sensitivity of the MTB-rt-MCDA assay was analyzed using serial dilutions of DNA templates extracted from pure MTB strains. The MTB-rt-MCDA reactions were repeatedly tested three times (A Ⅰ∼Ⅲ). In addition, the MTB-MCDA-LFB assay was performed in parallel for comparison and confirmation (B). Signals/strips1: 1 ng/ml, 2: 100 pg/ml, 3: 10 pg/ml, 4: 1 pg/ml, 5: 100 fg/ml, 6: 25 fg/ml, 7: 10 fg/ml, 8: blank control. TL, testing line; CL, control line.

Fig. 5

Specificity conformation for MTB-rt-MCDA assay. DNA templates from 4 MTB strains and 24 non-MTB strains were tested by the MTB-rt-MCDA assay (A) to confirm its specificity. In addition, the MTB-MCDA-LFB assay was performed in parallel for comparison and confirmation (B). Signals/tubes/strips 1 to 4 represented four MTB strains, and the other ones represented non- MTB strains. TL, testing line; CL, control line.

Fig. 6

Application of MTB -rt-MCDA assay in clinical specimens. DNA templates from 32 sputum samples were detected by the MTB-rt-MCDA assay (A) and the MCDA-LFB test (B). TL, testing line; CL, control line. Samples 1–24 displayed positive results, while the others were tested negative.

Fig. 7

Comparison of the performance of the MTB-rt-MCDA assay, MTB- MCDA-LFB assay, the Xpert MTB/RIF assay and sputum smear microscopy in detection of the 32 clinical specimens. 1–32 represented 32 sputum samples. +, positive; -, negative.