Two target genes based multiple cross displacement amplification combined with a lateral flow biosensor for the detection of Mycobacterium tuberculosis complex

Abstract

Background: Tuberculosis (TB) is a serious chronic infectious disease caused by Mycobacterium tuberculosis complex (MTBC). Hence, the development of a novel, simple, rapid and sensitive method to detect MTBC is of great significance for the prevention and treatment of TB.

Results: In this study, multiple cross displacement amplification (MCDA) combined with a nanoparticle-based lateral flow biosensor (LFB) was developed to simultaneously detect two target genes (IS6110 and mpb64) of MTBC (MCDA-LFB). One suite of specific MCDA primers designed for the IS6110 and mpb64 genes was validated using genomic DNA extracted from the reference strain H37Rv. The MCDA amplicons were analyzed using a real-time turbidimeter, colorimetric indicator (malachite green, MG) and LFBs. The optimal amplification temperature and time were confirmed, and the MCDA-LFB method established in the current report was evaluated by detecting various pathogens (i.e., reference strains, isolates and clinical sputum samples). The results showed that the two sets of MCDA primers targeting the IS6110 and mpb64 genes could effectively detect MTBC strains. The optimal reaction conditions for the MCDA assay were determined to be 67 °C for 35 min. The MCDA assay limit of detection (LoD) was 100 fg per reaction for pure genomic DNA. The specificity of the MCDA-LFB assay was 100%, and there were no cross-reactions for non-MTBC strains. For sputum samples and MTBC strain detection, the positive rate of MCDA-LFB for the detection of MTBC strains was consistent with seminested automatic real-time PCR (Xpert MTB/RIF) and higher than acid-fast staining (AFS) and culture assays when used for sputum samples. The MCDA-LFB assay was a rapid tool, and the whole procedure for MCDA-LFB, including DNA template preparation, MCDA reaction and amplification product analysis, was completed within 70 min.

Conclusion: The MCDA-LFB assay targeting the IS6110 and mpb64 genes is a simple, rapid, sensitive and reliable detection method, and it has potential significance for the prevention and treatment of TB.

Keywords: Detection; Diagnosis; Lateral flow biosensor; Multiple cross displacement amplification; Mycobacterium tuberculosis complex.

Fig. 1

Confirmation and detection of MTBC-MCDA products. A The LFB and MG methods were applied for IS6110 gene amplification. The products of the MTBC-MCDA assay were visually analyzed by observation of TL1 and color change. Tube 1/biosensor 1: positive amplification of M. tuberculosis; Tube 2/biosensor 2: black control of DW. B The LFB and MG methods were applied for mpb64 gene amplification. The products of the MTBC-MCDA assay were visually analyzed by observation of TL2 and color change. Tube 3/biosensor 3: positive amplification of M. tuberculosis; Tube 4/biosensor 4: black control of DW. C The LFB and MG methods were applied for both IS6110 and mpb64 gene amplification. The products of the MTBC-MCDA assay were visually analyzed by observation of TL1, TL2 and color change. Tube 5/biosensor 5: positive amplification of M. tuberculosis; Tube 6/biosensor 6: black control of DW

Fig. 2

Optimization of reaction temperature for MTBC-MCDA primers. Both the IS6110 gene (A1-A8) and mpb64 gene (B1-B8) were detected at different temperatures. The genes were detected for different temperatures (63–70 °C, 1 °C intervals) with template DNA at the level of 100 pg per reaction. The standard MCDA reactions for the detection of M. tuberculosis were monitored by a real-time turbidimeter, the threshold value was 0.1, and a turbidity > 0.1 was set as positive. The M. avium genomic templates were used as a negative control

Fig. 3

The optimal duration of time required for the MTBC-MCDA-LFB method. Four distinct reaction times (20, 30, 40, and 50 min) were examined and compared at 67 °C. Biosensors represent DNA levels of 100 pg of M. tuberculosis templates

Fig. 4

Sensitivity of the MCDA method using serially diluted genomic DNA extracted from M. tuberculosis (H37Rv, ATCC27294). A total of two detection techniques, including the lateral flow biosensor (LFB) and colorimetric indicator (MG) methods, were applied to analyze the amplification products. Serial dilutions of target templates were subjected to standard MCDA reactions. DNA levels of 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg per reaction. Double distilled water was the template for the blank control. A Singly, the IS6110 gene at genomic DNA levels of 100 ng per reaction, 10 ng per reaction, 1 ng per reaction, 100 pg per reaction, 10 pg per reaction, 1 pg per reaction, 100 fg per reaction and 10 fg per reaction produced positive reactions. B The mpb64 gene at genomic DNA levels of 100 ng per reaction, 10 ng per reaction, 1 ng per reaction, 100 pg per reaction, 10 pg per reaction, 1 pg per reaction, 100 fg per reaction and 10 fg per reaction produced positive reactions. C Both the IS6110 gene and mpb64 gene at genomic DNA levels of 100 ng per reaction, 10 ng per reaction, 1 ng per reaction, 100 pg per reaction, 10 pg per reaction, 1 pg per reaction and 100 fg per reaction produced positive reactions

Fig. 5

Sequences, modifications and locations in the expression sites of the IS6110 and mpb64 genes. A Location and nucleotide sequence of the M. tuberculosis IS6110 gene used to design the MCDA primers. B Location and nucleotide sequence of the M. tuberculosis mpb64 gene used to design the MCDA primers. All sequences of the primer sites are underlined. Right and left arrows indicate sense and complementary sequences that are used