Reagents and Apparatus
DNA isothermal amplification kits, Colorimetric indicator (Malachite Green, MG), biotin-14-dCTP and nanoparticles-based lateral flow biosensor (Disposable Lateral Flow Biosensor, LFB#2) were purchased from Bei-Jing HaiTaiZhengYuan. Co., Ltd. (Beijing, China). The DNA extraction kits (QIAamp DNA minikits; Qiagen, Hilden, Germany) were purchased from Qiagen (Beijing, China).
Design of MCDA primer
There are two specific MTC-MCDA targets gene of the IS6110 (GenBank, Sequence ID: CP053903.1) and mpb64 (GenBank, Sequence ID: CP053903.1) were chosen. The 10 specific primers were designed by primer software PRIMER PREMIER 5.0 and Primer Explorer V4 [13] for each of them. In addition, FITC (fluorescein isothiocyanate) was labeled at 5ʹ end of the IS6110-C1* primer, digoxigenin (Dig) was labeled at 5ʹ end of the mpb64-C1* and biotin labeled at 5ʹ end of the mpb64-C1 mpb64-D1* primers. The details, including primer design, sequences and modifications, locations in the expression site of the IS6110 and mpb64 genes were listed in Table 4 and Fig. 6. All of the primer sequences were synthesized and purified through Tian-Yi Biotech (Beijing, China) at HPLC purification grade.
Bacterial Strains and Genomic DNA Preparation
There were 60 strains among MTBC 38 strains, Non-tuberculous mycobacterium (NTM) 12 strains and other bacteria 10 strains, the detail in Table 1. The bacterial strains were stored in 10% (w/v) glycerol broth at −80 ℃, then were refreshed and cultured. According to the instruction book, the genomic templates were extracted from the cultured strains by the QIAamp DNA Mini Kit (Qiagen, Germantown, MD, USA). Sputum samples were treated with 4% NaOH solution and genomic templates were extracted by the Kit. Then the genomic templates were tested by ultraviolet spectrophotometer at A260/280, and stored at -20℃ before the templates were used. The above experiments are required to be carried out in the biosafety Level II laboratory.
MCDA Reactions and Detection
Three detection methods, including real-time turbidimeter (LA-320C), disposable lateral flow biosensor and colorimetric indicator (MG), were applied to detecting the MCDA amplification products. The suitability of two target genes of MCDA primers were examined through MCDA reaction of the single IS6110 gene either for mpb64 gene. Then both of the two target genes were detected at the same time. The standard strain M. tuberculosis (H37Rv, ATCC 27294) genomic templates were used for MCDA assay according to the DNA isothermal amplification kits. Briefly, 12.5 μl 2 ×Buffer, 1 μl Bst 2.0 DNA polymerase (10 U), 1 μl biotin-14-dCTP, 1 μl malachite green, 2 μl genomic templates, 5.3 μl DW and 2.2 μl amplification primers or mix primers containing 0.4 μM each of displacement primers F1 and F2, 0.4 μM each of amplification primers C1* and C2, 1.2 μM each of amplification primers R1, R2, D1 (D1*) and D2, 2.4 μM each of cross primers CP1 and CP2, the reaction mixtures of MCDA were in a final volume of 25 μl. Then they were reacted 35 min on the 67 ℃ by PCR, and 1 μl reaction products were used for LFB detection (Fig. 1). Double distilled water was used as the template for the blank control, the M. avium genomic DNA was used as the templates in the negative control.
Optimizing the reaction temperature and time of MCDA assay
Both IS6110 gene and mpb64 gene were detected at different temperatures by real-time turbidimeter (LA-320C). We examined the effect of different temperatures, from 63 ℃ to 70 ℃ with 1 ℃ intervals for MCDA amplification. The M. avium genomic templates were used as negative control. Subsequently, the two target genes were confirmed at different amplification reaction times, 20 min, 30 min, 40 min and 50 min, detected by LFB and MG.
Analytical Sensitivity and Specificity of MCDA Assay
The genomic DNA templates of MTB (H37Rv, ATCC 27294) were serially diluted (100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg) for sensitivity analysis by MCDA-LFB detection for single gene and double genes. And double distilled water was the template for the blank control. MTBC 38 strains, Non-tuberculous mycobacterium (NTM) 12 strains and other bacteria 10 strains (Table 1) were used for specificity assay. The genomic DNA from the strains was amplification by MCDA reactions, then assayed through LFB. The experiments were repeated at least two times.
Culture strains detecting by MCDA-LFB
In this study, 34 MTBC strains were isolated and cultured by Guizhou Provincial Center for Disease Control and Prevention (GZCDC). The semi-nested automatic real-time fluorescence quantitative PCR (Xpert MTB/RIF) and MCDA-LFB method were applied for the culture strains. In short, in the biological safety cabinet, a small amount of bacteria were scraped and placed in the sample reagent following instructions of GeneXpert MTB/RIF kit. Then they were mixed well, reacted and stand for 15 min. The 2 ml reacting solution was added into the Cartridge reaction box, and was tested by GeneXpert system. All the experiments needed the biosafety Level II laboratory.
Sputum samples detecting by MCDA-LFB
The research had 51 sputum samples (provided by pulmonary hospital of Guiyang), which were detected by acid-fast staining (AFS) and MCDA-LFB for MTBC. AFS and microscopy examination, briefly, a small amount of sticky phlegm were selected and spread evenly on the glassslides to form a film. Then the flame was fixed for 30 seconds, and acid-fast staining was carried out. Glass slides were dried naturally and microscopy. The above experiments are required to be carried out in the biosafety Level II laboratory.