Primer design
Pneumolysin, a virulence factor, encoded by the ply gene, is a specific target for the detection of S. pneumoniae. Five pairs of MCDA primers were designed on the ply gene from the S. pneumoniae standard strain of ATCC49619 according the principle of MCDA developed by Wang et al [13, 14]. Primer Primer 5.0 and PrimerExploer V4 were used. The pneumolysin (ply)-encoding primer sets are listed in Table 3 and Figure 5. The 5'- ends of the primers C1 and D1 were labeled with fluorescein isothiocyanate (FITC) and biotin, respectively. Primers were synthesized in Aoke Dingsheng Biotechnology Co., Ltd. (Beijing, China).
Bacteria strains
The bacteria strains used in this study included 15 S. pneumoniae strains and 25 non-S. pneumoniae strains (Table 1). The QIAamp DNA minikits (Hilden, Germany) was used for the extraction of genomic DNA from bacteria strains. Protocols were carried out in accordance with the manufacturer’s instructions. The S. pneumoniae standard strain ATCC49619 was used for confirmation of the performance, determination of the optimal temperature and analysis of sensitivity. DNA of S. pneumoniae ATCC49619 was serially diluted (10ng, 10pg, 1pg, 100fg, 10fg, 1fg and 0.1fg/μL) for sensitivity analysis.
S. pneumoniae MCDA assay
The S. pneumoniae MCDA assay was conducted based on previously published methods of Wang et al. [13, 14]. The isothermal amplification kits (Haitaizhengyuan, Beijing, China) was used in this study. S. pneumoniae MCDA assay was carried out in a 25μl reaction mixture including 1 μl of DNA template, 12.5μl of 2×Reaction buffer, 0.4μM of F1 and F2,1.6μM of CP1 and CP2, 0.8μM of C1 (C1*), C2, D1 (D1*), D2, R1 and R2, and 1μl of Bst DNA polymerase (10U). DNA of Staphylococcus aureus and Salmonella typhii were used as negative controls. Distilled water was used as blank control. The reaction systems were isothermally amplified at 63°C for 40 min, followed by 85 °C for 5 min to stop the amplification.
In this study, four methods were used to determine the amplification results, including colorimetric indicator (Malachite Green, MG), turbidimeter (Loopamp Realtime Turbidimeter LA-320C), lateral flow biosensor (LFB) and 2% agarose gel electrophoresis. The above four methods were performed as previously described [14].
Optimal temperature of S. pneumoniae MCDA assay
The optimal amplification temperature of S. pneumoniae MCDA assay was determined at isothermal temperature ranging from 62 to 67°C with 1 °C intervals. DNA templates of Staphylococcus aureus and Salmonella typhi were used as negative control, and distilled water was used as blank control. The temperature that contributed to produce higher amplification products and occur turbidity earlier was considered for the optimal temperature. The experiment was repeated three times.
Sensitivity of S. pneumoniae MCDA assay
Sensitivity of MCDA assay was determined using serial dilutions of S. pneumoniae ATCC49619 genomic DNA. A total of 7 different concentrations of DNA templates (10ng, 10pg, 1pg, 100fg, 10fg, 1fg, 0.1fg/μL) were acquired. 1 μl of each dilution were added into the amplication mixture and then to be amplified at optimal temperature for 40min. Color indicator, real time turbidity, LFB detection and gel electrophoresis were conducted to analyze the MCDA products and then determine the limit of detection of S. pneumoniae MCDA assay. Distilled water was used as blank control. The sensitivity experiment was repeated in triplicate.
Eliminating carryover contamination by using AUDG enzyme
The S. pneumoniae MCDA products in the absence of AUDG enzyme were quantified using ultraviolet spectrophotometer (NanoDrop ND-1000, Calibre, China), and then to be serially diluted from 1×10−10 to 1×10−20 g/μL. These dilutions were served as the source of simulated carryover contamination and were used as templates in S. pneumoniae MCDA assay. To demonstrate that simulated carryover contamination (UTP-incorporated products) can contaminate the MCDA reaction and to confirm that AUDG enzyme can eliminate the false-positive results caused by carryover contaminants, the sensitivity of MCDA-AUDG and MCDA were compared by using the above serial diluted MCDA products (1 μl).
Specificity of S. pneumoniae MCDA assay
To determine the specificity of S. pneumoniae MCDA assay, DNA templates from 40 bacteria strains including 15 S. pneumoniae strains and 25 non-S. pneumoniae strains were applied to the MCDA assay (Table 1). The products were analyzed using color indicator and LFB detection. The specificity experiment was repeated three times.
Evaluation of clinical sensitivity of S. pneumoniae MCDA assay
The sensitivity of S. pneumoniae MCDA assay in clinical application was evaluated by artificially adding serial dilutions of colony forming units (CFUs) of S. pneumoniae ATCC49619 into the sputum samples. The single colony of ATCC49619 was enriched and cultured. Then, the number of ATCC49619 CFUs was counted, and cultured suspensions were added into the S. pneumoniae negative sputum samples. The concentration of ATCC49619 in sputum samples was adjusted to 4.47×100, 4.47×101, 4.47×102, 4.47×103, 4.47×104, 4.47×105, 4.47×106 CFU/mL. Then, 100 μL of artificially contaminated sputum samples were subjected to extract the genomic DNA, which were eluted in 10 μL of Qiagen elution buffer (Qiagen, Germany). Next, 1 μL of DNA templates were used for MCDA assay and the PCR method was also adopted. The experiment was performed three times independently.