PA-MCDA assay primer design
Based on the mechanism of MCDA, a set of MCDA primers used for P. aeruginosa. detection was designed that targeted the oprL gene, which encodes L-lipoprotein. The details of MCDA primers used in the report are shown in Figure 1 and Table 2. The primers were commercially synthesized and purified by Tsingke (Beijing, China).
MCDA reactions were performed in a one-step reaction in a 25 μl mixture containing 12.5 μl 2×the supplied buffer (BeiJing- Hai Tai Zheng Yuan Technology Co., Ltd.), 0.1 μl each of the displacement primers F1 and F2, 0.2 μl each of the amplification primers C1, C2, R1, R2, D1 and D2, 0.4μl each of the cross primers CP1 and CP2, 1μl (8U) of Bst 2.0 DNA polymerase, 1μl of the DNA template and 0.8 μl of the colorimetric indicator. Moreover, negative control mixtures contained 10 ng of the Staphylococcus aureus and Klebsiella pneumoniae genomic templates, and blank control mixtures contained 1μl of double distilled water (DW). To evaluate the feasibility of the MCDA primer set that was designed to detect P. aeruginosa, we initially conducted the MCDA reactions at 63ºC for 45 min and terminated the MCDA reaction by heating at 85ºC for 5 min. Then, the optimal amplification temperature of the MCDA primer set was examined at fixed temperatures from 59ºC-68ºC at steps of 1ºC intervals. In particular, MCDA products were detected using a colorimetric indicator and agarose gel electrophoresis.
Bacterial strains and genomic template preparation
A total of 124 bacterial strains and 14 fungi of positive culture were isolated in the clinical microorganism laboratory of the Third Hospital of Xiamen from 26th June to 26th July, 2017 The bacterial strains list of standard culture is detailed in Additional file 1. The positive bacterial strains including 6/124(4.84%) polymicrobial growth and 32/124(22.81%) Multidrug-Resistant (MDR) strains were isolated from 118 clinical samples in which the tracheal aspirate and BALF in 38 cases, the secretion and drainage in 33 cases, the blood in 16 cases, urine, faeces, catheter and other samples in 31 cases. We chose top 13 bacteria strains, 77 samples to design the PA-MCDA reactions (Table 3). The bacteria strains identified by conventional cultivation method，automatic bacterial identification system (VITEK 2，Bio-Merieux, France) were stored in a 15% (w/v) glycerol broth at -70°C. After refreshing the culture three times on a nutrient agar plate at 37°C, the genomic templates were then extracted from all cultured strains using DNA extraction kits Qiagen Co.,Ltd. Beijing, China), and subsequently tested with an ultraviolet spectrophotometer and stored under -20ºC before use.
Specificity of the PA –MCDA assay
To evaluate the analytical specificity of the PA-MCDA assay, MCDA reactions were conducted under conditions that were described above with the 77 P. aeruginosa and non-P. aeruginosa pure genomic templates that were derived from all pure bacterial strains.
Sensitivity of the PA-MCDA assay
The genomic templates of P. aeruginosa were serially diluted (10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg per microliter) with the intent of verifying the limit of detection (LoD), and 1μl of each serial dilution was then added to the MCDA reaction mixtures. The LoD of the MCDA assay was confirmed by the genomic DNA concentration of the template. MCDA results were detected using a colorimetric indicator, malachite green, MG (BeiJing- Hai Tai Zheng Yuan Technology Co., Ltd.) and 2.5% agarose gel electrophoresis.
Verification of the PA–MCDA assay
This study was conducted in the 30-bed ICU of The Third Hospital of Xiamen in Fujian province, which is a 1200-bed hospital in China. A total of 102 patients enrolled in this study who were suspected VAP from 1st January 2018 to 14th March 2020. Patients satisfied two or more of the following criteria: fever > 38.5°C, leukocytosis > 109/L or leukopenia < 4×108/L, purulent tracheobronchial secretions, and a new or persistent infiltrate on chest radiography. The following data were recorded: demographic characteristics, indication(s) for ICU admission, prior antimicrobial therapy within 2 months before VAP, duration of mechanical ventilation before VAP, Clinical pulmonary infection score (CPIS) including temperature, blood leukocytes, tracheal secretions, oxygenation and pulmonary radiography, usual biochemical and hematological tests.
The BALF were abstracted in one bottle, following which, one half (5ml) processed for standard culture by the clinical microorganism laboratory of the Third Hospital of Xiamen and the other 5ml stored at -70°C until the time of DNA extraction. BALF was plated on chocolate, sheep blood, and MacConkey agar plates and incubated for 48-72 h according to routine clinical protocol. The DNA extraction from BALF method was described before. In this procedure, 1μl of the extracted DNA template of the BALF specimen was added to the PA-MCDA assay and the reactions were performed at an optimal amplification temperature for 45 min. The products were detected by a color change and compared to the results of a standard clinical culture, which was blinded to the research investigators. This study was approved by the local ethics committee of the Third Hospital of Xiamen, and performed according to the ethical standards of the latest revision of the Declaration of Helsinki. Written and informed consent was obtained from family members or the appropriate responsible parties.
Continuous variables of patients’ characteristics were reported as the means±standard deviations (SD) or the medians (interquartile ranges (IQR)), and categorical variables were reported as numbers (%). The accuracy of the PA-MCDA assay was compared with the microbiological culture in a cross-sectional analysis. P value < 0.05 was considered significant. Statistical analysis was performed using SPSS version 20.0 for Windows (SPSS Inc., Chicago, IL, USA).