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Accurate, Sensitive, and Rapid Detection of Pseudomonas aeruginosa Based on CRISPR/Cas12b with One Fluid-Handling Step

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单位: [1]Chinese Ctr Dis Control & Prevent, Natl Inst Communicable Dis Control & Prevent, State Key Lab Infect Dis Prevent & Control, Beijing, Peoples R China [2]Wenzhou Med Univ, Sch Lab Med & Life Sci, Wenzhou, Peoples R China [3]Shanxi Med Univ, Hosp 3,Shanxi Bethune Hosp, Tongji Shanxi Hosp, Shanxi Acad Med Sci, Taiyuan, Peoples R China [4]Capital Med Univ, Beijing Friendship Hosp, Dept Clin Lab, Beijing, Peoples R China
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关键词: CRISPR Cas12b LAMP Pseudomonas aeruginosa nucleic acid detection diagnosis accurate diagnosis

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This study reports a P. aeruginosa CRISPR-top assay which can precisely identify P. aeruginosa using nucleic acids from pure cultures or clinical samples in one pot with one fluid-handling step. The P. aeruginosa CRISPR-top reaction is suitable for on-site testing, and its diagnostic performance can be compared with that of qPCR. Pseudomonas aeruginosa is a major bacterial pathogen causing nosocomial infections and accounts for morbidity and mortality among patients with cystic fibrosis. An accurate, sensitive, and rapid method to detect P. aeruginosa is critical for the early control of infection and patient management. In this study, we established a P. aeruginosa clustered regularly interspaced short palindromic repeats testing in one pot (CRISPR-top) assay which detected P. aeruginosa with one fluid-handling step in one tube. The reaction was performed isothermally within 1 h; thus, specific instruments were not required. The optimal reaction conditions of this assay were determined to be a temperature of 55 degrees C; working concentrations of 1 mu M for the forward inner primer and backward inner primer, 0.5 mu M for the loop forward primer and loop backward primer, and 0.25 mu M for the forward outer primer and backward outer primer; as well as a 2 mu M concentration single-stranded DNA reporter molecules. In terms of specificity, our assay showed 100% inclusivity and exclusivity among 48 strains, including 15 P. aeruginosa clinical isolates and 33 non-P. aeruginosa strains. The limit of detection of our method was 10 copies per reaction mixture. Forty-six human sputum specimens from patients with respiratory symptoms were tested. Using the results of quantitative real-time PCR as the gold standard, our method showed 85.3% (29/34) sensitivity, 100% (12/12) specificity, a positive predictive value of 100% (29/29), and a negative predictive value of 70.6% (12/17). In summary, the P. aeruginosa CRISPR-top assay developed in the present study is a high-efficiency alternative tool for the accurate and rapid detection of P. aeruginosa, especially in resource-limited settings.IMPORTANCE This study reports a P. aeruginosa CRISPR-top assay which can precisely identify P. aeruginosa using nucleic acids from pure cultures or clinical samples in one pot with one fluid-handling step. The P. aeruginosa CRISPR-top reaction is suitable for on-site testing, and its diagnostic performance can be compared with that of qPCR.

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出版当年[2022]版:
大类 | 1 区 生物学
小类 | 1 区 微生物学
最新[2025]版:
大类 | 2 区 生物学
小类 | 3 区 微生物学
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出版当年[2021]版:
Q1 MICROBIOLOGY
最新[2023]版:
Q2 MICROBIOLOGY

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第一作者单位: [1]Chinese Ctr Dis Control & Prevent, Natl Inst Communicable Dis Control & Prevent, State Key Lab Infect Dis Prevent & Control, Beijing, Peoples R China
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