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An Ultrasensitive PCR-Based CRISPR-Cas13a Method for the Detection of Helicobacter pylori

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机构: [1]Department of Clinical Laboratory, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China. [2]Department of Gastroenterology, Jingdong Medical Area, General Hospital of Chinese PLA, Beijing 101149, China. [3]Department of Gastroenterology, Emergency General Hospital, Beijing 100028, China. [4]Department of Gastroenterology, The Eighth Medical Center of PLA General Hospital, Beijing 100091, China. [5]Department of Clinical Laboratory, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, China.
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关键词: CRISPR-Cas13a fluorescence Helicobacter pylori in vitro transcription nucleic acid detection PCR

摘要:
The rapid and simple detection of Helicobacter pylori (H. pylori) is essential for its clinical eradication. Although various methods for detecting H. pylori have been well established, such as endoscopy in combination with histology or culture, rapid urease test (RUT) and molecular tests using clinical specimens, it is of great importance to develop an ultrasensitive and accurate nucleic acid detection platform and apply it to identify H. pylori. To meet these demands, a novel method based on PCR and CRISPR-Cas13a, called PCR-Cas13a, was developed and validated using the DNA of 84 clinical strains and 71 clinical specimens. PCR primers for the pre-amplification of conservative sequence and CRISPR RNA (crRNA) for the detection of specific sequence were designed according to the principle. The designed primers and crRNA were specific to H. pylori, and the assay showed a high degree of specificity compared with other common pathogens. Our detection system can screen H. pylori with a limit of 2.2 copies/μL within 30 mins after PCR amplification. Using a coincidence analysis with traditional methods, our method exhibited 100% accuracy for the detection of H. pylori. Furthermore, its diagnostic performance was compared, in parallel with a q-PCR. The PCR-Cas13a demonstrates 98% sensitivity and 100% specificity. Moreover, our approach had a lower limit of detection (LOD) than q-PCR. Herein, we present a diagnostic system for the highly sensitive screening of H. pylori and distinguish it from other pathogens. All the results demonstrated that this PCR-based CRISPR assay has wide application prospects for the detection of H. pylori and other slow-growth pathogens.

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出版当年[2023]版:
大类 | 3 区 医学
小类 | 3 区 医学:内科 4 区 卫生保健与服务
最新[2023]版:
大类 | 3 区 医学
小类 | 3 区 医学:内科 4 区 卫生保健与服务
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出版当年[2022]版:
Q2 HEALTH CARE SCIENCES & SERVICES Q2 MEDICINE, GENERAL & INTERNAL
最新[2023]版:
Q1 MEDICINE, GENERAL & INTERNAL Q2 HEALTH CARE SCIENCES & SERVICES

影响因子: 最新[2023版] 最新五年平均 出版当年[2022版] 出版当年五年平均 出版前一年[2021版] 出版后一年[2023版]

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第一作者机构: [1]Department of Clinical Laboratory, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China.
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