Department of Infectious Diseases, The Fifth Affiliated Hospital, Sun Yat-Sen University, Zhuhai, China.
Department of Gastroenterology, Guangdong Provincial Key Laboratory of Gastroenterology, State Key Laboratory of Organ Failure Research, Nanfang Hospital, Southern Medical University, Guangzhou, China.
PeerJ. 2024 Aug 30;12:e17776. doi: 10.7717/peerj.17776. eCollection 2024.
The gene codes for an important toxin produced by (), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the gene.
Three sets of primers were designed and optimized to amplify the gene in using a LAMP assay. To evaluate the specificity of the LAMP assay, VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The gene of was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction.
At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the primers for 26 pathogenic bacterial strains that do not carry the gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X = 47, < 0.01).
LAMP method is an effective technique for the rapid and visual detection of the gene of , and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The -LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of infection in populations at high risk.
该基因编码由 （）产生的一种重要毒素，但目前尚无简单且经济有效的方法来检测它。本文建立并验证了一种用于检测 基因的快速可视化环介导等温扩增（LAMP）检测方法。
使用 LAMP 检测法，设计并优化了三组引物以扩增 中的 基因。为了评估 LAMP 检测法的特异性，使用 VPI10463 作为阳性对照，而缺乏 基因的 26 种致病性细菌菌株和蒸馏水作为阴性对照。为了进行敏感性分析，将 LAMP 检测法与使用从 VPI10463 获得的 DNA 的十倍系列稀释液的 PCR 进行比较，范围从 207 ng/µl 至 0.000207 pg/µl。使用 LAMP 和聚合酶链反应（PCR）检测了 164 份粪便标本中的 基因。使用实时浊度监测和显色反应来区分阳性和阴性结果。
在 66°C 的温度下，用一组称为 的引物成功地扩增了目标 DNA，并且在 60 分钟内可视化。在相同条件下，对于不携带 基因的 26 种致病性细菌菌株，目标 DNA 未被扩增。LAMP 的检测限为 20.700 pg/µl，比常规 PCR 灵敏 10 倍。使用 LAMP 方法检测 164 份粪便标本中的 ，阳性率为 17%（28/164），明显高于 PCR 方法的 10%（16/164）（X=47， < 0.01）。
LAMP 方法是一种快速可视化检测 的 基因的有效技术，与 PCR 相比，在速度、简单性和敏感性方面具有潜在优势。-LAMP 检测法特别适用于资源有限的医疗诊断环境，是筛查和检测高危人群中 感染的有前途的诊断策略。
