Shanxi Eye Hospital, Taiyuan, Shanxi, China.
Transl Vis Sci Technol. 2021 Dec 1;10(14):26. doi: 10.1167/tvst.10.14.26.
Pseudomonas aeruginosa is the most common bacteria causing endophthalmitis after cataract surgery. Vitreous fluid culture and molecular studies are commonly used in clinical diagnoses, but have disadvantages, such as a long culture cycle and low detection sensitivity. Here, we report a loop-mediated isothermal amplification (LAMP) method combined with the nanoparticles-lateral flow biosensor (LFB) method for rapid and specific detection of P. aeruginosa.
A set of six primers was designed to target the OprL gene of P. aeruginosa. Genomic DNA extracted from several gram-negative and gram-positive bacteria was used to determine the sensitivity and specificity of the analysis. LAMP reactions were conducted at 65 °C for 50 minutes, and results were reported using the LFB method.
The DNA template of P. aeruginosa was specifically recognized by the P. aeruginosa-LAMP-LFB (PA-LAMP-LFB) method as no cross reactions were observed for non-P. aeruginosa templates. The analytical sensitivity of our assay was 100 fg per test for the pure cultured DNA template, and the result obtained using the LFB was consistent with that of colorimetric indicator detection. The whole test could be completed within 1h. This method was used to detect P. aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae; only P. aeruginosa was positive. The positive rates of P. aeruginosa detected by a traditional culture method, the LAMP-LFB method, and the fluorescence quantitative polymerase chain reaction method were 17.7%, 17.7%, and 13.3%, respectively.
The P. aeruginosa-LAMP-LFB method established here is a rapid, specific, and sensitive method for the detection of P. aeruginosa, which can be widely used.
铜绿假单胞菌是白内障术后眼内炎最常见的细菌。玻璃体液培养和分子研究常用于临床诊断，但存在培养周期长、检测灵敏度低等缺点。本文报告了一种环介导等温扩增（LAMP）方法与纳米颗粒侧向流生物传感器（LFB）联合快速特异性检测铜绿假单胞菌的方法。
设计了一组针对铜绿假单胞菌 OprL 基因的 6 个引物。从几种革兰氏阴性和革兰氏阳性细菌中提取基因组 DNA，以确定分析的灵敏度和特异性。LAMP 反应在 65°C 下进行 50 分钟，并用 LFB 法报告结果。
铜绿假单胞菌-LAMP-LFB（PA-LAMP-LFB）方法特异性识别铜绿假单胞菌的 DNA 模板，非铜绿假单胞菌模板无交叉反应。该方法的分析灵敏度为 100 fg/测试，使用 LFB 获得的结果与比色指示剂检测结果一致。整个试验可在 1 小时内完成。该方法用于检测铜绿假单胞菌、金黄色葡萄球菌和肺炎克雷伯菌，仅铜绿假单胞菌呈阳性。传统培养法、LAMP-LFB 法和荧光定量聚合酶链反应法检测铜绿假单胞菌的阳性率分别为 17.7%、17.7%和 13.3%。
本文建立的铜绿假单胞菌-LAMP-LFB 方法是一种快速、特异、敏感的检测铜绿假单胞菌的方法，具有广泛的应用前景。
