Department of Central Laboratory, Lianyungang Hospital Affiliated to Jiangsu University, Lianyungang, China; Department of Central Laboratory, The Second People's Hospital of Lianyungang City (Cancer Hospital of Lianyungang), Lianyungang, China; Department of Central Laboratory, Lianyungang Hospital Affiliated to Xuzhou Medical University, Lianyungang, China.
Department of Central Laboratory, Lianyungang Hospital Affiliated to Jiangsu University, Lianyungang, China; Department of Central Laboratory, The Second People's Hospital of Lianyungang City (Cancer Hospital of Lianyungang), Lianyungang, China; Department of Central Laboratory, Lianyungang Hospital Affiliated to Xuzhou Medical University, Lianyungang, China.
Anal Chim Acta. 2023 Sep 8;1273:341534. doi: 10.1016/j.aca.2023.341534. Epub 2023 Jun 15.
Staphylococcus haemolyticus (S. haemolyticus), which is highly prevent in the hospital environment, is an etiological factor for nosocomial infections. Point-of-care rapid testing (POCT) of S. haemolyticus is not possible with the currently used detection methods. Recombinase polymerase amplification (RPA) is a novel isothermal amplification technology with high sensitivity and specificity. The combination of RPA and lateral flow strips (LFS) can achieve rapid pathogen detection, enabling POCT. This study developed an RPA-LFS methodology using a specific probe/primer pair to identify S. haemolyticus. A basic RPA reaction was performed to screen the specific primer from 6 primer pairs targeting mvaA gene. The optimal primer pair was selected based on agarose gel electrophoresis, and the probe was designed. To eliminate false-positive results caused by the byproducts, base mismatches were introduced in the primer/probe pair. The improved primer/probe pair could specifically identify the target sequence. To explore the optimal reaction conditions, the effects of reaction temperature and duration of the RPA-LFS method were systematically investigated. The improved system enabled optimal amplification at 37 °C for 8 min, and the results were visualized within 1 min. The S. haemolyticus detection sensitivity of the RPA-LFS method, whose performance was unaffected by contamination with other genomes, was 0.147 CFU/reaction. Furthermore, we analyzed 95 random clinical samples with RPA-LFS, quantitative polymerase chain reaction (qPCR), and traditional bacterial-culture assays and found that the RPA-LFS had 100% and 98.73% compliance rates with the qPCR and traditional culture method, respectively, which confirms its clinical applicability. In this study, we designed an improved RPA-LFS assay based on the specific probe/primer pair for the detection of S. haemolyticus via rapid POCT, free from the limitations of the precision instruments, helping to make diagnoses and treatment decisions as soon as possible.
溶血葡萄球菌（S. haemolyticus）在医院环境中高度预防，是医院感染的病因。目前使用的检测方法无法对溶血葡萄球菌进行即时护理点快速检测（POCT）。重组酶聚合酶扩增（RPA）是一种具有高灵敏度和特异性的新型等温扩增技术。RPA 与侧流条（LFS）的结合可以实现快速病原体检测，从而实现 POCT。本研究开发了一种使用特定探针/引物对识别溶血葡萄球菌的 RPA-LFS 方法。进行基本的 RPA 反应，从靶向 mvaA 基因的 6 对引物中筛选出特异性引物。根据琼脂糖凝胶电泳选择最佳引物对，并设计探针。为了消除引物/探针对中副产物引起的假阳性结果，在引物/探针对中引入碱基错配。改进的引物/探针对可以特异性识别目标序列。为了探索最佳反应条件，系统研究了 RPA-LFS 方法的反应温度和持续时间的影响。改进的系统在 37°C 下 8 分钟即可实现最佳扩增，1 分钟内即可观察到结果。RPA-LFS 方法的溶血葡萄球菌检测灵敏度为 0.147 CFU/反应，其性能不受其他基因组污染的影响。此外，我们用 RPA-LFS、定量聚合酶链反应（qPCR）和传统细菌培养检测方法分析了 95 份随机临床样本，发现 RPA-LFS 与 qPCR 和传统培养方法的符合率分别为 100%和 98.73%，证实了其临床适用性。在本研究中，我们设计了一种基于特异性探针/引物对的改进的 RPA-LFS 检测方法，用于通过快速 POCT 检测溶血葡萄球菌，不受精密仪器限制，有助于尽快做出诊断和治疗决策。
