Abstract：
BACKGROUND: Antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), pose a significant threat to public health. Existing detection methods, like cultivation-based techniques, demand significant time and labor, while molecular diagnostic techniques, such as PCR, necessitate sophisticated instrumentation and skilled personnel. Although previous multiplex loop-mediated isothermal amplification assays based on fluorescent dyes (mfLAMP) offer simplicity and cost-effectiveness, they are prone to false-positive results. Therefore, developing a rapid and efficient multiplex assay for high-sensitivity MRSA is imperative to create a practical diagnostic tool for point-of-care testing.
RESULTS: Here, we developed a mfLAMP combined with a lateral flow assay (mfLAMP-LFA) for the visual and simultaneous detection of the mecA (PBP2a-specific marker) and nuc (S. aureus-specific marker) genes in MRSA. We optimized mfLAMP-LFA using graphene oxide (GO)-based purification and specific DNA probes and evaluated its sensitivity, specificity, and stability. Utilizing GO to mitigate false-positive results by acting as a trap for free DNA probes, the mfLAMP-LFA method successfully identified mecAf and nucf-probes, exhibiting distinct red, green, and yellow fluorescence signals. The detection sensitivity of the developed mfLAMP-LFA method (1 CFU mL-1 in phosphate-buffered saline (PBS)) was comparable to other highly sensitive MRSA detection methods (1 CFU mL-1 in PBS). Furthermore, the method demonstrated specificity for MRSA, detecting it in irrigation water samples within the desired range and achieving reliable recovery rates from spiked samples.
CONCLUSIONS: This novel strategy is the first to incorporate GO into mfLAMP-LFA, enabling specific and sensitive MRSA detection and advancing rapid bacterial detection. This assay facilitates MRSA diagnostics, contributing to improved public health and food safety by delivering rapid, cost-effective point-of-care results. It enables the simultaneous detection of multiple bacteria, even in irrigation water samples artificially inoculated with MRSA, which contain aerobic bacteria at 2.7 × 102 CFU mL-1.
RESULTS: Here, we developed a mfLAMP combined with a lateral flow assay (mfLAMP-LFA) for the visual and simultaneous detection of the mecA (PBP2a-specific marker) and nuc (S. aureus-specific marker) genes in MRSA. We optimized mfLAMP-LFA using graphene oxide (GO)-based purification and specific DNA probes and evaluated its sensitivity, specificity, and stability. Utilizing GO to mitigate false-positive results by acting as a trap for free DNA probes, the mfLAMP-LFA method successfully identified mecAf and nucf-probes, exhibiting distinct red, green, and yellow fluorescence signals. The detection sensitivity of the developed mfLAMP-LFA method (1 CFU mL-1 in phosphate-buffered saline (PBS)) was comparable to other highly sensitive MRSA detection methods (1 CFU mL-1 in PBS). Furthermore, the method demonstrated specificity for MRSA, detecting it in irrigation water samples within the desired range and achieving reliable recovery rates from spiked samples.
CONCLUSIONS: This novel strategy is the first to incorporate GO into mfLAMP-LFA, enabling specific and sensitive MRSA detection and advancing rapid bacterial detection. This assay facilitates MRSA diagnostics, contributing to improved public health and food safety by delivering rapid, cost-effective point-of-care results. It enables the simultaneous detection of multiple bacteria, even in irrigation water samples artificially inoculated with MRSA, which contain aerobic bacteria at 2.7 × 102 CFU mL-1.
摘要：
背景：抗生素抗性细菌，例如耐甲氧西林金黄色葡萄球菌（MRSA），对公众健康构成重大威胁。现有的检测方法，比如基于种植的技术，需要大量的时间和劳动力，而分子诊断技术，如PCR，需要复杂的仪器和熟练的人员。尽管以前基于荧光染料（mfLAMP）的多重环介导等温扩增试验提供了简单性和成本效益，他们容易出现假阳性结果。因此,开发用于高灵敏度MRSA的快速有效的多重检测方法对于创建用于即时检测的实用诊断工具至关重要.
结果：这里，我们开发了一种mfLAMP结合侧流测定（mfLAMP-LFA），用于视觉和同时检测mecA（PBP2a特异性标记）和nuc（S.MRSA中的金黄色葡萄球菌特异性标记）基因。我们使用基于氧化石墨烯(GO)的纯化和特异性DNA探针优化mfLAMP-LFA，并评估其灵敏度，特异性，和稳定性。利用GO作为游离DNA探针的陷阱来减轻假阳性结果，mfLAMP-LFA方法成功鉴定了mecAf和nucf探针，表现出明显的红色，绿色,和黄色荧光信号。开发的mfLAMP-LFA方法（磷酸盐缓冲盐水（PBS）中的1CFUmL-1）的检测灵敏度与其他高灵敏度的MRSA检测方法（PBS中的1CFUmL-1）相当。此外，该方法证明了对MRSA的特异性，在所需范围内的灌溉水样品中检测它，并从加标样品中获得可靠的回收率。
结论：这种新策略是第一个将GO纳入mfLAMP-LFA的策略，使特异性和灵敏的MRSA检测和推进快速细菌检测。该检测有助于MRSA诊断，通过提供快速、具有成本效益的即时护理结果。它可以同时检测多种细菌，即使在人工接种MRSA的灌溉水样中，其中含有2.7×102CFUmL-1的需氧菌。
结果：这里，我们开发了一种mfLAMP结合侧流测定（mfLAMP-LFA），用于视觉和同时检测mecA（PBP2a特异性标记）和nuc（S.MRSA中的金黄色葡萄球菌特异性标记）基因。我们使用基于氧化石墨烯(GO)的纯化和特异性DNA探针优化mfLAMP-LFA，并评估其灵敏度，特异性，和稳定性。利用GO作为游离DNA探针的陷阱来减轻假阳性结果，mfLAMP-LFA方法成功鉴定了mecAf和nucf探针，表现出明显的红色，绿色,和黄色荧光信号。开发的mfLAMP-LFA方法（磷酸盐缓冲盐水（PBS）中的1CFUmL-1）的检测灵敏度与其他高灵敏度的MRSA检测方法（PBS中的1CFUmL-1）相当。此外，该方法证明了对MRSA的特异性，在所需范围内的灌溉水样品中检测它，并从加标样品中获得可靠的回收率。
结论：这种新策略是第一个将GO纳入mfLAMP-LFA的策略，使特异性和灵敏的MRSA检测和推进快速细菌检测。该检测有助于MRSA诊断，通过提供快速、具有成本效益的即时护理结果。它可以同时检测多种细菌，即使在人工接种MRSA的灌溉水样中，其中含有2.7×102CFUmL-1的需氧菌。
