Multiplex fluorescence loop-mediated isothermal amplification with lateral flow assay for rapid simultaneous detection of mecA and nuc genes in methicillin-resistant Staphylococcus aureus.
Anal Chim Acta
; 1319: 342984, 2024 Aug 29.
Article
em En
| MEDLINE
| ID: mdl-39122282
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.SIGNIFICANCE:
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.Palavras-chave
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Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
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Técnicas de Amplificação de Ácido Nucleico
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Proteínas de Ligação às Penicilinas
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Staphylococcus aureus Resistente à Meticilina
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Nuclease do Micrococo
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article