Hemoculture and Direct Sputum Detection of mecA‐Mediated Methicillin‐Resistant Staphylococcus aureus by Loop‐Mediated Isothermal Amplification in Combination With a Lateral‐Flow Dipstick

Abstract

This study reports loop-mediated isothermal amplification (LAMP) for rapid detection of methicillin-resistant Staphylococcus aureus from direct clinical specimens. Four primers including outer and inner primers were specifically designed on the two target sequences-femB to identify S. aureus and mecA to identify antibiotic-resistant gene. Reference strains including various species of gram-positive/gram-negative isolates were used to evaluate and optimize LAMP assays. The optimum LAMP condition was found at 63°C within 70 min assay time (include hybridization with FITC probe for 5 min and further 5 min for reading the results on the lateral flow dipstick). The detection limits of LAMP for mecA was 10 pg of total DNA or 100 CFU/ml. The LAMP assays were applied to a total of 155 samples of direct DNA extraction from sputum and hemoculture bottles. The sensitivity of LAMP for mecA detection in sputum and hemoculture bottles was 93.3% (28/30) and 100% (52/52), respectively. In conclusion, LAMP assay is an alternative technique for rapid detection of MRSA infection with a technical simplicity and cost-effective method in a routine diagnostic laboratory.

Evaluation of the loop-mediated isothermal amplification assay for Staphylococcus aureus detection: a systematic review and meta-analysis

“…After removing duplicates, 594 studies were retained, of which 526 studies were excluded because of the title and abstract. The remaining 68 studies were further read in full text, and finally, a total of 11 studies [ 3 , 4 , 6 , 12 – 19 ] were included in our analysis (Additional file 1 : Fig. S1).…”

“…1 and 2 . In the aspect of patient selection, 6 studies [ 3 , 4 , 6 , 13 , 15 , 16 ] were considered as high risk of bias which was caused by didn’t avoid setting up a case–control trial. Furthermore, 3 studies [ 14 , 17 , 19 ] were evaluated as unclear risk of bias, because didn't mention whether improper exclusions had been avoided.…”

“…Furthermore, 3 studies [ 14 , 17 , 19 ] were evaluated as unclear risk of bias, because didn't mention whether improper exclusions had been avoided. In the aspect of the index test, 4 studies [ 4 , 13 , 16 , 19 ] were considered as high risk of bias. Because the index test results were interpreted in a situation where the results of the gold standard were already known.…”

“…With strong virulence, invasiveness, and antibiotic resistance, S. aureus has become the primary pathogen of hospital and community-acquired infections [ 2 ]. In particular, MRSA has become one of the most crucial epidemiological problems in hospitals worldwide, resulting in a large number of premature deaths [ 4 ]. A recent meta-analysis, focusing on the all-cause mortality of S. aureus and MRSA, found more than one-third of patients who have S. aureus will die within 3 months, and the index of MRSA was higher [ 5 ].…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…However, the biotinylated nontargeted product would not hybridize with FITC DNA probe (only specific to LAMP amplicon), nor forming complexes with goldlabelled anti-FITC antibody to react with streptavidin on the test line, generating no signal thus a negative result on the test line. This complex would then move through the test line and was trapped at the control line (Chowdry et al 2014;Yan et al 2015;Nawattanapaiboon et al 2016).…”