Rapid Detection of Staphylococcus Aureus by Loop-Mediated Isothermal Amplification

Abstract

Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is a major bacterial pathogen associated with nosocomial and community-acquired S. aureus infections all over the world. A rapid detection assay for staphylococcal gene of nuc and mecA is needed. In this study, a rapid identification assay based on the loop-mediated isothermal amplification (LAMP) method was established. PCR and LAMP assays were used to detect Staphylococcus aureus and other related species for nuc and mecA. With optimization of the primers and reaction temperature, the LAMP successfully amplified the genes under isothermal conditions at 62 °C within 60 min, of which the results were identical with those of the conventional PCR methods. The detection limits of the LAMP for nuc and mecA were 1.47 and 14.7 pg/μl DNA per tube, respectively, by naked eye inspections, while the detection limits of the PCR for nuc and mecA were 14.7 pg/μl and 147 pg/μl DNA, respectively. Finally, The LAMP method was then applied to clinical blood plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples with the culture assay. Together, the LAMP offers an alternative detection assay for nuc and mecA with a great advantage of the sensitivity and rapidity.

Loop-mediated isothermal amplification assay forEnterococcus sp., E. coliandS. aureusin chicken

“…The detection limits of E. coli -LAMP and S. aureus -LAMP were 2 pg/μl and 400 fg/μl, respectively; however, the detection limits of the PCR reactions were 50 pg/μl for E. coli and 10 pg/μl for S. aureus . Furthermore, the sensitivities of the E. faecalis -LAMP and S. aureus -LAMP assays were superior to those reported previously (9, 14–15, 27, 32). Collectively, these observations indicated that the sensitivity of the ten LAMP assays was much higher than that of conventional PCR and of previously devised LAMP assays.…”

GLAPD: Whole Genome Based LAMP Primer Design for a Set of Target Genomes

“…Staphylococcus aureus is one main type of foodborne pathogens around the world (Kadariya et al, 2014;Paudyal et al, 2017). Traditionally, its nuc, mecA genes were used to design the LAMP primer sets (Wang et al, 2015;Chen et al, 2017). But the two genes are not conserved among all S. aureus (Hoegh et al, 2014;Karmakar et al, 2016) and may exist in other Staphylococcus spp.…”

“…In addition, the primers based on traditional methods often can't meet the requirements in practice. For example, the LAMP primer set from Wang et al (2015) targeting Staphylococcus aureus was not able to amplify some S. aureus strains and might amplify some unexpected genomes (more details in results part). With more whole genomes available (O'Leary et al, 2016), it's a better method to design group-specific primers based on whole genomes (Treven, 2015;Demkin et al, 2017).…”

Research advance in rapid detection of foodborne Staphylococcus aureus

“…LAMP reactions usually result in about 10 3 -fold or higher levels of amplification product with stem-loop DNAs in 60 min than with conventional PCR. [36] The magnesioum ions react with pyrophosphate ions from the dNTP release and a precipitate is formed. Therefore, it is easily to identify whether the amplification occurred or not without the need for cumbersome electrophoresis and ultraviolet observation process.…”