Loop-Mediated Isothermal Amplification Assay for the Rapid Detection ofStaphylococcus aureus

Abstract

Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is an important human pathogen that produces a variety of toxins and causes a wide range of infections, including soft-tissue infections, bacteremia, and staphylococcal food poisoning. A loop-mediated isothermal amplification (LAMP) assay targeting the arcC gene of S. aureus was developed and evaluated with 119 S. aureus and 25 non-S. aureus strains. The usefulness of the assay was compared with the PCR method that targets spa and arcC genes. The optimal temperature for the LAMP assay was 58.5°C with a detection limit of 2.5 ng/μL and 102 CFU/mL when compared to 12.5 ng/μL and 103 CFU/mL for PCR (spa and arcC). Both LAMP and PCR assays were 100% specific, 100% sensitive, 100% positive predictive value (PPV), and 100% negative predictive value (NPV). When tested on 30 spiked blood specimens (21 MRSA, eight non-S. aureus and one negative control), the performance of LAMP and PCR was comparable: 100% specific, 100% sensitive, 100% PPV, and 100% NPV. In conclusion, the LAMP assay was equally specific with a shorter detection time when compared to PCR in the identification of S. aureus. The LAMP assay is a promising alternative method for the rapid identification of S. aureus and could be used in resource-limited laboratories and fields.

Evaluation of LAMP Assay Using Phenotypic Tests and Conventional PCR for Detection of nuc and mecA genes Among Clinical Isolates of Staphylococcus spp

“…Timely detection detection of very minute quantities of target DNA. The amplification occurs under isothermal conditions between 63°C [9,14] and time loss due to thermal changes is prevented [7]. In addition the reaction is not inhibited by the inhibitors in the sample [21].…”

“…There are very few studies using LAMP assays for the detection of Staphylococcal isolates, which include femA and mecA genes from food isolates [13], femA from clinical and food samples [14], spa and arc C genes from spiked blood specimens [7], spa and mecA gene from positive blood culture bottles [15], orfX (a highly conserved open reading frame of S.aureus) from clinical isolates [16].…”

“…Molecular methods like Polymerase chain reaction (PCR) and realtime PCR have been used for rapid identification of S. aureus, particularly for MRSA [7,8]. Loop-mediated isothermal amplification (LAMP) assay is a novel method for amplifying DNA and RNA with high specificity, sensitivity, and simplicity.…”

Novel primers for increased specificity and sensitivity for the detection ofStaphylococcus aureusby real-time LAMP

“…For comparison, the detection limit of the reported method by Lim et al (2013) was determined by carrying out reactions according to the conditions specified in their publication.…”

“…LAMP has also been broadly applied in pathogen detection, and has successfully detected Escherichia coli O157:H7 (Maruyama, Kenzaka, Yamaguchi, Tani, & Nasu, 2003), Actinobacillus actinomycetemcomitans (Osawa et al, 2007), Mycobacterium tuberculosis (Iwamoto, Sonobe, & Hayashi, 2003), Streptococcus pneumonia (Seki et al, 2005), and Listeria monocytogenes (Tang et al, 2011). More recently, LAMP has been used to successfully detect S. aureus (Lim, Ju Teh, & Thong, 2013).…”