Rapid detection of Staphylococcus aureus using a novel multienzyme isothermal rapid amplification technique

Abstract

Staphylococcus aureus is a common pathogen that causes various infections. Therefore, it is crucial to develop a fast and easy detection method for diagnosing and preventing S. aureus infections. In this study, MIRA assay was developed and validated (specificity; 100%) for the detection of S. aureus with nuc as the target gene. The reaction temperature and reaction time were then optimized, and the best reaction was at 40°C, 20 min. The assay could detect S. aureus in only 25 min. Additionally, the limit of detection of MIRA was 5 × 102 CFU/ml, 10-fold lower than that of the traditional PCR. Furthermore, this assay efficiently detected 219 S. aureus of 335 strains obtained from different bacterial samples (detection accuracy; 99.40%). In conclusion, this study provides a rapid and easy-to-operate method for the detection of S. aureus, and thus can be used for the timely diagnosis and prevention of S. aureus infection.

Development of Multienzyme Isothermal Rapid Amplification (MIRA) Combined with Lateral-Flow Dipstick (LFD) Assay to Detect Species-Specific tlh and Pathogenic trh and tdh Genes of Vibrio parahaemolyticus

“…However, recent studies using the MIRA assay have reported that the positive test line could be observed after 5 min for S. eriocheiris, A. baumannii, and Streptococcus agalactiae [33,34,36]. Since the MIRA assay applies a different source of recombinase (Streptomyces azure recA, SC-recA) compared to the RPA assay that uses the RecA/Rad 51 ortholog of bacteriophage T4, T4 UvsX, this may result in different incubation times for amplification [31].…”

“…Previous studies on the detection of V. parahaemolyticus using RPA assays have reported detection limits ranging from 76 to 2 CFU of bacterial culture and 10 pg of genomic DNA [27][28][29][30]. More recently, MIRA assays for various pathogens have shown detection limits ranging from 760 to 6 CFU of bacterial culture and ranging from 97 pg to 64 fg of genomic DNA [31,33,34,[36][37][38].…”

“…Previous studies on the detection of V. parahaemolyticus using RPA assays hav ported detection limits ranging from 76 to 2 CFU of bacterial culture and 10 pg of geno DNA [27][28][29][30]. More recently, MIRA assays for various pathogens have shown detec limits ranging from 760 to 6 CFU of bacterial culture and ranging from 97 pg to 64 f genomic DNA [31,33,34,[36][37][38]. Similar detection limit results were obtained with the qPCR assay, which could detect as little as 10 fg of gDNA for the tdh and trh genes and 1 fg of the tlh gene (Figure 6A).…”

“…Recently, a new RPA method, known as multienzyme isothermal rapid amplification (MIRA), has been introduced to achieve the rapid, sensitive, and specific amplification of the target gene through the synergetic action of various functional proteins, including helicase, recombinase, single-stranded binding protein, and DNA polymerase [31]. MIRA uses a different source of recombinase (Streptomyces azure recA, SC-recA), which may enhance the stability of the reaction and resistance to interference [31]. In the present study, the MIRA-LFD assay was developed to rapidly detect not only the species-specific tlh gene but also the two pathogenic trh and tdh genes of V. parahaemolyticus for field conditions with limited resources.…”

A novel detection method based on MIRA-CRISPR/Cas13a-LFD targeting the repeated DNA sequence of Trichomonas vaginalis

“…MIRA has been widely applied in diagnostic settings due to its advantages such as simplicity, rapidity, and sensitivity. It has been used for the detection of various pathogens, including bacteria and viruses (12,13).However, MIRA also has some limitations. One major drawback is the potential for nonspecific amplification, leading to false-positive results.…”