(163e) Colorimetric Detection of Methicillin-Resistant Staphylococcus Aureus Using DNA Nano-Sensing Probes

Colorimetric Detection of Methicillin-Resistant
Staphylococcus aureus Using DNA Nano-sensing Probes

Y.M. Garcia^{1, 2}, Y.C. Tung¹,
H. Sakamoto ³, and H.S. Chuang^{1, 4, *}

¹Department of Biomedical Engineering, National Cheng
Kung University, Tainan, Taiwan

²School of Chemical, Biological, Materials Engineering
and Sciences, Mapua University, Manila, Philippines

³Tenure-Track
Program for Innovation Research, University of Fukui, Fukui, Japan

⁴ Center for Micro/Nano Science and
Technology, National Cheng Kung University, Tainan, Taiwan

ABSTRACT

According
to the World Health Organization (WHO), 20% of all documented S. aureus infections
are attributable to Methicillin-resistant Staphylococcus
aureus (MRSA), but figures rise up to 80% in
some developing countries.¹ MRSA is still
considered as one of the major threats amongst hospital-acquired infections globally,
causing lots of life-threatening diseases, such as complicated wound abscesses,
pneumonia, endocarditis, and sepsis². Especially for those immunocompromised
patients who are vulnerable to bacterial infections, this may lead to higher mortality
since approximately 5% of patients in the hospitals are MRSA-carrier according
to the U.S. Center for Disease Control and Prevention’s report³. As
a result, preventing the spread of the disease and initiating a timely
treatment with an appropriate antibiotic, i.e. a simple, fast and sensitive
method of detection, becomes vital for saving more lives. In this study, we present a rapid diagnostic for MRSA
infection by targeting the organism’s biomarker, mecA gene. Detection of
mecA gene in the sample is tantamount to the presence of the resistant
strain of Staphylococcus aureus (SA) and that can selectively
distinguish MRSA from methicillin-sensitive Staphylococcus
aureus (MSSA) ⁴. The nano-sensing probes comprise a mixture of two designed DNA probes,
DNA Probe I and DNA Probe II, targeting a specific sequence in two different
regions in the MRSA mecA gene. The DNA Probe I is conjugated with carrier
polystyrene (PS) particles (d_p= 50 μm) while
the DNA Probe II is conjugated with gold nanoparticles (AuNPs). Pretreated liquid
samples containing the extracted MRSA mecA gene together with the
designed DNA sensing probes are allowed to mix and hybridized in the chamber
for a few minutes. After the reaction is done, the mixture will be pumped
through the filter adjacent to it that serves as an obstacle for the aggregated
hybridized PS/Probe I-MRSA mecA gene-Probe II/AuNP complex but will
allow other small particles (AuNPs and DNA) to pass through. When the MRSA mecA
genes are present, the two DNA Probes bind to its complementary DNA in the MRSA
mecA gene, forming the PS/Probe I-MRSA mecA gene-Probe II/AuNP
complex. MRSA mecA gene acts as a linker between the two DNA sensing
probes. As a result, the PS/Probe I-MRSA mecA gene-Probe II/AuNP complex
tends to accumulate in the filter paper, resulting in visible red color due to the
aggregation of AuNPs. Conversely, in the absence of MRSA mecA gene,
hybridization and linkage of the two DNA sensing probes are not possible, thus
allowing the AuNPs to be filtered out and washed away from the filter paper.
The absence of AuNPs then results in no visible red color in the filter paper. In summary, we proposed
an integrated platform combining  DNA nano-sensing probes that can detect a
specific target and that its presence can easily be evaluated by naked eye,
prompting its simple accessibility in point-of-care applications. This platform can address current challenges in rapid MRSA diagnosis with high
sensitivity and specificity through the detection of MRSA mecA gene.
Likewise, this platform can be extended in the diagnosis of other bacterial
diseases such as Mycobacterium tuberculosis by applying the same principle of
detecting a specific target using designed DNA nano-sensors.

Keywords - Methicillin-resistant Staphylococcus aureus, mecA gene, DNA
nano-sensing probes, colorimetry.

Figure 1:
Scheme and experimental setup. (a) Schematics. The extracted mecA gene
from the samples is allowed to react and hybridized with two kinds of DNA
nano-sensing probes Probe I and Probe II. The mecA gene act as a linker
between the two probes forming the PS/Probe I-MRSA mecA gene-Probe
II/AuNP complex. (b) In the presence of the target mecA gene in the
samples, there will be the hybridization of the DNA Probes I and II with mecA
gene forming the PS/Probe I-MRSA mecA gene-Probe II/AuNP complex that
aggregates and are trapped in the filter paper after filtration. Conversely, if
the mecA gene is absent, then no complex will be formed, allowing the
small AuNP/DNA Probe to pass through the filter and red filtrate is observable
due to the color of AUNP that was filtered out.

References:

1.    
WHO. Antimicrobial resistance: global report on surveillance 2014. http://www.who.int/drugresistance/documents/surveillancereport/en/on

2.     CDC.
Healthcare-associated Infections: Staphylococcus aureus in healthcare settings http://www.cdc.gov/HAI/organisms/staph.html

3.     CDC. Methicillin-resistant Staphylococcus aureus: General
information. https://www.cdc.gov/mrsa/community/index.html

4.    
Watanabe K, Kuwata N, Sakamoto H,
Amano Y, Satomura T, Suye S (2015) A smart DNA sensing system for detecting
methicillin-resist Staphylococcus aureus using modified
nanoparticle probes. Biosens Bioelectron 15:419–423