Escherichia Coli ve Salmonella Enteritidis Tayinine Yönelik Yatay Akış Analiz Sistemi Geliştirilmesi
Özet
In recent years, rapid and accurate detection of pathogenic bacteria has been a current
research area in terms of public health and food safety. One of the effective methods for
this purpose is Surface-enhanced Raman scattering (SERS), its sensitivity, signal
amplification capability, portability, and paper-based horizontal flow immunoassay
(LFIA) system; Due to their high analysis rate and low cost, they stand out as a powerful
and effective technique for the detection of pathogenic microorganisms.
In the first step of this study, E. coli and S. enteritidis strains selected and known common
pathogenicity were used to generate the SERS-based LFIA system using the raman tag
DTNB; 5,5 ́-Dithiobis(2-Nitrobenzoic acid). Quantitative analysis and rapid detection of
selected bacteria was performed after pre-enrichment with Fe3O4/Au-PEI nanoparticles.
For this purpose, 20 nm gold nanoparticle (AuNP) and 15 nm Fe3O4/Au-PEI were
synthesized. In the next stage, optimized conditions for detection of selected
microorganisms were investigated by using rennet enzyme cleaving casein.
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In the second stage of our study, Fe3O4/Au-PEI nanoparticles coated selected bacterial
antibody were interacted with E. coli concentrations of 101-107 cfu/mL with used of
enzyme based magnetic extraction LFIA system. Then, when the casein was hydrolyzed
with the help of rennet, E. coli bound to Fe3O4/Au-PEI nanoparticles was separated by
magnetic field. Free bacteria were carried out on nitrocellulose membrane with DTNB
raman label bound to AuNPs on conjugation pad (AuNP DTNB antibody complex), and
the paper-based LFIA system was then allowed to be detected bacteria concentration on
the test line of the nitrocellulose membrane. Tracking the SERS signals of DTNB
molecule, LOD: 0,52 cfu/mL and R2: 0,98 values were determined from a linear
calibration curve against the increasing concentration of bacteria. These values have been
shown to work exclusively for E. coli against different bacterial species such as B. subtilis,
M. luteus, S. enteritidis strains.
In the third stage of our study, synthetic urine, reference blood and commercial milk
samples were used as biological samples. With the developed system, the efficiency of
binding of selected E. coli was determined as 86% or more.
In the last stage of our study, the effectiveness of E. coli was investigated in synthetic
urine, reference blood and commercial milk known as biological samples. Here, this study
showed that the synthetic urine, reference blood and milk, valuable nutrients especially
indicator E. coli tested as biological samples confirmed the idea that we will detect other
pathogen bacteria.
In this study, an alternative method was considered due to the expensive antibody used
in the determination of pathogen microorganisms. In accordance with this purpose,
instead of the antibody used in LFIA system, it is aimed to use bacteriophages as an
alternative method because of their natural affinity and high specificity of host selectivity.
For this reason, the P22 phage selecting S. enteritidis as the host, and its antibody were
used to compare. As a result of bacteriophage study, S. enteritidis (101-107 cfu/mL) was
determined from SERS calibration curve as LOD: 7 cfu/mL and R2: 0,98 whereas LOD:
6 cfu/mL and R2: 0,98 were found in our antibody study. These results showed that
bacteriophage would be an alternative and inexpensive method compared to antibody in
the detection of pathogenic microorganisms. In our in vivo study, egg and chicken
samples infected with S. enteritidis were found to bind and separate 90% or more bacteria
when using our P22-bacteriophage-based LFIA system. As a result, it was found that our
LFIA system works effectively and also bacteriophages can be an alternative method.
