Aptasensors for rapid detection of Escherichia coli O157:H7 and Salmonella typhimurium
Wen-he Wu
1
Min Li
1
Yue Wang
1
Hou-xian Ouyang
1
Lin Wang
1
Ci-xiu Li
1
Yu-chen Cao
1
Qing-he Meng
0
1
Jian-xin Lu
1
0
Department of Laboratory Medicine, The University of Texas, MD Anderson Cancer Center
,
Houston, Texas 77030, USA
1
Key Laboratory of Laboratory Medicine
, Ministry of Education,
Zhejiang Provincial Key Laboratory of Medical Genetics, Wenzhou Medical College
, Wenzhou, Zhejiang 325035,
China
Herein we reported the development of aptamer-based biosensors (aptasensors) based on label-free aptamers and gold nanoparticles (AuNPs) for detection of Escherichia coli (E. coli) O157:H7 and Salmonella typhimurium. Target bacteria binding aptamers are adsorbed on the surface of unmodified AuNPs to capture target bacteria, and the detection was accomplished by target bacteria-induced aggregation of the aptasensor which is associated as red-to-purple color change upon high-salt conditions. By employing anti-E. coli O157:H7 aptamer and anti-S. typhimurium aptamer, we developed a convenient and rapid approach that could selectively detect bacteria without specialized instrumentation and pretreatment steps such as cell lysis. The aptasensor could detect as low as 105colony-forming units (CFU)/ml target bacteria within 20 min or less and its specificity was 100%. This novel method has a great potential application in rapid detection of bacteria in the near future.
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Background
Diarrheal diseases caused by a range of enteropathogenic
bacteria represent a major health threat in developing
countries. According to the data from the World Health
Organization, there are almost two million deaths per
year (1.7-2.5 million deaths) caused by diarrhea.
Emerging and known enteropathogenic bacteria are, among
others, Escherichia coli (E. coli) O157:H7 and Salmonella
typhimurium (S. typhimurium). E. coli O157:H7 has
been recognized as a major intestinal flora for a long
time since it was detected as the pathogen that causes
foodborne disease outbreaks in the USA [1,2]. In the
past decades, E. coli O157:H7 has received tremendous
attention, because it becomes an important pathogenic
cause for several severe illnesses in human beings such
as gastrointestinal disease and bloody diarrhea which is
a root cause of hemolytic uremic syndrome [3]. In
addition, the incidence of S. typhimurium outbreaks is
on the rise. In 2010, the outbreak of S. typhimurium
resulted in 85 infections according to the data from the
Centers for Disease Control and Prevention (CDC,
USA). Most recently, a multi-state outbreak in the USA
was associated with contaminated ground beef.
With the health risks of enteropathogenic bacteria,
various methods have been developed for their analysis.
Traditional culture-based methods for assay of E. coli
O157:H7 and S. typhimurium, however, are
timeconsuming and are unable to meet the needs of
realtime bacteria detection. Other detection technologies
such as immunoassays [4,5] and polymerase chain
reaction-based assay [6] require either long time spans
or specialized instrumentation. Therefore, it is critical to
develop a fast and simple method to detect
enteropathogenic bacteria for the diagnosis and treatment.
Aptamer is single-stranded nucleic acid (DNA or RNA)
ligand that usually possesses high affinity and results in a
significant conformation change upon binding with a wide
range of targets. Aptamers are generally selected from the
pools containing randomly created sequences through an
in vitro systematic evolution of ligands by exponential
enrichment (SELEX) [7]. Compared to antibody-based
biosensors, aptamer-based biosensors (aptasensor) [8,9]
possess unprecedented advantages with high productivity,
affinity, selectivity, and stability.
Recently, several aptasensor using gold nanoparticles
(AuNPs) [10,11] that act as signal transducer element of
the biosensor [12] have been developed. The application
of single-stranded DNA-modified AuNPs for the highly
selective colorimetric detection has been conducted, in
which it can result in an aggregation of AuNPs with red
to pinkish/purple color change in the presence of target
molecules in solution [13]. Wei et al. reported a simple
and sensitive aptamer-based colorimetric sensor of
thrombin using unmodified AuNPs [14]. The thrombin
aptamer (TBA) was used to form aptamer-AuNPs.
Introduction of thrombin leads to the conformation change
of aptamer and increases the repulsion between TBA
and AuNPs causing salt-induced aggregation.
Nevertheless, most of AuNPs-based aptasensors were
developed to detect proteins and small molecules
[15,16]. To the best of our knowledge, no work exists in
AuNPs-based aptasensors for bacteria detection. Herein,
we reported the development of aptamer-based biosensors
(aptasensors) based on label-free aptamers and AuNPs for
the detection of E. coli O157:H7 and S. typhimurium, with
the aim of establishing a preliminary method to evaluate
the utility of aptamer-AuNPs assay for enteropathogenic
bacteria. Two species of bacteria (E. col (...truncated)