Aptasensors for rapid detection of Escherichia coli O157:H7 and Salmonella typhimurium
- Wen-he Wu†1,
- Min Li†1,
- Yue Wang1,
- Hou-xian Ouyang1,
- Lin Wang1,
- Ci-xiu Li1,
- Yu-chen Cao1,
- Qing-he Meng1, 2Email author and
- Jian-xin Lu1Email author
© Wu et al.; licensee Springer. 2012
Received: 9 October 2012
Accepted: 12 November 2012
Published: 29 November 2012
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.
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 . 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 time-consuming and are unable to meet the needs of real-time bacteria detection. Other detection technologies such as immunoassays [4, 5] and polymerase chain reaction-based assay  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) . 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  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 . Wei et al. reported a simple and sensitive aptamer-based colorimetric sensor of thrombin using unmodified AuNPs . 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. coli O157:H7 and S. typhimurium) were examined in this work.
Reagents and chemicals
Sequences of oligonucleotides employed in this work
A1: anti-E. coli O157:H7 aptamer
A2: anti-S. typhimurium aptamer
R1: random sequence
Bacterial strains and cultivation
E. coli O157:H7 (CICC21530) was purchased from China Center of Industrial Culture Collection (CICC, Beijing, China). E. coli O111 (CMCC44151) was purchased from National Center for Medical Culture Collections (CMCC, Beijing, China). S. typhimurium (CMCC50115), Shigella flexneri (CMCC51571), Salmonella paratyphi A (CMCC50001), S. paratyphi B (CMCC50004), E. coli (ATCC25922), E. coli (CMCC44825), Pseudomonas aeruginosa (ATCC27853), Listeria monocytogenes (ATCC19115), and Staphylococcus aureus (ATCC25923) were gifts from the Central Laboratory of Biology of Wenzhou Medical College (Zhejiang, China). Stock cultures in 25% glycerol were maintained frozen at −80°C.
All bacteria were inoculated into lysogeny broth (LB) and grown for 6 h at 37°C with shaking at 165 rpm. The cultures containing bacteria were centrifuged at 3,000 rpm for 5 min and washed with phosphate-buffered solution (PBS) (10 mM, pH 7.4) three times. The pellets were then dispersed in PBS. Serial dilutions of cultures were made in PBS, 50 μl diluted suspension was inoculated onto agar plates for enumeration. The bacterial densities were determined using a scattered light turbidimeter. The actual amount of bacteria was then determined based on the bacterial density.
Ultraviolet spectroscopy was performed on aqueous solutions of DNA aptamer using Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, USA) at 260 nm. Colorimetric assays were recorded on Varioskan Flash spectral scan multimode plate reader (Thermo Fisher Scientific, USA). Scanning electron microscopy (SEM) images were taken on the ZEISS-ULTRA 55 (ZEISS, Oberkochen, Germany) with an accelerating voltage of 5.0 kV. The pH measurements were carried out on model PHS-3E digital ion analyzer (Jiangsu Instruments, China). Zeta potential measurements were performed using the NICOMP 380ZLS zeta potential/particle sizer (Agilent Technologies, CA, USA).
Preparation and characterization of aptasensors
Aptamers play an important role in colorimetric detection. Since aptamers can absorb AuNPs and stabilize AuNPs against aggregation upon addition of high level salts, it is essential to determine an optimal amount of aptamers. In our study, aptamer-AuNPs were prepared according to previous work . Briefly, aptamers (A1 or A2) of different concentrations were mixed with 50 μl AuNPs thoroughly and allowed to react at room temperature for 25 to 30 min. The above solution was incubated at room temperature for 10 min following addition of 50 μl PBS. Subsequently, 5 μl of NaCl (1.7 M) was mixed with the resulting solution. After equilibrating for 10 min, the UV–vis absorption spectrum was measured at the wavelength range from 450 to 750 nm with Varioskan Flash spectral scan multimode plate reader. All assay procedures were performed at room temperature. The quality of aptamer-AuNPs was monitored with SEM and zeta potential analyzer.
Colorimetric detection of E. coli O157:H7 and S. typhimurium
Various amounts of target bacteria of 50 μl were added to the 50 μl aptamer-AuNPs solution. After incubating for 10 min, 5 μl of NaCl (1.7 M) was introduced to the mixtures, followed by either visual observation or UV–vis characterization. A series of bacterial exposure to the aptamer-AuNPs were carried out to explore the specificity of the aptamers. The bacteria were as follows: E. coli (ATCC 25922, CMCC44825, and CMCC44151), S. paratyphi A, S. paratyphi B, S. flexneri, P. aeruginosa, L. monocytogenes, S. aureus, S. typhimurium, and E. coli O157:H7.
Results were expressed as mean ± SD. Comparisons between two groups were made by unpaired two tailed Student’s t test using SPSS 15.0 software. The P value of less than 0.05 was taken to indicate statistical significance.
Results and discussion
Optimal condition screening of aptamer for binding with AuNPs
Characterization of aptasensors
Colorimetric detection of E. coli O157:H7 with aptamer-AuNPs
By using both anti-E. coli O157:H7 aptamer and anti-S. typhimurium aptamer as models, we have developed aptasensors-based, unmodified AuNPs that can rapidly and selectively detect bacteria with the naked eye. The aptasensors take only 20 min or less to complete bacteria detection, which greatly reduces the time compared with conventional bacterial cultivation and other rapid detection methods [23, 24]. In addition, The detection limit (105 CFU/ml) was significantly lower [25, 26] than or comparable  to the presently available antibody-based biosensors with the same bacteria. In order to meet lower levels of bacteria detectable, sample preconcentration steps will be necessary prior to analysis, such as microfiltration and immunomagnetic separation. In addition, this method avoids the labeling of aptamers or modification of AuNPs, which significantly reduces the cost. This novel method is simple and can be used for a rapid diagnosis for diarrheal diseases. However, the most important strength of this aptasensor is its direct detection of the whole bacteria without specialized instrumentation and pretreatment steps such as cell lysis. Moreover, this method could be potentially applied in principle to detect other bacteria by substituting the anti-E. coli O157:H7 aptamer or anti- S. typhimurium aptamer.
We are grateful to Professor Da-xiang Cui for the helpful suggestions and English revision of the manuscript. This work was financially supported by the National Natural Science Foundation of China (81101148), Key Science and Technology Innovation Team of Zhejiang (2010R50048), Zhejiang Province Science and Technology Planning Project (2009C33036), Zhejiang Provincial Natural Science Foundation (Y2081072), Zhejiang Province Innovative Scientific Research Project for Postgraduates (YK2008078), Major Project of Wenzhou science and Technology Foundation (Y20080080), and Wenzhou Science and Technology Planning Project (Y20110021).
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