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Córdova-Espinoza, M.G.; González-Vázquez, R.; Barron-Fattel, R.R.; Gónzalez-Vázquez, R.; Vargas-Hernández, M.A.; Albores-Méndez, E.M.; Esquivel-Campos, A.L.; Mendoza-Pérez, F.; Mayorga-Reyes, L.; Gutiérrez-Nava, M.A.; et al. DNA Aptamers to Detect Vibrio spp.. Encyclopedia. Available online: https://encyclopedia.pub/entry/55674 (accessed on 23 April 2024).
Córdova-Espinoza MG, González-Vázquez R, Barron-Fattel RR, Gónzalez-Vázquez R, Vargas-Hernández MA, Albores-Méndez EM, et al. DNA Aptamers to Detect Vibrio spp.. Encyclopedia. Available at: https://encyclopedia.pub/entry/55674. Accessed April 23, 2024.
Córdova-Espinoza, María Guadalupe, Rosa González-Vázquez, Rolando Rafik Barron-Fattel, Raquel Gónzalez-Vázquez, Marco Antonio Vargas-Hernández, Exsal Manuel Albores-Méndez, Ana Laura Esquivel-Campos, Felipe Mendoza-Pérez, Lino Mayorga-Reyes, María Angélica Gutiérrez-Nava, et al. "DNA Aptamers to Detect Vibrio spp." Encyclopedia, https://encyclopedia.pub/entry/55674 (accessed April 23, 2024).
Córdova-Espinoza, M.G., González-Vázquez, R., Barron-Fattel, R.R., Gónzalez-Vázquez, R., Vargas-Hernández, M.A., Albores-Méndez, E.M., Esquivel-Campos, A.L., Mendoza-Pérez, F., Mayorga-Reyes, L., Gutiérrez-Nava, M.A., Medina-Quero, K., & Escamilla-Gutiérrez, A. (2024, February 28). DNA Aptamers to Detect Vibrio spp.. In Encyclopedia. https://encyclopedia.pub/entry/55674
Córdova-Espinoza, María Guadalupe, et al. "DNA Aptamers to Detect Vibrio spp.." Encyclopedia. Web. 28 February, 2024.
DNA Aptamers to Detect Vibrio spp.
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Early and accurate diagnoses of pathogenic microorganisms is essential to correctly identify diseases, treating infections, and tracking disease outbreaks associated with microbial infections, to develop precautionary measures that allow a fast and effective response in epidemics and pandemics, thus improving public health. Aptamers are a class of synthetic nucleic acid molecules with the potential to be used for medical purposes, since they can be directed towards any target molecule. Currently, the use of aptamers has increased because they are a useful tool in the detection of specific targets. 

pathogens aptamers V. parahaemolyticus V. alginolyticus V. vulnificus detection

1. Introduction

Ninety-five years since penicillin was discovered [1], and despite the technological advances of the era, continued efforts are still being made to improve health systems worldwide due to emerging pathogen epidemics and the burden of hospital-care-associated infections (HCAIs), which today are a major public health concern globally [2][3]. In addition, the emergence of virulent and high-risk bacterial strains, such as “ESKAPE” pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species), represent a global threat to human health [4]. Therefore, the rapid detection, quantification, and adequate treatment of infectious microorganisms are challenges to protecting public health [5]. Traditional methods for the detection of pathogenic bacteria are culture-based methods and biochemical tests, which are low cost, easy to operate, and highly standardized, but they lack differentiation between the target and other non-target endogenous microorganisms, they produce false negative/positive results, they are time- and labor-consuming procedures, and they are unable to detect viable but nonculturable cells [6]. Antibodies have made tremendous contributions in a wide range of applications. However, there are certain limitations associated with their use; monoclonal antibodies generally are incapable of membrane penetration due to their larger size and hence are less ideal as carriers for the targeted delivery of cytotoxic molecules inside cells. The production of monoclonal antibodies is laborious, expensive, time consuming, and suffers from batch-to-batch variations; they are also immunogenic, temperature sensitive, and their target binding kinetics cannot be easily modified [7]. In addition, detection with antibodies is not accurate when there are minimal amounts of microorganisms. In some cases, the detection is not precise due to the null specificity of some antibodies or due to false positives [8]. Thus, aptamers are considered to be an alternative to antibodies in many biological applications [9]. Molecular approaches such as quantitative PCR are often used for the rapid and accurate enumeration of pathogen-derived nucleic acids; however, some nucleases may inhibit the enzymes of PCR resulting in false-negative results [10]. Aptamers are proposed as substitutes for traditional detection methods; they include having an oligonucleotide single-stranded DNA (ssDNA) or RNA with target-selective high-affinity features, considered as nucleic acid-based affinity ligands [11][12], and which do not differ in specificity or affinity. RNA aptamers have greater flexibility and produce a greater variety of possible structural configurations [13]. Aptamers also have high stability, rapid production (synthesis or modification), low immunogenicity, are economic—they can be used for a wide range of targets—there are no animals needed for their production, they can be created against targets such as toxic or non-immunogenic molecules, and no cold chain for transportation and maintenance is needed [14]. DNA or RNA aptamers in comparison to antibodies can undergo reversible folding and unfolding, leading to a greater stability and a simpler elution of the bound target from the aptamer [15]; additionally, aptamers bind to their targets with high affinity and specificity due to their three-dimensional structure [12]. The binding of aptamers to their target results from the structure’s compatibility, electrostatic interactions, Van der Waals forces, hydrogen bonds, or a combination of these [16]. The affinity of the aptamers for their target molecule is measured using their dissociation constant (Kd) [17]. In the treatment of microbial infections, the aptamer-based systems have been found to be talented tools, regarding their promising anti-biofilm and antimicrobial activities. Aptamers can reduce or inhibit the effects of bacterial toxins, inhibit pathogen invasion to immune cells, and they can also be used in drug delivery systems [18]. To design aptamers, SELEX (Systematic Evolution of Ligands by Exponential Enrichment) has been employed as a useful technique for selecting nucleic acid ligands that interact with the target molecule in a desirable manner. SELEX is an iterative process of selection and amplification, in which large pools of nucleic acid molecules (>1 trillion distinct sequences) are challenged to bind to a desired target under defined conditions such as temperature and salt concentration; later, these sequences are amplified to generate a new population of enriched molecules. In SELEX, a negative selection is completed, which involves the incubation of a cell or microorganism closely related to the nucleotide sequences previously selected and the removal of sequences that are non-specific to the target molecule [19]. Finally, using bioinformatics programs, with secondary structures from the sequences selected, Kd, the different probable binding sites, and the structure of tertiary bonding are determined [20].
V. parahaemolyticus, V. harveryi, V. alginolyticus, and V. vulnificus are important pathogens in the aquaculture environment [21][22][23], causing huge losses to the aquaculture industry; they can also infect humans through food and water and become a serious threat to public safety. Traditional methods have been used for the detection of these pathogens, which include microbial-testing techniques; instrumental analyses methods such as real-time polymerase chain reaction, molecular biology techniques, and immunological detection methods such as enzyme-linked immunosorbent assay; enzyme-linked fluorescence analysis; time-resolved fluorescence immunoassay; and chemiluminescence immunoassay [24]. However, these methods have some specific limitations/disadvantages for Vibrio detection. Aptamers have been proposed as an alternative to these limitations [25], which could be successfully selected using SELEX prior to knowing the corresponding target molecules [26].

2. Detection of Vibrio parahaemolyticus

V. parahaemolyticus is an important seafood-borne pathogen with a serious impact on human health [27]. Consumption of fresh fish and seafood causes acute gastroenteritis, and it can be associated with wound infections in humans worldwide [28]. Although culture-based biochemical identification is widely used and isolation of the bacteria is the gold standard, these methods require laborious steps and are time-consuming. Thus, novel sensitive and rapid detection methods have been developed such as polymerase chain reactions (PCRs) [29][30] and ELISA [31]; these methods are still restricted, since specialized equipment and qualified operators are needed. Rapid, simple, and sensitive methods for screening V. parahaemolyticus-contaminated foods are urgently needed to ensure food safety.
In a study by Duan et al. [32], with a cell-SELEX method, they selected ssDNA aptamers that bind specifically to the strain ATCC 17802. The purpose of using live bacterial cells is to avoid a priori purification of specific target molecules from cell surfaces; additionally, live bacteria can grow in suspension, allowing simple separation using centrifugation. Flow cytometry was used to identify the aptamer bounded with high affinity. Aptamers A3 and A3P showed higher binding affinity, with fluorescence values around 75%. The aptamers A1, A1P, A3, A3P, and A18P were incubated with L. monocytogenes, E. coli, S. typhimurium, S. aureus, C. sakazakii, and S. pneumoniae as the negative control. The aptamer A3P(5′-FAM-ATAGGAGTCACGACGACCAGAATCTAAAAATGGGCAAGAAACAGTACTCGTTGAGAACTTATGTGCGTCTACCTCTTGACTAAT-3′) showed a high binding affinity with a 76% affinity for V. parahemolyticus and low affinity for the other bacteria (4%). The Kd was 16.88 +- 1.92 nM. This research was the first to report on the use of whole-bacterium SELEX for selecting specific aptamers for V. parahaemolyticus, allowing detection even when it is applied to a complex sample matrix, such as food. Also, these sequences of aptamers can be linked to magnetic nanoparticles to capture and conserve the bacteria with a magnetic field, or they can be chemically modified and conjugated to sensitive detection probes, etc.

3. Detection of Vibrio alginolyticus

V. alginolyticus is a pathogenic bacterium widely distributed in ocean, coastal, and estuarine areas, and it poses a threat to public health due to its significant impact on morbidity and mortality [33][34]. Consumption of raw seafood contaminated with V. alginolyticus or even exposure to contaminated water could result in bacterial infections such as gastroenteritis, otitis media, and chronic diarrhea. Diseases are reported worldwide including in Europe, Asia, North America, and South America. Moreover, indiscriminate use of antibiotics can lead to an increase in bacterial antibiotic resistance, so rapid, sensitive, and accurate methods for detecting this bacterium are needed [33].
The conventional methods for detection of V. alginolyticus comprise culture-based traditional microbial methods including biochemical identification, selective cultivation, serotype identification, toxin detection, and a wide range of PCR methods. However, those methods are still complicated, time consuming, expensive, and require expertise and advanced laboratory systems. New methods for providing a simple and efficient way to detect the bacterium for accurate diagnoses and treatment are being developed [33][35].
In a study conducted by Tang et al. [36] using formaldehyde-inactivated bacteria, aptamers against this bacteria were selected from an 82 bp random library of ssDNA using cell SELEX. All sequences obtained were divided into nine families according to their homology, and some conserved sequences were found in each of the six families (GCACAAGAGGGA), suggesting that the conserved sequence could be important to form the secondary structure of the aptamers.
After 15 rounds of selection, the final group of aptamers was highly specific for V. alginolyticus, with a dissociation constant of 27.5 ± 9.2 nM. The specificity was determined by contacting the aptamers with other bacteria (V. alginolyticus, E. tarda, A. hydrophila, and V. harveyi) showing greater specificity towards V. alginolyticus. Qualitative detection of the inactive microorganism at low concentrations (100 cells/mL) was demonstrated using the family of aptamers with a higher affinity and PCR. Thus, the successful identification of aptamers that can be used in the selective detection of V. alginolyticus may be more useful in the detection of pathogenic microorganisms in aquatic environments.
In another study, Zhao et al. [37] developed a novel method for the detection of V. alginolyticus utilizing a specific aptamer for recognition and signal amplification via a hybridization chain reaction (HCR) and horseradish-peroxidase-conjugated streptavidin. The biotin–aptamer conjugate binds to streptavidin on horseradish-peroxidase-conjugated streptavidin (SA-HRP), which allows for a highly sensitive and specific detection method. The development of these methods allows the detection of a linear range from 10 to 107 CFU/mL with an LOD of 3 CFU/mL, which has a high potential for being used to monitor V. alginolyticus in aquaculture environments.

4. Detection of Vibrio vulnificus

V. vulnificus is a Gram-negative pathogenic bacterium that is motile, curved, and halophilic, inhabits estuarine or marine coastal areas, is one of the most important marine pathogens, and is highly pathogenic to humans [38]. Yan et al. [38] identified a highly specific DNA aptamer for Vibrio vulnificus using SELEX coupled with asymmetric PCR. After 13 rounds of cross-selection, the authors identified a novel DNA aptamer (Vapt2), which was characterized in terms of Kd and LOD; they also suggest that their work is a framework for the rapid detection of pathogenic bacteria and water pollution.

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