DNA aptamer selection and detection of marine biotoxin 20 Methyl Spirolide G
Introduction
Cyclic imines are an emerging group of marine biotoxins that contaminate seafood. 20 Methyl spirolide G (SPX G), a cyclic imine is a neurotoxin produced by dinoflagellate Alexandrium ostenfeldii / A. peruvianum. Spirolides were first identified in toxic digestive gland extracts of mussels and scallops found in the Atlantic coast of Nova Scotia, Canada. Sixteen different types of spirolides have been identified and characterized in contaminated shellfish and phytoplankton extracts from coasts of Europe, North and South America (Farabegoli, Blanco, Rodríguez, Vieites, & Cabado 2018). Accumulation of toxin in marine foods occurs as a result of ingestion of spirolide-producing dinoflagellates by the aquatic invertebrates. SPX G has been isolated from various species of shellfish, especially mussels and scallops (Guéret & Brimble, 2010). The toxin has been reported to bind neuronal nicotinic acetylcholine receptors and block skeletal muscles in mice models (Couesnon et al., 2016). Toxicity studies of SPX G, show an intraperitoneal LD50 value of 8 µg/kg body weight in mice (Munday et al., 2012). At present, there are no regulatory levels set by European Union (EU) or any other regulatory authorities for SPX G or other spirolide toxins in sea foods because of lack of conclusive data regarding their human poisoning (Rambla-Alegre et al., 2018). However, their toxicity and potential risk to consumers have raised growing concerns among public and policy makers to address food safety issues related to these biotoxins.
The present methods used for the detection of spirolides are classical analytical techniques such as high performance liquid chromatography, mass spectrometry and liquid chromatography – tandem mass spectrometry (Otero et al., 2011, Aasen et al., 2005, Gerssen et al., 2009). Mouse bioassay is the only official method accepted by EU for the determination of marine toxins including SPX G (European Commission, 2011). Intraperitoneal injection of shellfish extracts to mice in mouse bioassay has led to sacrifice of large number of laboratory animals leading to ethical concerns. Fluorescence polarization and solid-phase receptor-based assays are also reported for spirolide detection. Fluorescence polarization method works on the principle of quantitative spirolide binding to the fluorescently labelled nicotinic acetylcholine receptor resulting in the change of fluorescence polarization signal (Otero et al., 2011). Solid-phase receptor-based assays use the competition of spirolide with biotin labelled α-bungarotoxin for binding to nicotinic acetylcholine receptor and the immobilization of the former complex on streptavidin coated surface (Rodríguez et al., 2011). Although, these methods are accurate and reliable, they suffer from disadvantages such as long processing time, complex sample pretreatment and preparation steps, photobleaching, need for specially trained personnel and use of antibodies which restricts their wide scale application.
Aptamers are short oligonucleotide or peptide sequences generated in vitro against target molecules. They are screened, designed and evolved by an in vitro selection process known as systematic evolution of ligands by exponential enrichment (SELEX). Aptamers fold into specific three dimensional structures and bind to their ligands by complementary shape interactions and can incorporate small molecules into their folded structure or integrate into the structure of larger molecules (Hermann & Patel, 2000). The idea behind aptamer as recognition element comes from their ability to fold into three dimensional structures which facilitates their interaction with target molecules. Aptamer today are used in a wide range of applications, which comprise in situ bio-imaging, drug delivery and therapy, disease diagnosis, hazard detection, food monitoring, etc. (Wang et al., 2017, Jahangiri-Dehaghani et al., 2020). Relative to antibodies, aptamers have several advantages, including stability, low cost, and ease of synthesis (Jayasena, 1999). Aptamer-based biosensing platforms can be developed for both qualitative and quantitative measurements.
In this study, we report for the first time, the generation and selection of an aptamer for marine biotoxin SPX G. The selected aptamer showed high binding affinity to SPX G with dissociation constant (KD) in the nanomolar range. Microscale thermophoresis (MST) coupled aptamer-based detection of the target, indicated ultrasensitive detection with a broad linear range (from picogram to microgram level) and no significant interference of the matrix. The aptamer also was able to detect the toxin in real spiked samples with good recoveries. Apart from biosensing applications, the MST aptamer-based method can also be extremely useful in toxicological studies and to collect data on the occurrence of SPX G in marine products.
Section snippets
Chemicals and reagents
All the analytical grade chemicals were purchased from SRL (Mumbai, India). 20 methyl spirolide G (SPX G) was supplied by CIFGA laboratories, Spain. Polymerase chain reaction (PCR) reagents, streptavidin immobilized on agarose CL-4B and okadaic acid was purchased from Sigma-Aldrich (Bangalore, India). Aflatoxin B1 (AFB1) was purchased from Himedia Laboratories (Mumbai, India). All HPLC grade solvents were purchased from Merck (Bangalore, India). Immobilization buffer, regeneration buffer and
Results and discussion
Aptamers have proven to be one of the best suitable ligands for the detection of small molecules. Aptamers as bio-recognition elements have various advantages over antibodies, especially for development of biosensors. Aptamers can be synthesized in vitro without requiring the need for experimental animals. This reduces the cost, time and batch-to-batch variation during production. Aptamers can be developed against small molecules and toxins which is difficult in case of antibodies. They are
Conclusion
To summarize, highly specific DNA aptamers that bind to SPX G were selected by conventional SELEX. After sequence analysis of the DNA pool, three different aptamers were further studied for their binding affinities to the toxin. SPX 7 aptamer with KD in the nanomolar range had a better half-life, faster on-rate and slow off-rate for binding to the toxin and was therefore selected for further studies. A label-free MST coupled aptasensing using SPX7 aptamer showed a broad detection range and good
CRediT authorship contribution statement
Monali Mukherjee: Investigation, Formal analysis, Methodology, Writing - original draft. Srinivas Sistla: Investigation, Data curation, Writing - review & editing. Shivakumar R. Veerabhadraiah: Investigation, Formal analysis, Methodology. B.K. Bettadaiah: Validation, Writing - review & editing. M.S. Thakur: Conceptualization, Writing - review & editing. Praveena Bhatt: Supervision, Resources, Project administration, Validation, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors thank the Director, CSIR- Central Food Technological Research Institute for his constant encouragement. The authors are thankful to Prof. Loius Botana and Prof. Eva Cagide, CIFGA laboratories (Spain), for providing 20 methyl spirolide G (SPX G) toxin for the work. The authors duly acknowledge the funds provided by Department of Biotechnology (DBT) to carry out the studies. MM is thankful to DST-INSPIRE for providing Junior/Senior Research Fellowship.
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