Emerging Pathogen-Detection Techniques in Agro-Food Sector

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1		Virendra Kumar	--	1606	2022-07-21 17:18:00	\|
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This entry is adapted from the peer-reviewed paper 10.3390/bios12070489

The agro-food sector is one of major contributors to the economy of a developing country. This sector offers a primary source of nutrition for livestock and also more than 80% of the food consumed by human beings. The monitoring of agro-food products is essential to maintain our civilization with food security via reducing the risk of infections. The diagnosis of pathogens can be carried out in plants themselves, in obtained food products, or in humans after the consumption of contaminated agro-foods.

agro-food pathogen disease emerging pathogen detection

1. Molecular Imprinting

Molecular imprinting is a comparatively new technique that comprises the integration of molecularly imprinted polymers (MIPs) with different transducer platforms such as optical, electrochemical, and mass-sensitive transducer platforms ^[1]^[2]^[3]. This technology can offer highly selective and sensitive detection of pathogens in an ultra-rapid manner ^[4]. MIPs are artificial receptors that are prepared by the polymerization of monomers and crosslinkers along with other initiators and promotors (if required). During this process, the presence of the target analyte works as a template and can be further removed to create free cavities in MIPs with conserved shape, size, and functionality (refer to Figure 1) ^[5]. Therefore, MIPs can easily recognize the target analyte in the sample and can easily discriminate it from other co-existing structures through a combination of size, morphology, and different chemical interactions (such as covalent, semi-covalent, and non-covalent).

Figure 1. Schematic representation of fabrication of molecularly imprinted polymers (MIPs) for recognition of pathogens.

In comparison to other receptors such as enzymes or antibodies, MIPs offer several interesting properties in terms of chemical and physical stability and reusability ^[6]. In the case of food products, MIPs have been employed for the determination of a large variety of hazards such as small molecules (mycotoxins), macromolecules (allergenic proteins), and larger analytes (pathogenic bacteria) ^[7]. In comparison to small molecules, bacterial detection faces several scientific challenges due to their intrinsic properties such as fragile structure, large size, poor stability in organic solvents, and fluidity ^[8]. In order to improve the rational design of MIPs at a molecular level for pathogen detection, several new imprinting strategies have been reported, e.g., cell membrane molecular imprinting and stamp imprinting ^[9]^[10]. Moreover, pathogen bacterial detection using MIPs is based on the assumption of a non-covalent interaction between the polymer and the pathogen, which needs to be elucidated further. Additionally, the impact of food components on MIP-based platforms needs to be tested ^[11].

2. DNA Microarray

DNA-based methods can overcome the limitations of culture-based methods via a faster response and offering more information. The expression and silencing of genes are the basic functional switches for all the biological processes in living organisms. Various developmental, etiological, temporal, and physiological stages are governed by the differential expression of genes in an organism. Further, microarray technology offers an expansion of capabilities of DNA-based methods (such as PCR) in terms of the analysis and molecular identification of multiple pathogens in a single array assay ^[12]. The DNA microarray provides information on the differential expression of genes between samples. It uses small DNA probes that can hybridize with the complementary DNA (cDNA) produced from the extracted mRNA from each sample. The cDNA from the samples under study are tagged with fluorescent tags that help in the study of the differential expression of genes. The relative expression of genes by estimating the copy number of genes can be studied using this technique. Schena et al. introduced the first DNA microarray by printing cDNA on a glass slide as a probe in order to understand the differential expression of genes of Arabidopsis thaliana ^[13].

The primary use of microarrays was in the field of transcriptomics ^[14]^[15]; nowadays, there is an availability of huge collections of annotated whole-genome sequencing (WGS) data, which facilitated the use of microarrays in other fields of biological sciences, including proteomics and diagnosis ^[16]. Besides the DNA microarray, nowadays, other microarrays (such as the antibody microarray and the small molecule microarray) have also been developed ^[17]^[18]. The DNA microarray also offers differentiation benefits in the same species ^[19]. Through multiplex analysis using microarray technology, a deeper understanding of the resistance profiles and the metabolic differentiation of pathogens can be gained ^[20]. No doubt, this multiplex approach can save time with a simultaneous analysis of multiple pathogens; however, designing probes for custom identification profiles is somewhat lengthy and expensive. Recently, a customized chip with desirable probes has been developed for the detection of multiple bacterial species and profiling ^[21].

3. Aptamer-Based Immunoassay

Aptamers are usually synthetic DNA or RNA molecules with appropriate secondary structures that have emerged as new analytical reagents. These offer several interesting properties such as binding specificity for targets with high affinity, liable modification, high stability, ease of labeling, and low production costs ^[22]. They are slightly more stable than antibodies after correcting for non-specificity, salinity, and pH range in the working matrices. In recent years, aptamers have been heavily used in bacterial detection as receptors against bacteria. However, aptamers cannot be directly labeled with enzymes ^[23]. Therefore, different fluorophores can be attached to DNA sequences for the detection of pathogens (refer to Figure 2) ^[24]. Further, the use of fluorescent or electrochemically active molecules can help in the design of self-reporting aptamer-based assays ^[25]^[26]. Pathogenic strains have been successfully detected by aptamer-NanoZyme-mediated sensing platforms (both electrochemical and colorimetric) ^[27].

Figure 2. Schematics represent the detection of Staphylococcus aureus (S. aureus) using a specific aptamer, a fluorescent molecular beacon, and signal amplification. The chimera aptamer sequence forms a hairpin structure with the aptamer sequence of S. aureus. In the presence of bacterial pathogens (i.e., S. aureus), it becomes bound to the aptamer region of the chimera sequence, which results in the opening of the hairpin structure and unlocking of molecular beacon, restoring the fluorescence ^[24].

However, the applicability of aptamer-based immunoassays is not as wide as antibodies due to the complexity in the selection of highly-specific aptamers ^[28]. The production of target-specific aptamers via the systematic evolution of ligands by exponential enrichment (SELEX) and post-SELEX is a time-consuming process ^[29]. In developing specific aptamers, microbial pathogens and their structural/non-structural proteins and enzymes are the targets. Any significant charge changes born by pathogens can enhance the complexity of a SELEX process ^[30]^[31]. The integration of aptamer-based assays with microfluidics, lateral flow, and surface-fabrication techniques can offer multiplexing capabilities along with a high degree of automation ^[32]. An aptamer-based lateral flow assay (LFA) has been reported for the simultaneous detection of Escherichia coli (E. coli), Salmonella Typhimurium (S. typhimurium), and Staphylococcus aureus (S. aureus) ^[33]. The developed sensing platform combined the advantages of both aptamer (high specificity and affinity) and LFA (simplicity, shorter assay time, low cost, and portability). Besides significant advantages, the aptamer-based LFAs have not been commercialized yet due to several limitations such as the poor affinity of the aptamer in the case of a change in buffer composition and the isoelectric point of the target proteins ^[33].

4. Omics- and CRISPR-Based Technologies

To meet the market demand for the efficient detection of food pathogens with high speed and high throughput, advanced research strategies deal with the integration of bioinformatics and food science, e.g., next-generation sequencing (NGS) ^[34], proteomics ^[35], transcriptomics ^[36], and CRISPR ^[37]. NGS instruments have been adopted in laboratories due to their smaller size and less expansive nature than conventional instruments ^[38]. In comparison with traditional microbial tests, NGS can offer the identification of a wide range of bacterial strains and even non-culturable or fastidious pathogens. Moreover, it needs much less or no prior knowledge of the pathogen to be detected. Besides these benefits, NGS suffers from several limitations, e.g., a low abundance of pathogens can be covered by other sequence information in the case of insufficient samples. Further, proteomics and transcriptomics may help in understanding the behavior of pathogens at a molecular level.

Emerging CRISPR-based technologies offer several interesting features such as ease of use, accuracy as PCR, cost-efficiency, various novel detection platforms, and visual strategies for the sensitive and specific detection of pathogens. The CRISPR-Cas system is a programmable universal genome editing tool that offers cleavage of invasive DNA/RNA through the CRISPR RNA (crRNA)-guided Cas protein. In a CRISPR-Cas 13a system, the “collateral effect” of RNase activity can be activated upon the crRNA-mediated detection of target RNA ^[39].

5. Integrated Biosensing Approaches

The new generation of pathogen detection is based on miniaturized integrated biosensing technologies that can offer reliable, sensitive, cost-effective, and rapid detection without the need for sophisticated instruments ^[40]. Moreover, the evolution of diagnostic assays has varied from conventional culture-based techniques to molecular immunological detection, and recently biosensors. Biosensing approaches (such as colorimetric, fluorescent, electrochemical, and others) require a small sample volume, thereby decreasing the experimental setup, analytical time, and expenses ^[41]^[42]^[43]. The immobilization of target pathogens on the surface of the bioreceptor can be strengthened by nanomaterials ^[44]^[45]. Nanomaterials in biosensors not only enhance sensitivity and selectivity but also reduce the risk of cross-contamination for the quick detection of pathogens ^[46].

Recently, several efforts have been made to develop innovative integrated biosensing methodologies, including microfluidic platforms, lateral flow tests, and smartphone-assisted sensors ^[47]^[48]. Digital microfluidics with loop-mediated isothermal amplification (LAMP) has been reported for the multiplex detection of different pathogens ^[49]. The use of microfluidics technology requires a minimal sample amount to localize the target analyte under flow conditions in a sensing area of a chip with small dimensions. Electrochemical biosensors have gained considerable research interest in combination with microfluidics due to their compatibility with microfabrication approaches ^[50]. A microfluidics-based on-chip artificial pore has been reported to sense bacterial pathogens ^[51]. The on-site detection of pathogens using POC devices mostly comprises lateral flow tests and lateral flow assays ^[52]^[53]. Further, the integration of sensing modalities with smartphones has been easily implemented in pathogen detection ^[54]^[55]. This integration offers several interesting features such as the facile real-time detection of pathogens and the ability to connect with cloud data storage systems.

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