New Technologies from the Microbial World: Alternatives for Biomedical Surrogate Research
Francisco M Ochoa Corona*, Kitty F Cardwell and Andres S Espindola
Department of Food and Agricultural Biosecurity, National Institute for Microbial Forensics & Food and Agricultural Biosecurity (NIMFFAB), Oklahoma State University, USA
Submission: February 08, 2019; Published: March 21, 2019
*Corresponding author: Francisco M Ochoa Corona, Department of Food and Agricultural Biosecurity, National Institute for Microbial Forensics & Food and Agricultural Biosecurity (NIMFFAB), Oklahoma State University, Stillwater, OK 74078, USA
How to cite this article: Francisco M Ochoa Corona, Kitty F Cardwell, Andres S Espindola. New Technologies from the Microbial World: Alternatives for Biomedical Surrogate Research. Adv Biotech & Micro. 2019; 13(2): 555859. DOI: 10.19080/AIBM.2019.13.555859
Abstract
A brief description of a few amenable technologies developed for research in biomedical and agricultural diagnostics tested using plant pathogens as microbial surrogates are presented. Plant pathogen surrogate microbes can assist in easing compliance with regulations and reducing costs, ethical and biosafety concerns in biomedical research.
Keywords: Phytopathogens, plant pathogens, sample collection, microbe storage, PCR, NGS
Abbrevations: EDNA: Electronic-probe Diagnostic Nucleic-acid Analysis; NGS: Next Generation Sequencing; ONT: Oxford Nanopore Technologies
Introduction
Biomedical research relies heavily on model organisms as a surrogate for animals and human biological systems [1,2], and surrogate research requires a strong level of evidence that relates the technology, the surrogate, and the experimental outcome. To assess such strength of association the new technological contribution has to be subject of meta-analytic approaches including quantification of the relation between the proposed surrogate and the final outcome [3].
Most surrogate models for human medicine are vertebrates, such as rodents and primates, but even simple organisms such as yeast provide valuable insights and feedback by elucidating bioprocesses at the molecular and cellular levels [4, 5]. Similarly, research in food and agricultural microbiology uses surrogate organisms [6]. For example, plant pathogenic viruses [7], bacteria [8], fungi [9], protozoa, insects and nematodes are used as a proxy for zoonotic organisms [10]. The use of animals in biomedical experimentation has attracted negative public attention and the search for ‘alternative methods’ gave birth to new technology development [11]. We briefly describe a few technologies developed for agricultural purposes that are amenable alternatives for biomedical diagnostics research using plant pathogens as microbial surrogates.
Example 1
Collecting and archiving Nucleic Acids (NA) are key steps in detection and diagnosis when using PCR for health, biosecurity, or microbial forensics applications. The cotton-swipe and paper-based technologies used for sample collection offer advantages, such as storage of NA in the sample at room temperature. However, recovering NA using these collection technologies requires several (wet-lab) steps. In addition, the performance of PCR directly from the sample is hampered by the cotton fibers and residual paper matrix itself, which significantly limits its application in rapid disease diagnostics.
An elution independent collection device (EICD) [12, 13], Patent US 9,423,398 B2, was conceived to streamline sample collection and microbial processing directly into detection assays such as PCR or ELISA. The compact, easy-to-use EICD collects fluid specimens by contact and lateral flow. After samples are collected and aliquoted onto the EICD, minute pieces (1.2 mm diameter) of a built-in soluble element are excised and dissolved directly in commercial PCR or ELISA mixtures without intermediate elution steps, thereby streamlining diagnostic assays. Seventeen plant viruses, fifteen bacteria, one fungus, one insect and one plant gene (used as internal control) were assessed using one-step RT-PCR without an intermediate RNA extraction step. EICD prototypes have been proven ready for PCR processing within 3 minutes, far less time than the 10-30 minutes required using commercially available DNA elution kits. Scanning electron microscopy of pore spaces and crevices of the biomaterials either dry or wet, and with or without bacteria and stable storage of sample DNA and RNA in the EICD has been shown to last a year at room temperature. This technology has passed the proof of concept stage of development [12, 14].
Example 2
Positive controls of infectious disease can pose biosafety risks during transportation and manipulation, nonetheless, are essential for PCR reliability and are challenging to obtain for rare, exotic, contagious, and/or emerging pathogens. As an alternative to this problem custom synthetic DNA inserts were designed de novo in tandems of forward and reverse complement primer sequences to be inserted in circularized plasmid vectors [15]. To test this new concept an artificial positive control (APCs, 203 bp long) for use in PCR was synthesized using primer sequences targeting four plant viruses infecting wheat (Barley yellow dwarf, Soil-borne wheat, Wheat streak mosaic and Triticum mosaic viruses) and the internal control plant mitochondrial nad5 gene [15]. The plasmids were maintained dry in EICD to avoid aerosol contamination in the laboratory. Similarly, a second 1126 bp long multi-target APC (GenBank KC555272) including probes sequences in addition to the tandems of primers, was synthetically generated (GenScript USA Inc, Piscataway, NJ) and cloned into pUC57. This APC allowed quantitative PCR of the insects Liposcelis decolor, L. bostrychophila, L brunnea, L pearmani, L. obscura, L. decolor, Lepinotus reticulatus, and the plant viruses’ High plains wheat mosaic virus (formerly High plains virus), Wheat streak mosaic and Triticum mosaic viruses, Pythium aphanidermatum and Pythium deliense; [16]. These two arrays of APC priming sequences from different kingdom species demonstrated the advantage of using surrogates from the plant world while developing new technologies to be translated to the biomedical field.
Example 3
The Electronic-probe Diagnostic Nucleic-acid Analysis (EDNA) was reported in 2013 [17] and provided the framework for a new sequence-based detection system that eliminates the need for assembly of Next Generation Sequencing (NGS) data and eliminates big-data bioinformatic challenges. NGS suffers from a large amount of computational time and power needed to identify a pathogen sequence from the obtained NGS dataset. EDNA allows rapid identification and simultaneous characterization of multiple specific pathogens and changes the roles of NGS data from a query to the queried database relative to other tools. EDNA uses pathogen-specific sequences, known as electronic probes (e-probes), to detect specific viruses or organisms in metagenomic data. E-probes have been validated and generated using either complete or partial pathogen genomes [18].
Although developed with plant pathogens (RNA virus, a DNA virus, bacteria, fungi, and an oomycete), the technology would be also useful in metagenomic sequences from vertebrates [17,19,20]. Recently, to make EDNA easier to operate by nonskilled bioinformatic operators, an online platform named MiFi© was created. The online graphical user interface, MiFi©, was developed upon the concept of EDNA [20] and comprises two parts:
a) MiProbe© which houses all tools needed for building and validating E-probes, and
b) MiDetect© which is the diagnostic side of the program able to rapidly identify the genetic signatures of targeted pathogens in metagenomic datasets, which occur by E-probes matching of DNA or RNA sequence-data from host tissue and associated microbes. MiFi allows the flexibility of using any sequencing platform and users are expected to take advantage of portable sequencing devices like the Oxford Nanopore Technologies (ONT) MinION.
Early trials with ONT have been successful when assessing functional transcripts activation for aflatoxin production in soil [20]. Furthermore, the availability of the MiFi platform has permitted the development and curation of e-probes for pathogens of citrus, grapevine, roses, and blueberry allowing a host tailored diagnostic assay validation [21,22]. MiFi is rapidly evolving to be used in animal and water diagnostics and it is expected to be adopted in the biomedical field potentially using cancer diagnostics as the early proof of concept.
Conclusion
The use of plant pathogens as surrogates during a proof of concept stages of new technologies allows procedural flexibility and assists research and development by easing compliance to biosafety regulations, as plant pathogens are not infectious to humans or animals but share equivalent molecular and biophysical properties. We have presented three cases in which technological approaches translatable to biomedical were initially developed using insects, microbes, and viruses from the plant world. EICD, although developed with plant pathogens, is applicable to any field-side DNA collection and storage and molecular-clinical diagnostics for health, veterinary, plant health, biosecurity, forensics, microbial forensics, and food quality. The development of APC arrays of DNA priming sequences from different kingdom species demonstrated that surrogates from the plant world are useful for developing new positive controls. The new APC concept can be translated to new biomedical situations or assays improving reliability and biosafety. The development of technologies such as APC create opportunities for development and commercialization of new non-contagious products synthetic positive controls [15,16,23]. EDNA was initially tested with plant pathogens. E-probes, carefully designed unique nucleic acid signatures, are used to identify microbes and viruses in NGS generated metagenome databases, can easily be translatable to human, animals and food-borne pathogen genomes. The unique, pathogen-specific sequences (E-probes), are designed for searches of unassembled, unchecked raw base-call read sequence data (i.e. Illumina or MinION) and are validated for sensitivity, and specificity [20,21,24].
Therefore, plant pathogens used as proxies would also contribute by reducing costs. In general, the low risk and biosafety level of plant pathogenic viruses, bacteria, and fungal species while testing new technologies has a positive impact speeding up research. Moreover, surrogate plant microbes are easy to maintain, manipulate and add high research value as demonstrated during the development of EICD, APCs and EDNA. However, additional corroboration is needed for these new technologies with human biomedical research validation. The merge of research in biomedical and agricultural diagnostics aiming the use of plant pathogens and insects as microbial surrogates is creating new research and development possibilities and products. Scientists in agricultural diagnostics walking into this new research space are seeking for partners in biomedical research to further validate these technologies in ways that will be meaningful to them.
References
- Yamamoto S, Jaiswal M, Charng WL, Gambin T, Karaca E, et al. (2014) A Drosophila genetic resource of mutants to study mechanisms underlying human genetic diseases. Cell 159(1): 200-214.
- Wangler MF, Yamamoto S, Chao H-T, Posey JE, Westerfield M, et al. (2017) Model Organisms Facilitate Rare Disease Diagnosis and Therapeutic Research. Genetics 207(1): 9-27.
- Ciani O, Buyse M, Drummond M, Rasi G, Saad ED, et al. (2017) Time to Review the Role of Surrogate End points in health policy: State of the art and the way forward. Value Health 20(3): 487-495.
- Barrientos A (2003) Yeast Models of Human Mitochondrial Diseases. IUBMB Life 55(22): 83-95.
- Sherman MY, Muchowski PJ (2003) Making Yeast Tremble: yeast models as tools to study neurodegenerative disorders. Neuro Molecular Medicine 4(1-2): 133-146.
- Pérez-Rodríguez, Valero FA, Carrasco E, García R Ma, Zurera G (2008) Understanding and modelling bacterial transfer to foods: a review. Trends in Food Science & Technology 19(3): 131e144.
- Bae J, Schwab KJ (2008) Evaluation of Murine Norovirus, Feline Calicivirus, Poliovirus, and MS2 as Surrogates for Human Norovirus in a Model of Viral Persistence in Surface Water and Groundwater. Appl Environ Microbiol 74(2): 477-484.
- Hu M, Gurtler JB (2017) Selection of surrogate bacteria for use in food safety challenge studies: A review. J Food Prot 80(9): 1506-1536.
- Halstensen AS (2008) Species-specific fungal DNA in airborne dust as surrogate for occupational mycotoxin exposure? Int J Mol Sci 9(12): 2543-2558.
- Steinert M, Leippe M, Roeder T (2003) Surrogate hosts: protozoa and invertebrates as models for studying pathogen-host interactions. Int J Med Microbiol 293(5): 321-332.
- Collins FS, Green ED, Guttmacher AE, Guyer MS (2003) A vision for the future of genomics research. Nature 422(6934): 835-847.
- Donna CR, Ochoa-Corona F, (2011) An elution-independent collection device for rapid sampling of microorganisms and nucleic acids for PCR assays. Phytopathology 101: S44
- Ochoa Corona, Francisco M (2016) Apparatus and method for biologic sample rapid collection and recovery device, and convenient storage. United States Patent: US 9423398 B2.
- Caasi DRJ (2012) Assessment of soluble biomaterials and innovation of an elution-independent collection device for rapid collection, detection, and storage of plant pathogens. Oklahoma State University. Doctoral Dissertation 116pp.
- Caasi, DRJ, Arif M, Payton M, Melcher U, Winder L, et al. (2013) A multi-target, non-infectious and clonable artificial positive control for routine PCR-based assays. J Microbiol Methods 95(2): 229-234.
- Arif M, Opit G, Mendoza-Yerbafría A, Dobhal S, Li Z, et al. (2015) Array of Synthetic Oligonucleotides to Generate Unique Multi-Target Artificial Positive Controls and Molecular Probe-Based Discrimination of Liposcelis Species. PLoS ONE 10(6): e0129810.
- Stobbe AH1, Daniels J, Espindola AS, Verma R, Melcher U, et al. (2013) E-probe diagnostic nucleic acid analysis (EDNA): A theoretical approach for handling of next generation sequencing data for diagnostics. J Microbiol Methods 94(3): 356-366.
- Bocsanczy AM, Espindola AS, Norman DJ (2019) Whole-Genome Sequences of Ralstonia solanacearum Strains P816, P822, and P824, Emerging Pathogens of Blueberry in Florida. Microbiol Resour Announc 8(3): e01316-18.
- Espindola AS, Schneider W, Cardwell KF, Carrillo Y, Hoyt PR, (2018) Inferring the presence of aflatoxin-producing Aspergillus flavus strains using RNA sequencing and electronic probes as a transcriptomic screening tool. PLoS One 13(10): e0198575.
- Espindola AS, Schneider W, Hoyt PR, Marek SM, Garzon C (2015) A new approach for detecting fungal and Oomycete plant pathogens in next generation sequencing metagenome data utilizing electronic probes. Int J Data Min Bioinfomatics 12(2): 115-128.
- Bocsanczy AM, Espindola A, Norman DJ, Cardwell KF (2018) E-probes development for rapid, sensitive and specific pathogen detection in blueberries. Phytopathology 108: S1 301.
- Pena-Zuniga L, Espindola A, Klein P, Debener T, Rees J, et al. (2017) EDNA-Rose a novel approach for detecting rose viruses combining next generation sequencing and bioinformatics. Phytopathology 107(12): S5 58.
- Dobhal S, Olson JD, Arif M, Garcia Suarez JA, Ochoa-Corona FM (2016) A simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries. J Virol Methods 232: 47-56.
- Espindola AS, Cardwell KF (2018) Third generation sequencing and EDNA for detection of aflatoxin production in the soil. Phytopathology108(10): S169.