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A large proportion of major crops currently grown in the US have been enhanced with one or more traits through the use of modern biotechnology methods. These crops are the result of the incorporation of specific agronomic, crop protection or quality traits into plants grown for agricultural production. These modified or transgenic plants differ from conventional plants in that they contain additional genes which produce a corresponding number of new proteins, the presence of which give the modified plants the desired beneficial traits. A gene for a specific trait is introduced by insertion of the gene DNA fragment into the plant genome via recombinant DNA methods. The introduced DNA fragment produces a protein which is expressed in the plant tissues. A particular introduced protein may produce a desired agronomic trait in the plant, or it may confer insect, disease or herbicide resistance to the plant, thus improving crop production. An introduced protein may also produce a quality trait such as enhanced flavor or nutritional value of the plant. With the introduction of transgenic plants, analytical technology is rapidly evolving to measure the introduced proteins. Among the most useful techniques for ag biotech applications are immunoassays. Immunoassays offer simple, specific and sensitive protein detection to address a wide range of needs of technology providers, food and commodity distribution channels, and regulatory bodies.

Accuracy – The closeness of individual predicted concentrations to the true concentration in a sample. Note: this definition combines the idea of bias (systematic error) and precision (random error). Assay – Qualitative or quantitative analysis of a substance.

Bias –
I. Recovery bias (absolute) – Bias with respect to the actual concentration of analyte – the degree to which the predicted concentration of an analyte differs, on the average, from the actual (or true) concentration.

False Negative – The percent test negativity in a population of true positives.

Interferences – Effects on the analytical performance of an immunoassay caused by substances other than the analytes(s) of interest.

Kit – A test kit is a packaged system of the key or principal components to detect or measure a specific analyte(s) in a given matrix(ces) within a laboratory or non-laboratory environment. The key components include antibodies, enzyme conjugates, etc. that may only be readily prepared by the purveyor of the kit. Test kits include directions for use and are often self-contained, complete, analytical systems, but they also may require supporting supplies and equipment.

Method – The entire system of procedures that describe how a final reassure of analyte concentration is obtained for a target matrix.

Precision – The extent to which replicate analyses of a sample agree with each other. Usually expressed as the standard deviation or percent coefficient of variation (%CV) of a population of values: %CV = (Standard deviation / Mean) X 100.

Reproducibility – The ability of the analytical method to yield the same result within analyses, between analyses and between operators.

Sensitivity –
I. Detection Sensitivity (Limit of Detection) – The smallest concentration of analyte that can be statistically significantly distinguished from zero for a given sample matrix with a stated degree of confidence.

II. Limit of Quantitation – The smallest concentration of analyte that can be measured in samples and yield a predicted concentration with stated relative precision or accuracy (or both).

Stability – 1. Kit Stability (Shelf Life) – The length of time that a complete kit can be stored under given conditions and still yield results that are within the stated performance parameters. 2. Sample Stability – the length of time that a target analyte level remains stable in a specific matrix under a given set of storage conditions.

PCR is a process which mimics the natural process of DNA replication occurring in all cellular organisms from bacteria to humans. The DNA molecules of a cell are duplicated prior to every cell division during DNA replication. In a PCR reaction, DNA replication is catalyzed by heat-stable DNA polymerase enzymes.

PCR differs from the natural process as the DNA molecules produced are restricted to target specific, relatively short regions of the original template DNA molecule. Short, single-stranded, synthetic complementary DNA molecules called primers or oligonucleotides confer specificity to the reaction. A single cycle of the PCR reaction is divided into three phases: denaturation, annealing and elongation, each with a different temperature regime. At the end of two cycles of these phases, the targeted DNA region has been replicated into two copies of the original double-stranded DNA molecule. This process of selective duplication per cycle is repeated multiple times in a cyclic reaction using a thermocycler and resulting in trillions of copies. The kinetics of the DNA reproduction resemble an exponential amplification in which the replicas of distinct length (amplicons) accumulate quickly and outnumber the original template molecules. The process has become the basis for several advanced technologies including: isothermal PCR [FAQ3], digital PCR [FAQ4], and sequencing [FAQ2], each with distinct and special advantages.

Quantitative Real-Time PCR or qPCR, in contrast to end-point PCR, enables the exponential accumulation of target amplicons to be monitored in real-time over the course of the amplification. The method uses specialty primers with fluorescent labels and an instrument with a laser or LED to measure the amount of an unknown target relative to a similar target of known concentration.

The biological sciences industry applies PCR and qPCR in several ways, for example, as the basis for diagnostic tools, for identity preservation, in forensics evidence support and in the development process of bioengineered products. Numerous diseases can be routinely detected at an early stage using signatures found in DNA called markers. In agriculture, PCR is utilized during gene discovery, cloning, vector construction, transformant identification, genotyping plants, characterization, quantification and seed quality control. Commodity crop companies, food companies and third-party diagnostic testing laboratories also rely on PCR for a number of purposes throughout the value chain. Quantitative Real-Time PCR has been used extensively for the study of human, animal, plant, and microbial biology. It presents a wide variety of applications including clinical diagnosis, gene expression analysis, determination of gene copy number variation, genotyping and allelic discrimination, zygosity testing, and GMO detection and quantification. Illustrations of the use cases for qPCR include verifying the presence or absence of biological material in a product, quantifying the amount of unwanted biological material in a product, and to certify compliance contracts between buyers and sellers of commodity grain.

1. Immunoassay as an Analytical Tool in Agricultural Biotechnology. Grothaus, G.D., Bandla, M., Currier, T., Giroux, R., Jenkins, G.R., Lipp, M., Shan, G., Stave, J.W., and Pantella, V., Journal of AOAC International, Vol. 89, No. 4, 2006, pp. 913-928.

2. Polymerase Chain Reaction Technology as an Analytical Tool in Agricultural Biotechnology. Lipp, M., Shillito, R., Giroux, R., Spiegelhalter, F., Charlton, S., Pinero, D., Song, P., Journal of AOAC International, Vol. 88, No. 1, 2005, pp. 136-155.

3. New Frontiers in Agrochemical Immunoassay, Kurtz, D.A., Skerritt, J.H., Stanker, L. (eds), AOAC International, Arlington, VA, 1995

4. Immunoanalysis of Agrochemical: Emerging Technologies, Nelson, J.O., Karu A.E. and Wong R. (eds.), ACS Symposium Series No. 586, American Chemical Society, 1995

5. Immunoassay for Trace Chemical Analysis: Monitoring Toxic Chemical in Humans, Food and the Environment, Vanderlaan, M.V. Stanker, L.H., Watkins B.E. and Roberts D.W. (eds.), ACS Symposium Series No. 451, 1991

6. Mihaliak, C.A. and Berberich
S. A. “Guidelines to the Validation and Use of Immunochemical Methods for Generating Data in support of Pesticide Registration”, in: Nelson, J.O., Karu A.E. and Wong R.B. (eds.), Immunoanalysis of Agrochemical: Emerging Technologies, ACS Symposium Series No. 586, American Chemical Society, 1995, pp. 288 – 300.

7. Rittenburg J. and Dautlick J. “Quality Standards for Immunoassay Kits” in: Nelson, J.O., Karu A.E. and Wong R.B(eds.), Immunoanalysis of Agrochemical: Emerging Technologies, ACS Symposium Series No. 586, American Chemical Society, 1995, pp. 301-307.

8. IUPAC Pesticides Report #33. Immunoassays for Residue Analysis ofAgrochemicals: Proposed guidelines for precision, standardization, and quality control. Pure & Appl. Chem., Vol. 67, No. 12, pp. 2065-2088, 1995.

9. Lipton, C.R., Dautlick, J.X., Grothaus, G.D., Hunst, P.L., Magin, K.M., Mihaliak, C.A., Rubio, F.M. and Stave, J.W. 2000. Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients. Food and Agricultural Immunology 12: 153-164.

Next generation sequencing (NGS) refers to a massively parallel sequencing technique that revolutionized sequencing capabilities. Compared to Sanger sequencing which uses capillary electrophoresis, NGS provides orders of magnitude more data at much lower cost.

NGS can be used to analyze DNA and RNA samples. NGS-based approaches have several advantages including i) prior knowledge of the genome or transcriptome is not required, ii) It offers single-nucleotide resolution, making it possible to detect genetic variants and single nucleotide polymorphisms, iii) mixture of nucleic acids from different sources can be analyzed all together simultaneously, and iv) can produce sequencing data with different sequencing lengths from 75bp to 20kb based on different technologies and platforms. Some of these platforms are:

Illumina sequencing platforms (Short reads): for more information please check this video -> https://www.youtube.com/watch?v=womKfikWlxM
454 Pyrosequencing platform (Short reads): for more information please check this video -> https://www.youtube.com/watch?v=bNKEhOGvcaI
Ion Torrent platforms (Short Reads) for more information please check this video -> https://www.youtube.com/watch?v=zBPKj0mMcDg
PacBio platforms (Long reads) for more information please check this video -> https://www.youtube.com/watch?v=_lD8JyAbwEo
Oxford Nanopore (Long reads) for more information please check this video -> https://www.youtube.com/watch?v=RcP85JHLmnI

Next Generation Sequencing applications in agriculture

The advent of NGS platforms has made it possible to rapidly and cheaply sequence whole genomes (i.e. whole genome sequencing or WGS) and transcriptomes (RNA Seq). In the field of plant sciences and agriculture, NGS is being used for the sequencing and assembly of large and complex genomes. NGS data has been used in fields ranging from comparative genomics and evolution of cultivated plants, to breeding needs (e.g. identification and mapping of genetic mutations, marker discovery and genotyping). Although lagging behind of analytical technologies that are currently the gold standard (i.e. real-time PCR), NGS has also been used and it is an upcoming technology for the analysis and detection of genetically modified products.

Genotyping using NGS has also become an effective tool in breeding programs for Marker Assisted Selection (MAS) , Genomics Selection (GS) , Genome Wide Association mapping (GWAS) and molecular marker discovery. Genotyping by sequencing (GBS) can be done through reduction of complexity using restriction enzymes, probes to capture specific sequences or parallel amplifications of specific targets. Recently, whole genome resequencing using low coverage NGS can be used for genotyping as well. Skim sequencing or low pass sequencing are such applications which require imputation in conjunction with resequencing.

NGS platforms have also been used for the analysis of RNA (i.e. RNAseq) to study the transcriptome of plant cells. The transcriptome includes all RNA molecules( messenger RNA, transfer RNA, ribosomal RNA and non-coding RNAs), and NGS data can be used not only to identify gene expression patterns, but also so study gene structure, including splicing, and post-transcriptional modifications. All these end points can be used to improve our knowledge in plant biology and ultimately facilitate crop breeding.

Isothermal PCR (isoPCR) is the broad term for amplifying nucleic acids utilizing a single temperature, while PCR is a generalized term for specific amplification of DNA for analytical purposes. Along with single-temperature amplification, a notable difference between the nucleic acid amplification technologies (NAAT), is the lack of a complex nucleic acid purification when using isoPCR.

IsoPCR is used to describe a wide range of strategies using different enzymes, including polymerase, temperatures, measurement methods, and detection methods. Depending on the specific isoPCR method, either or both DNA and RNA can be amplified. Detection of the result can be performed by turbidimetry, chromatography, gel electrophoresis, and fluorescence, for example, and can, in some applications, be achieved in a closed lateral flow device system.
Common types of isoPCR are:

LAMP - RNA & DNA, many visualization and detection strategies
RCA - RNA & DNA, simple, but may require a denaturation step
HDA - RNA & DNA, rapid, but can increase sensitivity with two-step process utilization
RPA - RNA & DNA, rapid, utilizes a crude sample extraction
SDA - RNA & DNA, useful for long templates
NASBA - RNA & DNA, simple primer design, DNA amplification can be inconvenient
CRISPR-Cas proteins: Cas9nAR, Cas12a, Cas13a - Can distinguish single-nucleotide differences

Standardization of the requirements for isoPCR are described, along with many of the approaches in the standard ISO 22942-1.

Isothermal PCR applications in agriculture

Because of isoPCR’s general simplicity, fewer equipment needs, and broad strategies, PCR level sensitivity can be achieved in laboratory and field site settings. In addition to the laboratory or field settings, the simplicity of isoPCR facilitates utilization without extensive training. isoPCR reactions are less susceptible to inhibitors than traditional PCR and thus can in many cases be used on crude lysates. Because of this and the fact that isoPCR can be performed using only simple temperature-controlled equipment, these methods can be employed in non-laboratory situations. Their main application has been qualitative detection although many of these approaches have now been developed into quantitative applications. While early applications were not particularly sensitive, isoPCR methods are now being deployed that have sensitivities that rival real-time PCR and look poised to replace it in some applications. In agriculture, isoPCR has been utilized in the detection of plant hormones, plant pathogens, plant sex identification, and GM trait detection.

Genome editing (also referred to as gene editing) is a technique which enables modification or change to the DNA sequence of an organism. The mechanism for such changes is derived from proteins called enzymes that are part of the natural machinery of an organism used to repair DNA. Two examples of these enzymes are: TALEN which stands for transcription activator-like effector endonucleases, and CRISPR-Cas which refers to clustered regularly interspaced short palindromic repeat associated protein. The enzymes can be directed to a targeted location in the genome, to make specific changes. The change could be as small as one single DNA base pair (out of the 3.2 billion base pairs in a human or 1.12 million base pairs in a soybean plant!), a short piece of DNA, or even an entire gene. It can also be used to add or remove a piece of sequence from the genome. The developers of the CRISPR-Cas 9 application, Emmanuelle Charpentier and Jennifer Doudna were awarded a Nobel Prize in 2020.

One important feature of genome editing is that the changes made are often the same as those found naturally or made by traditional breeding. This enables the user to leverage the beneficial effects of such DNA modifications in a very rapid and applied manner.

Genome editing applications in agriculture

Genome editing is used to improve the agronomics of crops and livestock by creating value-added traits such as herbicide tolerance, disease resistance, enhanced flavor and food/feed quality, and lack of horns in cattle. Genome editing has been successfully conducted in over fifty different plants and many animals. Commercially available products include: High Oleic Soybean Oil Calyno™ developed by Calyxt produced with a TALEN system, Cibus’ herbicide tolerant canola via Oligonucleotide Directed Mutagenesis (ODM), and DuPont’s waxy corn generated through a gene deletion with CRISPR-Cas9. The technology has been applied in medicine to provide more accurate diagnoses and correct undesired genetic defects in animals including human beings. Several companies are working on anticancer therapeutic agents and ways to modify blood disorders such as hemophilia and sickle cell disease and risks of heart disease.

Methods for detection of gene edits

“Gene edits” is a general term that includes simple base changes all the way to complex genome rearrangements. Therefore, the detection of gene edits depends on the type of edit that is intentionally made. Traditional PCR-based detection methods will suffice for simple edits. For larger and more complex gene edits, sequencing is definitely needed to characterize the complete region of an edit or series of edits. However, once new edits have been selected to move forward in a product pipeline, these edits can be tracked through detection of the new chromosomal junctions on both sides of the edit(s) using traditional PCR-based detection methods.

Although transgenic gene products are used to intentionally make gene edits, none of the machinery or the transgenes are incorporated into the host genome. This means that while we can detect gene edits, it is impossible to determine whether these changes are derived from man-made intervention or selected from natural sources.

Digital PCR (dPCR) is a recently developed PCR technology that has revolutionized quantitative PCR. dPCR is a refinement of conventional PCR methods [FAQ1] in which specialized equipment compartmentalizes the PCR reactions into thousands of partitions. The reaction is carried out in each partition such that each theoretically contains a single copy of the target DNA, which then is amplified using either a fluorescent dye hydrolysis probe (TaqMan) or an intercalating double strand binding dye (EvaGreen) assays. After amplification, reaction partitions containing templates produce positive fluorescence and those without templates remain negative (i.e. endpoint PCR). Absolute copy numbers of target DNA can be calculated by applying Poisson statistical analysis to whether amplification has taken place (positive) or not (negative). Poisson statistics is a distribution that expresses the probability of a given number of events occurring in a fixed interval of time or space.

dPCR offers some advantages over qPCR [FAQ1]. qPCR typically uses either a standard curve or calibrator run within the experiment to estimate relative and absolute values. dPCR differs from qPCR, in that amplification efficiency is not a major factor in whether a partition is positive or negative. dPCR does not rely on references or standards and the desired precision can be achieved by adjusting the total number of PCR replicates. dPCR is also more tolerant to PCR inhibitors in a reaction compared to qPCR.

Different types of dPCR and its applications

There are two basic dPCR systems; droplet-based dPCR systems (https://www.youtube.com/watch?v=PC8qgy50klk) and chip-based dPCR systems (https://www.youtube.com/watch?v=Hb81JD3Kho0). Droplet digital PCR (ddPCR) is a type of dPCR that partitions the PCR reaction mix into thousands of droplets. The target DNA is subsequently amplified in each of the droplets, resulting in either positive or negative samples. A flow cytometer is then used to count and record the number of droplets in each category. Other forms of digital PCR utilize partition (or microchambers) chips instead of droplets. The main advantage of these platforms is their ease of use and the ability to automate the system for all steps from sample injection to the analysis. The trade-off between the degree of precision, throughput and the cost of the system and the assay affects the choice of dPCR systems.

Digital PCR is used in a wide range of applications such as rare event mutations, single nucleotide polymorphisms (SNPs), gene expression analysis, copy number variations (CNVs), and pathogen detection where absolute quantification is needed is where this technology is particularly useful. dPCR is becoming widely used in detection and event specific quantification of GM (Genetically Modified) organisms.

Due to the higher cost of consumables and equipment, digital PCR is less often used for higher throughput applications where qPCR can be employed. The balance between detection and quantification of multiple targets from a single reaction (multiplex) coupled with the relative cost of generating the data will determine the wider applicability of dPCR across the world.

Online Resources

AEIC Publications

Wang, R., Zhang, J.,Cheever, M., Geng, T., Herman, R., Pence, M., Schafer, B., Verhalen, B., Vigeolas,H., Wang, Y., Wu, J., Wu, P-Y., Ament, C. 2024. Considerations for safety assessment of intractable proteins expressed in genetically modified crops. (PDF)

Serrano, H., Jiang, X., Ghoshal, D., Wang, Y., Komorek, J. Ahi, L.,Gao, A., Kouba, K., Atkinson, T., Ament, C., Birukou, I., and Cummings, S. 2024. White Paper: Methods to Support Protein Extraction Efficiency from Plant Tissues.. AEIC. (PDF)

Wang, Y.,Bednarcik, M., Ament, C., Cheever, ML., Cummings, S., Geng, T., Gunasekara, DB., Houston, N., Kouba, K., Liu, Z., and Shippar, J. 2024. Immunoassays and Mass Spectrometry for Determination of Protein Concentrations in Genetically Modified Crops. J. Agric. Food Chem. 2024, 72: 8879-8889. (PDF)

Shillito, R., Whitt, S., Ross, M., Ghavami, F., De Vleesschauwer, D., D'Halluin, K., Van Hoecke, A., Meulewaeter, F. 2021. Detection of genome edits in plants--from editing to seed. In Vitro Cellular and Developmental Biology - Plants; (Detection of genome edits in plants—from editing to seed (springer.com)

Wang, R., Houston, N., Cheever, M., Geng, T., Gillikin, N., McDonald, J., Serrano, H., Shippar, J., Tetteh, A., Wang, Y., Liu, Z. 2020. Can mass spectrometry analysis of in vitro digestion products improve the assessment of allergenic potential of a newly expressed protein? J. Regulatory Science 9(1) (2020): 76-83.

Alarcon, C., Shan, G., Layton, D.T., Bell, T.A., Whipkey, S. and Shillito, R.D. 2019. Application of DNA- and protein-based detection methods in agricultural biotechnology. J. Agric. Food Chem. DOI: 10.1021/acs.jafc.8b05157.

Breeze, M.L., Leyva-Guerrero, E., Yeaman, G.R., Dudin, Y., Akel, R., Brune, P., Claussen, F., Dharmasri, C., Golbach, J., Guo, R., Maxwell, C., Privalle, L., Rogers, H., Liu, K., Shan, G., Yarnall, M., Thiede, D. and Gillikin, N. 2015. Validation of a method for quantitation of soybean lectin in commercial varieties. J. AOCS 92(8): 1085-1092.

Privalle, LS, Chen, J, Clapper, G, Hunst, P, Spiegelhalter, F and Zhong, C. 2012. Development of an Agricultural Biotechnology Crop Product: Testing from Discovery to Commercialization. J of Agric. and Food Chem. 60(4): 10179-10187 - PDF

Pantella, V. 2007. Testing Oilseeds for GMOs. AOCS 18(3): 154-156.

Grothaus, GD, Bandla, M., Currier, T, Giroux, R, Jenkins, R, Lipp, M, Shan, G, Stave, S and Pantella, V. 2006. Immunoassay as an Analytical Tool in Agricultural Biotechnology. J. AOAC 89(4): 913-928. - PDF

Lipp, M, Shillito, R, Giroux, R, Spiegelhalter, F, Charlton, S, Pinero, D and Song, P. 2005. Polymerase Chain Reaction Technology as Analytical Tool in Agricultural Biotechnology. J. AOAC 88(1): 136-155 - PDF

Lipton, CR, Dautlick, JX, Grothaus, GD, Hunst, PL, Magin, KM, Mihaliak, CA, Rubio, FM and Stave, JW. 2000. Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients - PDF

Reeves, SG, Berberich, S, Dautlick, J and Mihaliak, C. 1996. The Analytical Environmental Immunochemical Consortium. Food Agric. Immunol. 8: 137-139.

Mihaliak, C., Nardone, D and Nugent, P. 1996. Why are Immunochemical Tests Important? Imunochemical Methods Food Testing and Analysis 2: 8-9.

Mihaliak, CA and Berberich, SA. 1994. Guidelines to the Validation and Use of Immunochemical Methods for Generating Data in Support of Pesticide Registration. In: Immunoanalysis of Agrochemicals: Emerging Technologies. Eds. Nelson, JO, Karu, AE and Wong, RB. ACS Symposium Series 586: 288-300.

A USER'S GUIDE TO ENVIRONMENTAL IMMUNOCHEMICAL ANALYSIS - An excellent resource put together by the Environmental Protection Agency, Environmental Sciences Division AEIC Technical Comments Regarding Publication on "Performance Assessment of Rapid Immunological Test" - PDF

Education

Education is a top priority in gaining broader understanding and acceptance of the application of immunoassay and nucleic acid-based detection methods to agricultural and environmental analytical applications. Thus, on behalf of AEIC, individuals from member organizations make numerous presentations on educational, technical and regulatory subjects at regional, national and international meetings. Workshops and training sessions have been designed, developed and presented to state and federal regulatory agencies. Members of AEIC also participate in training workshops sponsored by American Association of Cereal Chemists International (AACCI) which are held to train scientists from various geographies on ELISA and PCR methods for agricultural biotechnology. The AEIC Meetings (Spring and Fall) also provide members information on new detection technologies and challenges for detection methods via talks and discussions sessions.

The following slide decks were created by AEIC and are historical. These slide decks are in the process of being updated.

Methods for Detecting and Measuring Ag Biotech Products -

Full Presentation	PDF	PPT
Introduction	PDF	PPT
Applications	PDF	PPT
Intro to PCR (2023)	PDF	PPT
Immunoassays	PDF	PPT
Validation	PDF	PPT

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