United States

Seafood substitution poses a significant threat to the global seafood market, driven by increasing consumer demand and fraudulent practices like species mislabeling. Among seafoods, crustaceans are valuable commodities and are particularly vulnerable to substitutionary activities due to their morphological similarities. This study focuses on developing rapid and accurate identification methods to combat seafood fraud. To address this, we developed species-specific C-LAMP and mPCR-LFD assays targeting the COI gene of three mussel species (Perna canaliculus, Mytilus galloprovincialis, and Perna viridis) and three crab species (Portunus pelagicus, Portunus sanguinolentus, and Charybdis feriata). The C-LAMP and mPCR-LFD assay conditions were optimized by varying the temperature and time. The C-LAMP assay demonstrated high accuracy at 63°C for 30 mins. The C-LAMP exhibited high sensitivity for P. viridis and P. canaliculus, with limits of detection of 0.003 and 0.01 ng/reaction, respectively. Similarly, the mPCR-LFD assay achieved high sensitivity, detecting DNA concentrations as low as 0.1 ng, 0.006 ng, and 0.01 ng for blue swimming crab, three spotted crab, and crucifix crab, respectively. Further, in-house validation using various processing methods (boiling, steaming, frying, and canning) using the C-LAMP and mPCR-LFD assay demonstrated high sensitivity and robustness. Out-house validation of crab species revealed a 28% mislabeling rate. Therefore, the C-LAMP and mPCR-LFD assays developed in this study are simple, rapid, and cost-effective for authenticating seafood products. Hence, this novel technology offers a promising solution for routine fishery examinations to stakeholders, industry people, and regulatory authorities, which contributes to seafood safety and authenticity. 

2025 AOAC INTERNATIONAL Annual Meeting &amp; Exposition

C-LAMP and mPCR – LFD assay: A novel approach for combatting seafood fraud in crustaceans

#### The AOAC INTERNATIONAL Annual Meeting: A Unique Analytical Science Opportunity

The AOAC Annual Meeting & Exposition provides unparalleled professional development, networking, and collaboration in methods-based science. 

* **Businesses**: Meet scientific and regulatory experts and engage with new trends and standards.

* **Scientists**: Build professional expertise and network with your community to share information and best practices.

* **Regulators**: Leverage unprecedented opportunity for stakeholder collaboration on complex testing and analysis challenges, helping improve compliance and public safety.

* **Academia**: Engage with colleagues – both students and faculty – at all career and research levels.


#### Keep Pace with Innovation
* Take advantage of emerging methods, best practices, and trending topics like food fraud and cannabis purity and potency. 
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This multi-faceted “idea incubator” focuses on regulatory changes and emerging food safety issues. A plenary and two breakout sessions feature thought-provoking ideas to identify and meet analytical needs.

The 2025 AOAC INTERNATIONAL Annual Meeting & Exposition was held in person in San Diego, USA.

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2025 AOAC INTERNATIONAL Annual Meeting & Exposition

The AOAC Annual Meeting & Exposition provides unparalleled professional development, networking, and collaboration in methods-based science.

Molecular confirmation methods have revolutionized microbiological testing, offering speed, sensitivity, and specificity in pathogen and spoilage organism detection. Techniques such as PCR and whole genome sequencing enable rapid identification, bypassing traditional culture-based approaches. However, while these methods enhance detection capabilities, they also introduce challenges in colony confirmation workflows.
One key advantage of molecular confirmation is its ability to detect viable but non-culturable (VBNC) organisms, providing a more accurate assessment of contamination risks. It reduces time to results, allowing for quicker decision-making in food safety interventions. Additionally, molecular methods can differentiate closely related species or serotypes that may be indistinguishable through biochemical or phenotypic tests.
Conversely, molecular approaches present limitations. PCR and sequencing may detect DNA from dead cells, leading to potential false positives. The lack of direct phenotypic confirmation can also create discrepancies between molecular and culture-based results, challenging data interpretation. Moreover, reliance on molecular techniques necessitates specialized training, higher costs, and stringent contamination controls to prevent cross-reactivity or false amplification.
For users, balancing molecular confirmation with traditional colony confirmation is essential. Integrating both approaches provides a comprehensive assessment, ensuring that molecular positives are verifiable through culturing when required for regulatory or investigative purposes. As industry and regulatory bodies evolve, understanding the strengths and weaknesses of each method is critical for developing robust, actionable microbiological testing strategies. This presentation will explore practical considerations, method selection criteria, and real-world applications to optimize confirmation workflows in food microbiology.

The Pros and Cons of Molecular Confirmation and its Impact on Colony Confirmation: A User's Perspective

Traditional Salmonella detection methods, including ISO 6579-1:2017 and ISO/TR 6579-3:2014, rely on enrichment and culture-based workflows that require 3–14 days to generate results. These conventional methods may not detect viable but non-culturable (VBNC) cells. Commercial PCR kits, while faster, often target highly variable virulence genes, leading to potential false negatives when applied to genetically diverse regional strains.
This study presents a custom TaqMan real-time PCR assay specifically designed for Salmonella strains isolated across Indonesia. Primers and probes were developed to target genetically stable regions: the AHS-like conserved domain and iclR gene for Salmonella spp., and a Helix-Turn-Helix domain-containing protein for S. Enteritidis. A rapid DNA extraction protocol using lysis buffer and PBS wash was employed, eliminating the need for column purification and reducing sample preparation time.
The assay was evaluated using 20 local Salmonella strains obtained from various food matrices, including egg, meat, dairy, fish, and processed products. While five strains were undetectable by commercial PCR kits, the custom assay accurately detected all 20. It demonstrated a limit of detection of 0.0001 ng/μL and produced results in less than 24 hours. The assay enables subspecies-level discrimination through primer design and includes internal controls for quality assurance.
By targeting conserved genomic regions and avoiding culture dependency, this method enables the detection of VBNC strains and offers enhanced sensitivity, speed, and inclusivity. It represents a practical molecular tool for food safety surveillance, regulatory screening, and trade assurance, particularly in regions with unique pathogen profiles.

Custom TaqMan PCR for Indigenous Salmonella Strains: A Rapid, Sensitive, and Robust Alternative to Conventional and Commercial Assays

As molecular methods like PCR continue to reshape microbiological testing, the preparation of cannabis samples for molecular quantification of tragets like total yeast and mold (TYM) and pathogens is critical to ensuring accurate, reliable results. This session will explore best practices for sample preparation, including homogenization techniques, enrichment strategies, and extraction methods that optimize sensitivity and specificity. We will discuss challenges unique to cannabis matrices, such as sample inhibition and variability, and how proper preparation can enhance detection while maintaining regulatory compliance. Attendees will gain insights into refining molecular workflows to improve both efficiency and data integrity in cannabis microbiology testing.

Looking to the Future: Molecular Approaches for Cannabis Microbiology—Preparing Samples for Accurate Detection

Current state-of-the-art methods require multiple procedures, which are expensive, complex, and time-intensive. Additionally, many laboratories lack the necessary infrastructure to perform comprehensive analyses in-house, relying instead on third-party service providers. We have developed MLSTnext technology, a single-tube-PCR, with simple workflow for microbial pathogens, capable of serotyping, subtyping, surveillance, identifying multiple serotypes as well as an integrating data analysis software without the need for bioinformatics expertise. MLSTnext has been applied to a wide range of food safety applications such as Salmonella, Listeria, Legionella, HAV, STEC/E. coli, Campylobacter, Cyclospora and more. All assays have generated high-resolution results, accurately serotyping to the subtype level. For instance, the Salmonella assay distinguished between two S. typhimurium strains by identifying sequence variations in four loci, revealing the same serotype but different strains. The same assay demonstrated the ability to detect the co-presence of multiple serotypes of Salmonella or Listeria (up to nine serotypes), within a single sample. Furthermore, the unique loci profile enabled effective traceability for contamination surveillance and outbreak management. MLSTnext is versatile and the entire workflow takes 10-24 hours (depending on the sequencing platform) without the need for pure colonies/isolates.

MLSTnext: High-Resolution Bacterial, Parasitic, and Viral Genotyping, Subtyping, and Surveillance in a Single PCR-Ready-to-Sequence Format

Probiotics are among the most widely used specialty supplements in the United States according to a recent consumer survey conducted by the Council for Responsible Nutrition.  A continued increase in probiotic usage is expected as companies develop probiotic strains for uses beyond gut health to include sport nutrition, upper respiratory infections, and sleep.  Like many natural products used in dietary supplements, probiotics are presumed to be safe.  This presumption of safety is not completely unfounded as there is global consumption of common probiotics and a lack of reported serious adverse events.  However, as probiotic manufacturers are increasingly seeking to use new strains, species, or even novel probiotics (human commensals but not currently found in the food supply), justification based on a significant history of use may be challenged.  Furthermore, the availability of safety and efficacy data supporting the widespread use of probiotics has been questioned by some healthcare professionals.  Additional criticisms have been directed towards the dietary supplement industry for circumventing FDA review of new dietary supplement products by utilizing the self-GRAS approach. There are efforts underway by a variety of stakeholders including global pharmacopeia and various probiotic and dietary supplement trade associations to develop best practices guidelines for assessing the quality and safety of probiotics.  This presentation will focus on efforts underway to define safety standards for probiotics and justification for continued self-regulation by industry through a self-GRAS path.

An Industry Perspective on Assessing Safety of Probiotic Dietary Supplements

Consumer awareness of probiotics and their connection to overall health is driving significant growth in global probiotics markets. Projections estimate a market size of USD 105.7 billion by 2029. In this evolving landscape, probiotics have been integrated into both human and animal diets and have transitioned from being primarily powder, tablet, and capsule supplements to being incorporated into a wide array of foods. Regardless of product format, probiotics manufacturers follow current good manufacturing practices (cGMP) to ensure quality and safety. However, these guidelines do not contain the specifics necessary to meet the unique challenges of working with live microorganisms. One challenge that the industry routinely encounters is the quantification of viable probiotic microorganisms. These counts impact the efficacy, safety, and quality of products. Managing, maintaining, and improving quantification is essential to manufacturing consistent ingredients and finished products.
Analytical procedure lifecycle management (APLM) provides flexible solutions for quantification challenges. The approach is based on the alignment of data uncertainties and an organization's tolerance for risk. APLM can be used to transparently manage an analytical procedure or method from selection, fit-for-purpose validation/verification, and performance monitoring via control charting, through changes and improvements made to the procedure, ending with termination of use. This presentation will provide an overview of APLM and give examples of how the information and data gathered through its application will help those tasked with managing, maintaining, and improving the quality of analytical procedures, quantification data, and probiotic products.

A Solution for Managing, Maintaining, and Improving the Quality of Probiotic Products: Analytical Procedure Lifecycle Management (APLM)

In a rapidly growing probiotic market, it is essential for probiotic products to meet their label claims of strain content and viable cell counts throughout shelf life to ensure efficacy. DNA based methods are the most commonly used methods for probiotic identification, and plate count methods are the gold standard methods for enumeration. Probiotic health benefits are strain specific. Thus, it would be ideal to use methods that can identify and quantify probiotics at the strain level such as real-time PCR (qPCR) methods. However, developing strain specific qPCR methods can be challenging especially when the target strain belongs to a highly isogenic taxon. Additionally, these methods should be developed and validated according to the validation guidelines including inclusivity, exclusivity, sensitivity, efficiency and accuracy, to ensure reliability of the methods. Here, we describe the process of developing and validating strain specific qPCR methods for probiotic identification and enumeration. Method performance including specificity, efficiency, accuracy and applicability to different product types and product matrices are evaluated. The ability of the enumeration methods to assess strain viability in multi-strain finished products throughout shelf life, and the accuracy of the enumeration methods to estimate the ratios of the target strain in experimental mixtures are assessed. The strain specific qPCR methods demonstrated high specificity, sensitivity, accuracy and applicability. The methods were able to assess strain viability in multi-strain finished products as well as to accurately estimate the ratios of the target strain in experimental mixtures, offering reliable and efficient tools for probiotic product authentication.

Redefining Probiotic Quality: Strain-Specific qPCR Solutions for Accurate Identification, Enumeration, and Viability Assessment

AOAC 2017.16, for the measurement of total dietary fiber, in alignment with CODEX definitions, achieved final action status in 2020, and replaced AOAC 2009.01 as a CODEX Type 1 method in 2021.  AOAC 2022.01, for the measurement of soluble, insoluble and total dietary fiber is following a similar track with AOAC final action status obtained in 2024 and it is expected to replace AOAC 2011.25 as a CODEX Type 1 method this year. As these methods are implemented across the industry, new insights into analytical challenges continue to emerge. This poster highlights chromatography-related insights , particularly the performance of recommended columns (Waters SugarPak, AOAC 2009.01; Tosoh TSKgel, AOAC 2001.03/2017.16/2022.01) alongside alternative columns sourced globally. A comprehensive carbohydrate dataset was analysed across these columns to evaluate their performance. The comparative chromatographic performance results will be outlined herein.
Additionally, regulatory developments in pet food labelling are driving a transition from crude fiber to dietary fiber analysis, as signalled by the Association of American Feed Control Officials (AAFCO). By 2029, pet food labelling will require dietary fiber determination by approved analytical methods, necessitating a major shift in analytical approaches. To support this transition, we generated relevant data using these methods, assessing their applicability to pet food matrices, the results of which will also be described.

Chromatographic Considerations and Emerging Applications in Dietary Fiber Analysis

The intensive use of pesticides in agriculture has led to the need for rigorous and extensive use of analytical technologies to ensure that there is no impact on human populations.  Maximum residue limits (MRL) are set for regulated residues that define the highest level of a pesticide or mycotoxin residue that is legally tolerated in food such that it is safe for consumers. Often these MRLs are set at low ppb level, to ensure highest safety, very sensitive, robust instrumentation and fast workflows are require quantify these compounds down to their MRL. 
When performing a method with such a vast number of compounds it is important to ensure that the quality of the data is not compromised and that every compound can be effectively quantified. Methods show very high sensitivity to meet different regulations and guidelines. For an assay with over 1500 MRM transitions to analyze, time scheduling of MRMs and instrument switching speed are keys to maintain high quantitative quality. Fast polarity switching time of the instrument ensures good data sampling rates are obtained across the LC peaks for different retention times. Accurate, robust fast quantitation results are presented align with SANTE guideline. Multiple MRMs per analyte also enabled ion ratio monitoring to ensure confident detection. Long batch of several days sample measurements with the representative matrices was run to demonstrate robustness of the method. Improved polarity switching speed allowed for improved quantitation quality for early eluting, polar pesticides. 
 

Ultra-fast MRM acquisition and quantitation of food contaminants in multiple food matrices

Solid Fat Content (SFC) analysis is a cornerstone in the characterization and quality control of fats, fat blends, and finished food products. Understanding the melting behavior of fats is essential for optimizing texture, stability, and processing performance, as well as for timely quality control decision-making. Time-Domain Nuclear Magnetic Resonance (TD-NMR) has emerged as the internationally recognized standard for SFC determination (ISO / IUPAC / AOCS), offering rapid, non-destructive, and solvent-free analysis.
Different SFC workflows based on TD-NMR benchtop technology will be highlighted — including direct and indirect modes for non-invasive determination of solid fat fractions, and parallel and serial tempering techniques for workflow optimization. SFC versus temperature curves obtained from samples of pure and oil-mixed cocoa butter, as well as a fat composite sample are presented. Fully automated SFC workflows, from sample tempering until readings through real time reporting, are highlighted to ensure the strictest compliance with all established international standards, as well as to maximize throughput and results reliability.
Finally, the Dynamic Fat Crystallization (DFC) method, will be discussed as a fundamental technique for the development of new fat substitutes, allowing real time tracking of crystallization behaviors, providing a detailed timeline of initial, main, and final crystallization phases.  Both SFC and DFC techniques empower food manufacturers to ensure consistent product quality, optimize processing conditions, and accelerate R&D cycles. A comparative between DFC curves obtained from samples of cocoa butter and a cocoa butter substitute (palm mid-fraction) are shown.

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The Pros and Cons of Molecular Confirmation and its Impact on Colony Confirmation: A User's Perspective

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