Technical Tools and Guidance for the Detection and Identification of LMOs

Minimum performance criteria for sample handling, extraction, detection and identification methodology

By: Natalhie Campos Reales Pineda, and Mojca Milavec

An important aspect when building up capacity for the detection, identification and quantification of living modified organisms (LMOs) is to apply minimal standard criteria to ensure the adequate handling and processing of samples, as well as the quality and confidence in the results obtained. A wide variety of methodologies and instruments can be put in place by a molecular biology laboratory that wishes to undertake activities pertaining to the detection and identification of LMOs. Deciding which of the available methods is sufficiently reliable, accurate and more suitable to each laboratory’s needs, including considerations such as costs of implementation, can sometimes be challenging. In addition, ensuring that the selected methods produce reliable and consistent results, while, at the same time, meeting minimum performance criteria, is essential.

Minimal performance criteria represent an advantageous set of parameters for selecting and implementing routine methodologies within each laboratory. Inconsistencies in results can have a major impact on the reliability of data produced. For example, the use of different measurement platforms, whether for the analysis of protein or nucleic acids involving the use of different instruments, or different algorithms to interpret the data, can cause serious discrepancy if no basic quality and performance criteria are established upfront. Understanding the relevance of minimal performance criteria, and monitoring some of the method’s parameters on a routine basis, allows the lab-facility to establish a quality assurance and quality control system (QA/QC) and standard operating procedures (SOP) that can be used later on for corroborating laboratory proficiency. Additionally, when installing performance quality control measures, the procedure facilitates compliance with the requests from accreditation bodies. To ensure consistency, it is important that the analyte intended to be measured is first defined and that the units in which the measurement is expressed are common standard as well as ensuring the use of common vocabulary for better understanding. Use of certified reference materials is fundamental for protocol harmonization between laboratories and method validation. This is of particular interest when it becomes necessary to allow for result comparison and harmonized interpretation.

International guidance and useful standards about method performance criteria are well documented and available from several sources. These documents include ISO guidelines, Codex Alimentarius standards, guidelines from international associations (ISTA, AOAC), metrology institutions, as well as official and peer reviewed references regarding proficiency trials for method validation procedures. The following is an overview of some topics on requirements for minimal performance criteria that are needed in a laboratory performing detection and identification of LMOs:

A. GENERAL PERFORMANCE CRITERIA

The performance of the selected methods is crucial for the production of reliable and reproducible data. This encompasses measuring, amongst other things, a method’s robustness, precision, trueness, sensitivity and specificity and this can only be achieved in a well-organized laboratory. As such one of the most important precautions in an LMO testing laboratory is to prevent cross-contamination of the samples and organize the unidirectional flow of the sample. Separate rooms or chambers for each testing step should be assured wherever possible. This includes a separate area for the receiving samples, sample preparation including homogenization, analyte extraction, reagent preparation, addition of the analyte to the reagents, and analysis. A number of quality control measures can be applied during the testing procedure, including the use of (certified) reference materials, control samples and duplicates to monitor the performance of a method and ensure the validity of analytical results. All steps in the testing procedure, from the first contact with the customer to issuing the test report should be documented in a traceable manner while ensuring data protection and confidentiality.

In working towards achieving these criteria, it is crucial to have competent and trained personnel to operate equipment and perform the technical steps of the testing procedure. Managerial and technical tasks and their associated responsible personnel should be clearly defined, including substitutes in the case the primary contact is absent. All personnel should be continuously educated and trained.

Understanding these parameters and being able to detect any deviations from set criteria during routine testing ensures that results are consistent and remain within acceptable ranges of variability.

B. PERFORMANCE CRITERIA FOR THE DETECTION AND IDENTIFICATION OF LMOs

Procedures can be regarded as the complete process from sampling to detection and analysis for data interpretation, or be approached in a modular manner. While method validation has relied on considering the process as a whole for determining the global uncertainty, a current trend to adopt a modular approach allows for setting step-wise criteria that permit corrective actions before proceeding to the next step if criteria are not met. Decisions over which of these approaches is to be taken into the lab depends on utility and particularities of the type of sample being analysed (e.g. food matrices, commodities, vegetative tissue, environmental, etc.). For facility, the following section describes procedures using a modular approach. The procedures include laboratory-related issues related to sample handling and homogenization and extraction for LMO detection. These steps are extremely important in the detection and identification of LMOs as significant analytical errors can be introduced at these stages. This section does not involve a description of sampling, which is an extremely important step of analytical procedure, but it is beyond the scope of this document.

1. Sample handling The laboratory sample is the portion of material to be used in the laboratory for the analyses. Sometimes mass reduction (sub-sampling) has to be done and the procedure has to be documented. The vast majority of samples must be homogenized prior to analysis. In general the whole laboratory sample is homogenized to obtain a test sample for the analysis. Homogenization is required for two reasons: to achieve sufficient efficiency of analyte extraction and, above all, to ensure homogeneity and an equal representation of LMO-derived particles in the test samples. The homogenization of samples may be achieved with mills, homogenizers, immersion blenders, coffee grinders, or a suitable equivalent device, depending on the size and the structure of the laboratory sample. After grinding, the laboratory sample should be thoroughly mixed to obtain a very homogeneous analytical sample. In cases where the laboratory sample is flour or liquid, homogenization is not needed but thoroughly mixing or shaking the sample is necessary. Homogenization is the step with the highest contamination risk. During grinding, a fine dust often appears that could contaminate subsequent laboratory samples, if appropriate laboratory practices are not in place. All the steps for sample preparation should be done under stringent conditions to avoid cross-contamination and minimize degradation of target analyte, be it DNA or proteins, in the test sample. Storage of the samples prior to and during sample preparation, as well as after its analysis should be performed under appropriate conditions (e.g. at room temperature, refrigerated, frozen).

2. Extraction Analyte extraction implies that the processed amount of sample remains representative. The choice of extraction methods must ensure high yield and quality of the analyte, and must be carefully selected, since components of the extraction solutions can affect the efficiency of subsequent detection steps. The principal criteria for quality which should be taken into account are the analyte’s integrity, quantity and the absence of inhibitors.

3. Validated methods of analysis As mentioned in previous sections of this Guidance, there are several types of analytical methods that target different analytes and use a wide variety of chemistries and strategies. For instance, for DNA based molecular methods, detection may involve the amplification of a GM event or its genetic markers by simple PCR reaction, hydrolytic probes, isothermal amplification or high resolution melting strategies amongst others either in single reaction, arrays or in a multiplex. The laboratory may select from this wide array of test methods based on, among other things, each country’s specific regulatory requirements, operational costs, infrastructure and technical capability as well as ensuring that the needs of the customer are met. The methods need to have been validated. The goal of the validation is to evaluate the performance characteristics and limitations of an analytical method. The validation process takes place either within each laboratory or, ideally, through inter-laboratory comparisons to ensure that they meet minimum performance criteria and that they provide accurate results that are, preferably, published in international, regional, or national standards. Specifically, during a validation process, laboratories participate in the study which is organized in accordance with internationally accepted requirements, such as the format described in ISO5725. Some of the parameters reported during the validation process are used to determine the acceptability of the method and include parameters such as applicability, specificity, dynamic range, robustness, limits of quantification, limits of detection and efficiency, amongst others. These elements also allow for measuring the method’s level of uncertainties, determining its overall performance, for which, precision and trueness complement the set of measured parameters. It is important to mention that the method used is validated with appropriate reference samples, which have been critically evaluated and a proper value assigned by metrologically sound procedures, which is normally available through National Metrology Institutes (NMI) or Designated Institutes (DI).

C. PROFICIENCY TESTING AND VALIDATION

Participation in proficiency testing schemes is crucial for the independent assessment of laboratory performance. Participation in proficiency tests depends on the methods introduced in the laboratory and the type of samples analyzed. Laboratories can use many methods for testing however not all of them can be assessed as each proficiency test covers only a limited number of LMOs. Therefore it is recommended to prepare a plan for the participation in proficiency tests and to assess individual methods periodically. It is also important to cover different types of matrices that are subject to testing during routine analyses.