A Protocol for organisation and operation of PCR laboratories

(Prof. Anna Maraz, Corvinus University Budapest, Hungary)

Introduction Traditional microbiological methods for the isolation and identification of the targeted (mostly pathogenic) microorganisms from foods depend on obtaining pure cultures. Enrichment and selection steps are often time-consuming and biochemical identification of particular species may add several days to the procedure. For ensuring the safety of consumers, the rapid detection of pathogens and other microbial contaminants in food is critical. One of the more and more widely used techniques is the Polymerase Chain Reaction (PCR). Beside several advantages, this biochemical reaction has a strong cross contamination risk. Therefore, out of the general microbiology laboratory requirements, management of a PCR laboratory has to meet special requirements. Several deliverables worked out within WP15 which provide relevant information for the companies helping in the decision making process for implementing microbiological analysis of food for different purposes. List of the relevant deliverables: D15.16: Overview of traditional methods as compared to new PCR-based techniques from FSMS verification perspective. D.15.18: Overview of laboratory management requirements (ISO) for new PCRbased techniques D.15.23: MAS analysis method selection tool. D15.24: Validation protocol to support FSMS D15.25: Verification protocol to support FSMS The present protocol is based mainly on D.15.18: Overview of laboratory management requirements (ISO) for new PCR-based techniques but an additional aim of this document is to outline the process and the required condition for establishing and operating molecular laboratories for food microbiological analytical purposes. Aim of protocol: The aim of this protocol is to provide aids for the microbiological testing laboratories for upgrading their capabilities into the application of molecular based techniques beside or instead of the conventional (culture-based) techniques in food microbiological analysis. 1. General requirements for microbiological testing laboratories: The general requirements for the competence to carry out tests in laboratories are specified in ISO/IEC 17025:1999 standard. It covers testing performed using standard methods, non-standard methods, and laboratory-developed methods. It is important to note that this standard incorporates all those requirements of ISO 9001 that are relevant to the scope of testing and calibration services that are covered by the laboratorys quality system. In this way testing laboratories that comply with this ISO standard also operate in accordance with ISO 9001. The ISO 17025 standard covers the management requirements of testing laboratories including organization, quality system, document control, review of

requests, tenders and contracts, subcontracting questions, purchasing services and supplies, services, control of nonconforming testing work, corrective action, preventive action, control of records and internal audits and management reviews as well. The internal quality control (IQC) is advisable not only to food control laboratories, but to other food laboratories as well, in order to ensure that the data produced in the laboratory are of known quality and certainty. The IQC procedure includes the assessment of a laboratory according to international accreditation standards, participating also in proficiency testing schemes (Wood and Corry, 2003). Technical requirements according to ISO 17025 standard involve the following: personnel, accommodation and environmental conditions, methods and method validation, equipment, measurement traceability, sampling procedures, handling and assuring the quality of test and calibration items and reporting requirements. The laboratory has to ensure the competence of all personnel operating equipments, perform tests, evaluate results and sign test reports and calibration certificates. The test or calibration laboratory shell use appropriate methods which meet the needs of the client. The selected method preferably should be published in international, regional or national standard. When using non-standard, laboratory-designed or developed methods and standard methods used outside their intended scope, the laboratory shall validate them as being fit for purpose before use. The validation can be performed by following the relevant ISO, CEN (European Committee for Standardisation), AOACI (Association of Analytical Communities International) or AFNOR (Association Francaise de Normalisation) standard procedures. PCR-based methods have to be validated according to the EN ISO 1640:2003. For performance testing of a given method one of, or a combination of, the following should be used: a) calibration using reference standards or reference materials; b) comparison of results achieved with other methods; c) interlaboratory comparisons; d) systematic assessment of the factors influencing the result; e) assessment of the uncertainty of the results based on scientific understanding of the theoretical principles of the method and practical experience (ISO 17025). As it is also recommended by the ISO 17025 standard, laboratories wishing to demonstrate the reliability of the data they are producing, should participate in proficiency testing schemes. These schemes are defined as a system for objectively checking laboratory results by an external agency. The internationally recognised protocol to which proficiency testing schemes should comply is the IUPAC/AOAC/ISO Harmonised Protocol. All the proficiency testing schemes are based on the regular circulation of homogeneous samples by a co-ordinator, analysis of samples and an assessment of the results (Wood and Corry, 2003). 2. Infrastuctural needs for PCR laboratories: One of the most important characteristics of the PCR-based techniques is their exquisite sensitivity. However, this high sensitivity renders it prone to false positive results because it is highly susceptible to contamination from its own products (i.e. the PCR amplicon itself). During the whole process it has to be guaranteed that contamination from the environment would not occur and

influence the results. For that reason it shell be ensured that separate work areas with their own equipments, devices and reagents are available. The main source of contamination is the feedback of amplicons generated during the previous PCR reactions. Therefore by separating the area of activities with PCR reactions (post-PCR) from the previous activities (pre-PCR) the potential for contamination is significantly reduced. The most appropriate condition if there are separate rooms where these activities occur. Miffin (2007) outlines a possible separation of the pre-PCR and post-PCR laboratories in a way that the forward flow concept is implemented. Moreover, different air pressures inside the two laboratories decrease the risk of cross contamination. In the sample preparation room the pressure should be higher, while in the post PCR room it should be slightly reduced. It is ideal if each separated area has its own air supplier. For further reduction of the risk of contamination it is recommended to maintain the doors closed in all rooms of the PCR laboratory. Besides these requirements it is important to supply the separated PCR laboratory areas with equipments, devices and reagents which are used only in the allotted room. Such kind of organisation system is illustrated in Fig. 1.

Fig. 1. Organization of a PCR laboratory with separate pre- and post-PCR rooms. (Adopted from Miffin, 2007) For the prevention or reduction of potential contamination during the PCR detection of special nucleic acid sequences the unidirectional workflow must be applied in the molecular laboratory. It means that during the different work phases the steps of the analysis have to pass from the clean (pre-PCR) to the dirty (post-PCR) areas (referred to as forward flow). The theory of forward flow helps the food examining laboratory to avoid the contamination of PCR reaction mixture by previously amplified target sequence.

The closed-tube system represent an additional safe mode against crosscontamination. In this case the reaction tubes are not opened after the PCR processing, thus reducing the risk of contamination of the molecular laboratory by the amplicons; moreover elimination of laborious post-PCR sample processing enables high-throughput analysis. Real Time PCR representing a closed tube system, therefore it is less sensitive for the cross-contamination than the conventional PCR (D 15.16. gives details about the main differences between conventional and Real Time PCR). The ISO 22174:2005 summarises the requirements for PCR-based molecular techniques used for the detection of microorganisms in food samples. For the organisation of a PCR laboratory and for the sample handling the standard recommends the forward flow principle and the systematic containment of the methodological steps involved in the production of the results. By keeping these measures it is ensured that the DNA from the test sample and the amplified PCR product remain physically separated during the detection procedure. For this purpose it is recommended that minimum four distinct areas with their own working facilities should be separated. a) The first area should be a laboratory for nucleic acid preparation from the test material. b) The second area should be the work area for master mix preparation, where all the reaction components necessary for the PCR amplification (except nucleic acid) are mixed together. c) The third area serves for the addition of the separated nucleic acid to the reaction mixture d) The fourth area is for the detection and confirmation of PCR products. The PCR thermocycler can be placed in the c) (third) or in the d) (forth) work area, and so the amplification step has been separated from the nucleic acid extraction and from the master mix preparation. The ideal situation is if all the four work areas are separated physically as distinct rooms and the pre-PCR and post-PCR areas have slightly increased and decreased air pressures, respectively. i. Sample preparation The analysis of a food matrix starts with the sample preparation. The ISO 20837:2006 document provides criteria for producing samples which are compatible with PCR and for separation of nucleic acid suitable for PCR analysis. This area has to have its own devices which should not leave the room. In sample preparation area positive-displacement pipettes or pipettors with aerosolresistant tips, disposable, powder-free gloves and laboratory coats assigned to that room are suggested to use. Biosafety cabinet (for pathogens), refrigerator, freezer and dry heat block/water bath are needed to be placed in this area. Reagents and solid items destined for the sample preparation room have to be autoclaved separately from the other reagents and materials. ii. Preparation of the reaction mixture For preparation of reaction mixture which contains all the necessary reagents for the nucleic acid amplification filter tips, micropipettes, positivedisplacement pipettes or pipettors, amplification reagent, appropriate supplies, PCR-cabinet, glows and dedicated laboratory coats are needed. The reagents have to be kept in fridge or in freezer, thus these apparatuses are also important equipments of

this area. By applying a PCR-cabinet (PCR workstation) equipped with UV irradiation it is possible to have a safe, nucleic acid-free environment which minimises the potential for PCR reaction contamination, since the UV light destroys the contaminating DNA inside the cabinet. iii. Addition of nucleic acid to the reaction mixture For this step a clean area is recommended. If there is no place in the molecular laboratory for separating a dedicated room for nucleic acid addition, this procedure can be made in the area of sample preparation. It is essential that handling of post-PCR materials is not allowed in this part of the room. The amplification is preferably carried out in this room, or if it is not possible (no place is available) it can be done in the area of detection and confirmation of PCR amplified nucleic acid. iv. Detection and confirmation of PCR amplified nucleic acid The PCR thermocycler has to be placed in an area where only PCR products are going to be handled. The ISO 20838:2006 document defines the general requirements for the specific amplification of target nucleic acid sequences and describes the way of detection and confirmation of the amplified nucleic acid sequence. This ISO standard helps the food analytical laboratories for getting comparable and reproducible results. This standard concerns not only to the detection of pathogenic microorganisms from food and feed origin, but of pathogens from environmental samples or to the detection of other investigated microorganisms. The list below represents the most important devices and equipments for molecular biological work in a food analyzing laboratory: a) Sample preparation - stomacher for homogenisation - vortex - refrigerator - thermostats - gloves - laboratory coat b) Nucleic acid extraction - positive displacement pipettes or pipettors with aerosol-resistant tips - refrigerator - freezer - water bath / dry heat block - laminar flow biosafety cabinet - micro-centrifuge - vortex - equipment for determination of nucleic acid concentration - equipment for preparation of Milli-Q water - gloves - laboratory coat c) Preparation of the reaction mixture - positive displacement pipettes or pipettors with aerosol-resistant tips dedicated to this area - micropipettes dedicated to this area

PCR cabinet (with UV sterilisation) freezer gloves laboratory coats dedicated to this area

d) Addition of the nucleic acid and PCR amplification - positive displacement pipettes or pipettors with aerosol-resistant tips dedicated to this area - dead air box for addition of nucleic acid - micro-centrifuge - freezer - thermal cycler(s) (normal, gradient) - real-time PCR instrument - gloves - laboratory coats e) Detection and confirmation of PCR products - gel electrophoresis equipment - gel imaging system - PC with network connection - hybridisation oven - incubator - refrigerator - freezer - gloves - laboratory coats The PCR equipments require regular maintenance. ISO/TS 20836:2005 gives instructions for the installation, implementation and maintenance of thermal cyclers. PCR controls: The application of PCR controls improves further the reliability of results derived from PCR amplification. For the detection of pathogenic microorganisms from food and animal feeding stuffs the ISO 22174:2005 standard recommends the application of positive process control, negative process control, internal positive control, negative extraction control (extraction blank), positive extraction control, and positive and negative PCR control. The ISO 24276:2006 standard, which concerns to the analysis of genetically modified organisms from foodstuffs suggests the use of a PCR reagent and an environmental control additionally. Table 1 contains the controls to be applied during microbiological analysis of foodstuffs.

Table 1. PCR controls based on ISO 22174:2005 and ISO 24276:2006

No. 1. Name of the control Positive process control (Positive extraction control) Negative process control Negative extraction control (Extraction blank control) Internal amplification control External amplification control Characteristics of the control Sample spiked with the target organism, which should be treated in the same way as the test sample Target pathogen-free sample of the food matrix which is run together all stages of the analytical process Control carried out through all steps of the DNA extraction procedure in the absence of test sample DNA added to each reaction in a defined amount or copy number which serves as an internal control for amplification Control DNA added to an aliquot of the extracted nucleic acid in a defined amount or copy number which serves as a control for amplification in a separate reaction. Reaction containing the target DNA in a defined amount or copy number. Reaction performed with DNA-free water without any PCR inhibitors Role of the control Demonstration that the nucleic acid extraction procedure has been done in the way that would allow the extraction of the target DNA from the matrix of the sample. Demonstrating the absence of contaminating nucleic acid during the analytical process. Demonstrating the absence of contaminating nucleic acid during the extraction procedure. This control is not necessary when the negative process control is performed. The absence of false negative results can be demonstrated. The absence of false negative results can be demonstrated.

2.

3.

4.

5.

6. 7.

Positive PCR control (DNA target control) Negative PCR control (DNA target control) PCR reagent control (Blank)

8.

9.

Environment control (Premise control)

Contains all of the amplification reagents except template DNA extracted from the test sample. Instead of template DNA a corresponding volume of nucleic acid free water is added to the reaction A tube containing the master mix left open in the PCR setup room or in other working area to detect possible contaminating DNA in the environment Three to four samples containing serial 10-fold dilutions of a known number of target DNA copies in a range above the detection limit.

By the use of this control the proper acting of the PCR reagents can be demonstrated. The use of this control confirms that the results of analysis of the test samples which do not contain the target sequence are negative. This control is applied to demonstrate the absence of contaminating nucleic acids in the reagents.

10. Standard concentration

It is used to identify the sources of contamination and the contaminated working area. It has to be done in certain intervals as part of the quality assurance program of the laboratory. Control of quantitative PCR reactions

ISO 22174:2005 recommends the application of controls from No.1 to No.7, however, application of two additional controls (No. 8 and No 9) in regular intervals or if any of the regular negative controls does not yield the expected

results. Standard concentration should be applied only in the case of quantitative PCR reactions. Special consideration is needed when the PCR amplified nucleic acid and materials used for the detection and confirmation are disposed. The ISO 22174:2005 standard recommends the application of 3 % (m/m) hypochlorite solution for the demolition of amplification products. The gels which are stained with intercalating dyes (e.g. ethidium-bromide) and used for the detection of PCR products have to be considered as biohazard waste, thus appropriate storage, shipment and destruction is essential in such cases. The EN 12740:1999 standard gives guidance on methods for handling, inactivating and testing of waste containing organisms generated by clinical, molecular biology, microbiology and other laboratories. Waste management should describe and document the measures for the prevention, minimization, segregation, handling, storage, treatment, transportation and final disposal of biohazardous waste from laboratory activities. The management should identify wastes which need different treatment methods, methods for the segregation of biohazardous from non-biohazardous waste, methods for the segregation of other categories of waste. 3. Personnel needs: All personnel who perform aspects of the testing procedures shall be trained to work with PCR and microbiology as appropriate (ISO 22174:2005). 4. Performance characteristics of PCR methods A MAS selection tool has been provided in deliverable 15.23 that can be used by the companies to help in the selection of the most appropriate method of microbial analysis to be used for a specific application. A decision tree based on a techno-managerial concept has been worked out for the method selection and it consists of four main inputs of information to decide upon which method is most appropriate for a defined situation. Categories and sub-categories of the decision tree are coupled with the important characteristics of the method that has to be taken into consideration during the decision making process. Selection of the appropriate microbiological method for food analysis depends on the objective of microbiological analysis, technological and managerial considerations as defined by the decision free. If validated methods should be applied for microbiological analysis molecular techniques have to be validated according to the EN ISO 16140:2003. In food microbiological analysis PCR-based techniques can be used for qualitative purposes (checking for the presence or absence of a specific pathogen in a defined amount of sample) or for quantitative analysis (that refers on the enumeration of the microorganisms in the sample). Till now, however, international standards have been work out only for the qualitative PCR-based techniques regarding the sample preparation, amplification and detection of the PCR product (ISO 20837:2006 and ISO 20838:2006). As the aim of food microbiological analysis for food safety purposes is the detection of pathogens in the sample qualitative detection methods meet these requirements. Most important performance characteristics of qualitative diagnostic microbiological techniques comprise the accuracy (specificity and sensitivity),

detection limit and robustness. Specificity of a PCR-based assay is considerably dependent on the selection of the target gene and the design of appropriate primers and probes. The sensitivity of a PCR test, however, depends not only on the reaction conditions but the matrix of the food sample and the DNA extraction method used have a great influence as well. A high degree of accuracy means that it is able to detect, true and precisely the target microorganism in the presence of a biological (e.g. food) matrix without interference from non-target components. (Relative) selectivity and (relative) specificity of a PCR-based method have to be calculated to evaluate the closeness of agreement between results of the PCR-based method and the accepted reference traditional method. Detection limit for a PCR-based method should be low. It is required by the appropriate international standards to detect as low as one cell per 25 g of food sample by traditional method. Detection level of the PCR-based technique should be close to this value (Malorny et al, 2003). References: Malorny, B., Tassios, P.T., Radstrom, P., Cook, N., Wagner, M., Hoorfar, J. (2003) Standardization of diagnostic PCR for the detection of foodborne pathogens. Intern. J. Food Microbiol. 83:39-48 Mifflin, T. E. (2007). Setting up a PCR Laboratory. CSH Protocols 2007: doi:10.1101/pdb.top14. http://cshprotocols.cshlp.org/cgi/content/abstract/2007/14/pdb.top14 Wood, R. and Corry, J.E.L. (2003): Quality assurance of laboratory performance. In: McMeekin, T.A. (ed.): Detecting pathogens in food. Chapter 5. 93-118. Woodhead Publishing Limited, Cambridge, England. EN ISO 16140:2003. Protocol for the validation of alternative method. ISO 22174:2005. Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens General requirements and definitions. ISO/TS 20836:2005. Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection of food-borne pathogens - Performance testing for thermal cyclers ISO 20837:2006. Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection of food-borne pathogens - Requirements for sample preparation for qualitative detection. ISO 20838:2006. Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection of food-borne pathogens - Requirements for amplification and detection for qualitative methods.