How to implement genetically modified microorganisms for routine quality control of methods in labs?

Poster # P2-33 • IAFP 2023 – Aberdeen May 3-5, 2023

Introduction

According to ISO/IEC 17025, testing laboratories should ensure they operate competently and are able to generate valid results. Implementation of Quality Control or Verification of methods requires the use of strains. Genetically Modified Microorganisms (GMM) (e.g., GFP tagged organisms) offer a less risky approach minimizing the impact of cross contamination events securing laboratory results.

BIOBALL^® LUMINATE 2.0 is a range of microbiological reference material containing a precise number of viable bacterial cells, tagged with a Green Fluorescent Protein (GFP). The GFP, integrated in the chromosome, allows easy differentiation between accidental cross-contamination (linked to quality control artificial contaminations) and natural contaminants. The 2.0 range displays a high fluorescence and includes 5 different strains (Tab 1) with a target of 100 cfu adapted to various applications in the food industry.

Purpose

Strains should be rightly qualified prior to routine use in the lab. To qualify & demonstrate the suitability of GMM, BIOBALL^® LUMINATE 2.0 range is evaluated on pathogenic detection methods (Table 1), the most sensitive and suitable for each application. Such application includes (daily) positive control, verification of methods according to ISO 16140-3 and positive results confirmation.

Daily control

The daily positive control or quality control is an experiment involving test  repetition using a known positive working sample. The Food Analysis  Laboratories need to have daily positive control to be confident that the method used in routine worked well and that a positive result obtained on a sample may be reported. This is their insurance to provide reliable test results.

To evaluate the use of BIOBALL^® LUMINATE 2.0 for daily positive control, one BIOBALL^® LUMINATE 2.0 was dissolved into 1 tube of 14-Day Re-Hydration Fluid 1,1 mL (Ref# 410386). After stirring, 200 µL (about 18 cfu) were pipetted out and used to inoculate bags of food samples. 5 bags were spiked and 1 bag used as a negative control (only necessary for the evaluation, not in routine) (Fig 1a). Upon positive results, GFP confirmation tests were run using plate method with UV check control at 365 nm. Successful evaluation is achieved when all daily control replicates are positive & their fluorescence confirmed.

Food samples & method protocols evaluated for daily positive controls are described in Table 1 & Fig 2.  

To verify the precise count of BIOBALL® LUMINATE 2.0 used for each test,  200 µL aliquots were spread onto TSA plates in triplicates and incubated for 24h at 37°C.

Table 2 presents results obtained for daily control tests. All methods showed 5/5 confirmed positive at the targeted spiked level of about 18 cfu. This evaluation confirmed the suitability of BIOBALL^® LUMINATE 2.0 range for daily control application.