Horizontal Method For Detection Research Articles

Validation of PCR methods for confirmation and species identification of thermotolerant Campylobacter as part of EN ISO 10272 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp.

This article describes the outline and organisation of the validation of three multiplex PCR methods for species identification and/or confirmation of thermotolerant Campylobacter spp. The three PCR methods were validated against the reference method described in the EN ISO standard 10272:2017. The results of the PCR methods were compared against the reference method in a method comparison study and an interlaboratory study based on EN ISO 16140-6:2019.The performance, in terms of inclusivity and exclusivity, of each of the eight PCR targets were comparable to the performance of the reference method: close, equal, or better depending on the target. In total, all three PCR methods were concluded to be equally qualified as the reference method for molecular identification and/or confirmation of thermotolerant Campylobacter spp., C. jejuni, C. coli and C. lari isolated from the food chain and have been included in Amendment 1 of ISO 10272:2017.

ВЕРИФИКАЦИЯ МЕТОДА ВЫДЕЛЕНИЯ VIBRIO PARAHAEMOLYTICUS ИЗ МОРСКОЙ РЫБЫ И РЫБНОЙ ПРОДУКЦИИ

Analysis of microbiological safety of fish and fish products involves the assessment of the presence of a number of pathogenic bacteria in these products. One of these important bacteria is Vibrio parahaemolyticus – a gram-negative halophilic bacilus found in warm sea waters. The aim of this work was to verify the method of isolation of Vibrio parahaemolyticus from marine fish and fish products, as well as to study the presence of Vibrio samples of fish products from large retail chains. Verification of the method of detection of parahemolytic Vibrio from fish and fish products was carried out in accordance with GOST ISO/TS 21872-2-2013 "Microbiology of food and animal feed. Horizontal method for detection of potentially enteropathogenic Vibrio spp. Part 2. "Detection of bacteria species differs from Vibrio parahaemolyticus and Vibrio cholerae". As a result of the performed studies, the reliability of this method of detection of Vibrio parahaemolyticus on artificially contaminated samples was established. During monitoring fish products, parahemolytic vibrio was not detected.

Validation by interlaboratory trials of EN ISO 10272 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 1: Detection method

During the last decade Campylobacter has been the most commonly reported gastrointestinal bacterial infection in humans in the European Union. The use of a sensitive detection method based on enrichment of Campylobacter spp. is often needed when examining foods. However, as background flora developed resistance to third generation β-lactams used in selective culture media, the ISO method was adapted. It now consists of three different procedures (A, B, and C) depending on the expected concentration and condition of Campylobacter and the background microflora. As the diagnostic sensitivity of the detection test varies between laboratories, this justifies the validation of the method in an interlaboratory study.The matrices selected for testing in the collaborative trials were frozen spinach (procedure A, Bolton enrichment broth), minced meat (procedure A, Bolton enrichment broth), raw milk (procedure B, Preston enrichment broth), chicken skin (procedure B, Preston enrichment broth), and broiler caecal material (procedure C, direct plating on mCCD agar). Each matrix was artificially inoculated with a different Campylobacter strain at a low and high contamination level, and with sterile diluent for ‘blanks’. Seventeen laboratories participated in the interlaboratory study. The sensitivity and specificity of the methods for the five selected matrices were determined, as well as the level of detection (LOD50). Calculated LOD50 values ranged from 0.84 cfu/test portion in frozen spinach and 2.2 cfu/test portion in minced meat to 14 cfu/test portion in chicken skin and 57 cfu/test portion in raw milk, all based on test portions of 10 g. The test portion size for broiler caecal material was a 10 μl-loop, yielding a LOD50 of 6.1 cfu/test portion. The validation data were incorporated in the newly published ISO standard EN ISO 10272-1:2017 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter - Part 1: Detection method.

Validation by interlaboratory trials of EN ISO 10272 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 2: Colony-count technique

The validation in an interlaboratory study of the International Standards Organization standard method for the enumeration of Campylobacter in foods (ISO 10272-2) was performed after preparation of the revised Standard based on scientifically sound and validated methods of analysis.The matrices selected for testing in the collaborative trial were frozen spinach, minced meat, raw milk, chicken skin, and broiler caecal material. Each matrix was artificially inoculated with a different Campylobacter strain. Fifteen laboratories participated in the interlaboratory study.As a general indication of repeatability limit (r), the following overall values can be used when testing chicken skin samples:r = 0.98 (expressed as a difference between log10-transformed test results), orr = 9.52 (expressed as a ratio between test results).As a general indication of reproducibility limit (R), the following overall values can be used when testing chicken skin samples:R = 1.31 (expressed as a difference between log10-transformed test results), orR = 20.43 (expressed as a ratio between test results).The validation data for all matrices were incorporated in the newly published ISO standard EN ISO 10272-2:2017 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter - Part 2: colony-count technique.

Rapid detection and differentiation of important Campylobacter spp. in poultry samples by dot blot and PCR

The detection of Campylobacter, the most commonly reported cause of foodborne gastroenteritis in the European Union, is very important for human health. The most commonly recognised risk factor for infection is the handling and/or consumption of undercooked poultry meat. The methods typically applied to evaluate the presence/absence of Campylobacter in food samples are direct plating and/or enrichment culture based on the Horizontal Method for Detection and Enumeration of Campylobacter spp. (ISO 10272-1B: 2006) and PCR. Molecular methods also allow for the detection of cells that are viable but cannot be cultivated on agar media and that decrease the time required for species identification. The current study proposes the use of two molecular methods for species identification: dot blot and PCR. The dot blot method had a sensitivity of 25 ng for detection of DNA extracted from a pure culture using a digoxigenin-labelled probe for hybridisation; the target DNA was extracted from the enrichment broth at 24 h. PCR was performed using a pair of sensitive and specific primers for the detection of Campylobacter jejuni and Campylobacter coli after 24 h of enrichment in Preston broth.The initial samples were contaminated by 5 × 10 C. jejuni cells/g and 1.5 × 102C. coli cells/g, thus the number of cells present in the enrichment broth at 0 h was 1 or 3 cell/g, respectively.

Manual for Reporting on Zoonoses, Zoonotic Agents and Antimicrobial Resistance in the framework of Directive 2003/99/EC and on some other pathogenic microbiological agents for information derived from the year 2012

Manual for Reporting on Zoonoses, Zoonotic Agents and Antimicrobial Resistance in the framework of Directive 2003/99/EC and on some other pathogenic microbiological agents for information derived from the year 2012