Enumeration of Salmonella in eggs and egg products by quantitative PCR

Transcription

1 Enumeration of Salmonella in eggs and egg products by quantitative PCR D. JAKOČIŪNö 1, F. PASQUALI 2, C. SOARES DA SILVA 3, A. LUCCHI 1, A. DE CESARE 1, G. KLEIN 3, G. MANFREDA 2 and J. E. OLSEN 1 University of Copenhagen, Danmark; 2 University of Bologna, Italy; 3 University of Veterinary Medicine, Hannover, Germany

2 Eggs and egg products EU N=701 Bakery products 3,6 % Meat and meat products 15,5 % Sweets 3 % Cereal products 1,9 % Buffet meals 4,1 % Eggs and egg products 21,4 % Mixed food 13,7 % Staphylococca l toxins 0,7 % Bacillus 1,3 % N=150 Calicivirus 0,7 % Unknown 2 % Fruits and vegetables 7,7 % Crustaceans and products thereof Fish and fish products 10,1 % Other foods 13,1 % Salmonella spp. 95,4 % EFSA & ECDC, 2013

3 Compliance criteria Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs Food safety Criteria Food category Microorganism Sampling plan Limits egg products* Salmonella 5 0 Absence in 25 g n c Analytical reference method EN/ISO 6579 Stage where the criterion applies Retail *Food categories 1.14 and 1.15

4 Salmonella detection in foodstuffs ISO g ml BPW Stomacher 1 min 37 ± 1 C x 18 ± 2h 0.1 ml in 10 ml RVS 41,5± 1 Cx 24 ± 3h 1 ml in 10 ml MKTTn 37± 1 Cx 24 ± 3h BGA XLD BGA XLD 37± 1 Cx 24 ± 3h Nutrient Agar 37± 1 Cx 24 ± 3h Results in 4-5 days Only qualitative data API 20E 37± 1 Cx 24 ± 3h TSI 37± 1 Cx 24 ± 3h

5 What if we use Real-Time PCR

6 Real-Time PCR for Salmonella detection Real-Time PCR Enrichment DNA Extraction Master Mix preparation and amplification Results in 1-2 days Higher sensitivity qualitative and quantitative data possible

7 Key issues of Real-Time PCR assays Selection of the target gene Selection of the chemistry (Probe-based vs SyBrGreen) (ISO ISO 22119) Inclusion of an Internal Amplification Control (ISO 22119) Sensitivity and specificity Harmonisation and validation

8 Real-Time PCR assay for Salmonella detection Real-Time PCR Pre-Enrichment BPW 18 h, 37 C DNA Extraction Master Mix preparation amplification Master Mix Primers for ttrrsbca locus Probe for ttrrsbca locus Probe IAC DNA IAC Sample DNA

9 Real-Time PCR assay for Salmonella enumeration Real-Time PCR Pre-Enrichment 8 h, 37 C BPW DNA Extraction Master Mix preparation and amplification Master Mix Primers for ttrrsbca locus Probe for ttrrsbca locus Probe IAC DNA IAC Sample DNA

10 Pre-enrichment and enumeration by Real-Time PCR Growth curve of S. Enteritidis in pasteurized liquid egg, 24 C Real-Time PCR limit of detection No dataqualitative and quantitative data Only qualitative data Addapted from Sakha & Fujikawa, Biocontrol Science.

11 Ct value Salmonella Real-Time PCR assay Detection limit of the Real-Time PCR assay E = 94,019 % R²= 0, fold serially diluted plasmid vector containing the cloned 86-bp ttrrsbca target log 10 gene copy number/pcr 5 gene copies 5 x 10 6 gene copies

12 Enumeration of Salmonella on eggs, egg products and egg containing dishes Experimental plan S. Enteritidis or S. Tennessee saline Real-Time PCR (Malorny et al., 2004) 5x10 8 CFU/ml 1:10 1:10 1 1:10 2 1:10 3 1:10 4 1: BPW DNA extraction 37 C 8 h preenrichment

13 Enumeration of Salmonella on table eggs Ct value R² = 0, Log CFU/eggshell Standard curve on eggshells experimentally inoculated by CFU/eggshell of S. Enteritidis 10 eggshell per inoculum level; three PCR replicate per sample

14 Enumeration of Salmonella in pasteurized egg products whole egg egg yolk Ct value R² = 0,9376 R² = 0, CFU/ml 6h 8h Ct value R² = 0,9185 Standard curves on egg products experimentally inoculated by CFU/ml of S. Enteritidis For pasteurized egg white Ct values did not fit a linear correlation R² = 0, CFU/ml 6h 8h

15 Enumeration of Salmonella in a raw egg containing dish: Tiramisù Ct value R² = 0, Inoculated Salmonella (log 10 CFU/25g sample) Standard curve 25 g Tiramisù (sample) x 3 samples x 5 inoculum levels ( CFU/sample) x 3 qpcr repeats Bruxelles October 2012

16 Enumeration of Salmonella in raw egg containing dish: Tiramisù S. Enteritidis 10 3 CFU days 8 C qpcr 25g Bruxelles October 2012

17 Enumeration of Salmonella in raw egg containing dish: Tiramisù * * **** Survival of S. Enteritidis in Tiramisù during storage at 8 C Bruxelles October 2012

18 Enumeration of Salmonella in egg products When things might get complicated.. Addapted from Sakha & Fujikawa, Biocontrol Science. Addapted from Zhou et al, Applied and Environmental Microbiology

19 Enumeration of Salmonella spp. in egg products - Influence of the inoculated serovar Standard curve on light pasteurised whole egg experimentally inoculated by CFU/ml of S. Enteritidis (squares) or S. Tennessee (circles) 5 samples per inoculum level; three PCR replicate per sample

20 Enumeration of Salmonella in egg products standard vs stressed cells Ct value Log CFU/ml standard stress Standard curves of pasteurized egg yolk spiked with S. Tennessee heat-stressed cells (48 C for 30 min) or standard stationary cells 5 samples per inoculum level; three PCR replicate per sample

21 CONCLUSIONS What if we use qpcr? Standard curves on eggs spiked with stressed bacteria should be further evaluated Standard curves need to be matrix specific Results in 1-2 days High sensitivity and specificity

22 THANK YOU FOR YOUR ATTENTION Frédérique Pasquali

23 Detection of Salmonella on table eggs Experimental plan S.Enteritidis Real-Time PCR (Malorny et al., 2004) saline 5x10 8 CFU/ml 1:10 1:10 1 1:10 2 1:10 3 1:10 4 1: BPW DNA extraction ISO 6579: C 18 h preenrichment

24 Detection of Salmonella on table eggs Detection of Salmonella on eggshells by Real-Time PCR and ISO 6579:2004 Salmonella inoculum level (CFU/eggshell) N of tested eggs ISO 6579 Real-Time PCR 4,7 x * 10* 4,7 x ,7 x ,7 x ,7 x ,7 x *each egg was tested in quadruplicate by Real-Time PCR and ISO 6579 and assigned as positive if at least one repeat was positive

25 Experimental plan Ethanol 10 ;10 2 ;10 3 CFU of S. Enteritidis ISO 6579 BPW 37 C 18 h *15 repeats per pooling were tested Monte Carlo simulation with 1,000 iterations

26 Detection of Salmonella on pools of eggshells MonteCarlo simulation results on the number of pooled egg samples to be tested to detect Salmonella with 95% certainty by Real-Time PCR and ISO 6579 n. of n. of pools to be sampled eggshells per pool ISO 6579 Real-Time PCR

27 Future work What if we use an alternative method with higher sensitivity? Can we lower down the number of samples to be tested? Are these number of samples practical from a food industry point of view? What if we use a rapid and more sensible alternative method? Will these informations help food authorities to fill the gap between egg-borne Salmonellosis cases and incidence of Salmonella on table eggs?