Elisabetta Delibato

Evaluation of DNA Extraction Methods for Use in Combination with SYBR Green I Real-Time PCR To Detect Salmonella enterica Serotype Enteritidis in Poultry

ABSTRACT The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system. The specificity of the reaction was determined by the melting temperature (Tm) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles). Boiling was selected as the preferred extraction method because it is the simplest and most rapid. This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2. The specificity of the reaction was confirmed by the Tm, which was consistently specific for the amplicon obtained; the mean peak Tm obtained with curves specific for serotype Enteritidis was 82.56 ± 0.22°C. The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 103 to 108 CFU/ml) showed good linearity (R2 = 0.9767) and a sensitivity limit of less than 103 CFU/ml. The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples.

Comparison of PCR, electrochemical enzyme-linked immunosorbent assays, and the standard culture method for detecting salmonella in meat products.

ABSTRACT An electrochemical enzyme-linked immunosorbent assay (ELISA) coupled with flow injection analysis (ELISA-FIA) and a PCR-based method using ST11 and ST15 primers for detecting salmonellae in meat were evaluated in comparison with the International Organization for Standardization (ISO) culture method. The methods were applied to experimentally contaminated and naturally contaminated meat samples. The results showed that both ELISA-FIA and PCR allowed detection of salmonella in a product contaminated with a low number of the microorganisms (1 to 10 salmonellae/25 g) after only 5 h of incubation of preenrichment broth, and they were just as effective as the ISO method.

Multiplex PCR for Detection of Botulinum Neurotoxin-Producing Clostridia in Clinical, Food, and Environmental Samples

ABSTRACT Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.

SYBR Green Real-Time PCR Method To Detect Clostridium botulinum Type A

ABSTRACT Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 101 copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).

A RAPID ELECTROCHEMICAL ELISA FOR THE DETECTION OF SALMONELLA IN MEAT SAMPLES

A sandwich ELISA assay using monoclonal and polyclonal antibodies against salmonella has been proposed. The activity of the label enzyme (horseradish peroxidase) is measured electrochemically using 3,3′,5,5′ tetramethylbenzidine as substrate. The efficiency and specificity of the method were verified during preliminary studies on various serotypes of Salmonella and other bacteria commonly present in food (E. coli, Klebsiella pneumoniae, Morganella morganii, Citrobacter freundii, Yersinia enterocolitica). The assay was then used to analyse samples of pork, chicken and beef experimentally contaminated with different concentrations of salmonella and samples not experimentally contaminated. Analysis were performed in parallel following the traditional cultural method. The samples were seeded in pre-enrichment broth (buffer peptone water) and amounts were taken at different times and analysed by electrochemical ELISA to determine the minimum incubation time needed to detect salmonella. Results showed that the method was efficient, sensitive and rapid: after only 5 h of incubation in pre-enrichment broth it was possible to detect salmonella in meat experimentally contaminated with low concentrations of salmonella (1–10 cells/25 g).

European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese.

The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories.

Development and application of an electrochemical plate coupled with immunomagnetic beads (ELIME) array for Salmonella enterica detection in meat samples.

Salmonella is one of the main organisms causing outbreaks of foodborne illness, and meat is one of the major vehicles of salmonellosis throughout the world. A novel analytical immunosensor array, based on a 96-well electrochemical plate coupled with immunomagnetic beads (ELIME array), is proposed for the detection of Salmonella in meat samples. After an optimization study, using Salmonella enterica serotype Enteritidis as reference antigen, the ability of the method to interact with a large number of Salmonella serovars commonly present in food was evaluated. The assay was then used to analyze samples of pork, chicken, beef, and turkey experimentally inoculated with Salmonella as well as real samples. The results were compared with those from the International Standard of Organization (ISO) culture method. The comparison showed that the ELIME array is able to detect a low number of Salmonella cells (1-10 CFU per 25 g) after only 6 h of incubation in a pre-enrichment broth. The investigation revealed a very good agreement between culture and ELIME array methods for meat samples, reducing the time for performing the analysis and obtaining the results quickly.

Next day Salmonella spp. detection method based on real-time PCR for meat, dairy and vegetable food products

The microbiological standard for detection of Salmonella relies on several cultural steps and requires more than 5 days for final confirmation, and as consequence there is a need for an alternative rapid methodology for its detection. The aim of this study was to compare different detection strategies based on real-time PCR for a rapid and sensitive detection in an ample range of food products: raw pork and poultry meat, ready to eat lettuce salad and raw sheep milk cured cheese. Three main parameters were evaluated to reduce the time and cost for final results: the initial sample size (25 and 50 g), the incubation times (6, 10 and 18 h) and the bacterial DNA extraction (simple boiling of the culture after washing the bacterial pellet, the use of the Chelex resin, and a commercial silica column). The results obtained demonstrate that a combination of an incubation in buffered peptone water for 18 h of a 25 g-sample coupled to a DNA extraction by boiling and a real-time PCR assay detected down to 2-4 Salmonella spp.CFU per sample in less than 21 h in different types of food products. This RTi-PCR-based method is fully compatible with the ISO standard, providing results more rapidly and cost-effectively. The results were confirmed in a large number of naturally contaminated food samples with at least the same analytical performance as the reference method.

Development of SYBR‐Green Real‐Time PCR and a Multichannel Electrochemical Immunosensor for Specific Detection of Salmonella enterica

Abstract The objective of the present work was to develop and evaluate an SYBR Green real time polymerase chain reaction (PCR) method for the specific detection of Salmonella spp in broth cultures and meat samples experimentally contaminated. Also, a simple and rapid multichannel electrochemical immunosensor (MEI) for this pathogen is under study. The PCR was carried out using primers ttr6 and ttr4 for the amplification of a highly conserved DNA region (ttr sequence) specific for all Salmonella serovars. A boiling step, for the extraction of DNA, was combined with a real time PCR method based on the double‐stranded DNA (dsDNA) binding dye SYBR Green. The standard curve constructed using the mean threshold cycle and various concentrations of S. enteritidis (ranging fron 102 to 108 CFU mL−1) showed good linearity (R2=0.999) with the minimum level of detection of 102 CFU mL−1. The experiments were conducted analyzing 30 Salmonella strains and 20 non‐Salmonella strains. All Salmonella serotypes tested were ttr‐positive and all other bacteria yielded no amplification products. The specificity of the reaction was confirmed by the melting temperature (Tm) of the amplicon obtained (Tm=80.1±0.1). To verify the effectiveness of the assay, experiments were conducted on experimentally contaminated samples, which were also analyzed for comparison by the standard cultural method. A multichannel electrochemical immunosensor for detection of Salmonella also was developed. It consists of a disposable screen‐printed sensor array, coupled with a multichannel pulse monitor, which was assembled as an immunosensor through the use of specific monoclonal (MAb) and polyclonal (PAb) antibodies in a sandwich format. The limit of detection was calculated to be 2×106 UFC mL−1 with a working range between 5×106 to 5×108 UFC mL−1 and a total analysis time of about 3 h. This immunoelectrochemical system is economical, rapid, and easy to use but it is still under development in order to improve its analytical performance.

European validation of Real-Time PCR method for detection of Salmonella spp. in pork meat

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories.