Biomode - Biomolecular Determination

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University of Minho, University of Porto and Biomode - Biomolecular Determination | Date: 2016-03-09

PNA is a synthetic molecule analogue to DNA that, due to its physicochemical properties, allows a faster analysis with higher sensitivity and specificity than the DNA probes. These probes are combined with fluorescence in situ hybridization (FISH), a molecular biology technique that allows the direct visualization of the microorganisms in the sample. The combination of these two technologies rendered the FISH procedure to be faster, simpler and more efficient. This method can be applied to a great variety of samples such as food, blood, biopsies, feces, water and other clinical, environmental or agriculture and food industry samples. The present invention also includes the development of the kit of detection and respective procedure for the specific identification of Escherichia coli O157:H7 using the above referred sample types.


Almeida C.,IBB Institute for Biotechnology And Bioengineering | Almeida C.,University of Porto | Almeida C.,Biomode - Biomolecular Determination | Sousa J.M.,IBB Institute for Biotechnology And Bioengineering | And 6 more authors.
Applied and Environmental Microbiology | Year: 2013

Despite the emergence of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, E. coli serotype O157 is still the most commonly identified STEC in the world. It causes high morbidity and mortality and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain the gold standard. Here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157. Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. The method then was optimized for detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1 × 10-2 to 1 × 102 CFU per 25 g or ml of food. Samples were then preenriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrices with a detection limit of 1 CFU/25 g orml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% confidence interval [CI], 82.83 to 100), a sensitivity of 97.22% (95% CI, 83.79 to 99.85%), and an accuracy of 98.33% (CI 95%, 83.41 to 99.91%). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day. © 2013, American Society for Microbiology.


Almeida C.,IBB Institute for Biotechnology And Bioengineering | Almeida C.,University of Porto | Almeida C.,Biomode - Biomolecular Determination | Cerqueira L.,IBB Institute for Biotechnology And Bioengineering | And 4 more authors.
International Journal of Food Microbiology | Year: 2013

Several methods for the rapid and specific detection of Salmonella in food samples have been described. Here, we compare 4 of those methods in terms of assay time, procedure complexity, detection limit, sensitivity, specificity and accuracy. Milk, eggs and mayonnaise samples were artificially contaminated with Salmonella enterica serovar Enteritidis cell concentrations ranging from 1×10-2 to 1×102CFU per 25g or ml of food. Samples were then pre-enriched and analyzed by either: i) real-time PCR, using the iQ-Check Salmonella kit; ii) immunocapture, using the RapidChek SELECT Salmonella; iii) a peptide nucleic acid fluorescence in situ hybridization (PNA FISH) method and iv) the traditional bacteriological method ISO 6579:2002. All methods were able to detect Salmonella in the different types of food matrixes and presented a similar detection level of 1CFU per 25g or ml of food sample. The immunocapture and the PNA FISH methods proved to be very reliable, as their results were 100% in agreement with the ISO method. However, real-time PCR presented a significant number of false positives, which resulted in a specificity of 55.6% (CI 95%, 31.3-77.6) and an accuracy of 82.2% (CI 95%, 63.2-91.4) for this method. Sensitivity was 100% since no false negative results were observed. In conclusion, the implementation of these molecular techniques, mainly the immunocapture and PNA-FISH methods, provides a reliable and less time-consuming alternative for the detection of Salmonella spp. in food samples. © 2012 Elsevier B.V.


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