Qualitative detection of Salmonella Enteritidis and Salmonella Typhimurium by real-time PCR method

Salmonella Enteritidis and Salmonella Typhimurium are among the top 5 global serotypes responsible for human infections.

What are Salmonella Enteritidis and Salmonella Typhimurium?

In accordance with Commission Regulation (EC) No. 2073/2005 on microbiological criteria for food, it is prescribed that in fresh poultry meat obtained from breeding flocks of the species Gallus gallus, laying hens, broiler chickens, and breeding and fattening turkeys and poultry carcasses, broiler chickens, and turkeys, testing must be conducted to determine the absence of these serotypes.

Recently, rapid testing methods have been employed, which are significantly shorter in duration than traditional culturing methods, thereby speeding up the testing process and obtaining equally reliable results.

Using specific primers in real-time polymerase chain reaction (PCR) with a PCR instrument and necessary accompanying reagents, DNA sequences specific for Salmonella Enteritidis and Salmonella Typhimurium are amplified. The PCR instrument detects these amplified fragments through fluorescence induced by the cleavage of the hybridized probe due to the 5' nuclease activity of Taq DNA polymerase.

The probe is labeled at the 5' end with a reporter fluorophore ("reporter") and at the 3' end with a quencher ("quencher").

During the extension phase of each cycle, the probe hybridizes with the internal amplification sequence, which is cleaved by the 5' nuclease activity of Taq DNA polymerase.

This cleavage of the probe separates the reporter signal from the quencher signal, increasing the reporter signal. The PCR instrument measures the emitted fluorescence of the reporter.

The Molecular Testing Laboratory is accredited for testing food, environmental samples, and bacterial cultures. The procedure consists of preparing the initial suspension where a certain amount of the sample is added to buffered peptone water, followed by incubation for 18 hours and then DNA isolation. If Salmonella spp. colonies are confirmed on the culture medium, only the DNA isolation procedure is necessary.

Afterwards, the final preparation of the PCR master mix, standards, placing strips in the device, setting the necessary parameters in the appropriate program, and starting the instrument operation follow.

Within 2 hours, the results are read and expressed as positive if the product is detected, and all controls give expected results, or negative if the PCR product is not detected, and all controls give expected results.