| Salmonella is one of the most common and harmful foodborne pathogens in our daily life. It is Gram-negative enteric bacilli. Most major food poisioning issues were caused by the Salmonella enteritidis. It may cause human typhoid, gastroenteritis, infectious diarrhea, fever, vomiting and other symptoms.Traditional detection method of Salmonella in food according to the domestic standard, GB4789.4-2010, can be divided into3parts which are bacterial isolation, culture and identification. Its testing procedure has five steps, including pre-enrichment, enrichment, isolation, biochemical and serological identification. In will take4to7days generally, it is a long time span. Meanwhile, screening for suspected colonies is more relied on work experience, there exists some uncertainty. In order to adapt to the rapid development of the food industry and to meet the requirements of food safety inspection, which is rapidness and accuracy, we need a new way to monitor and control the spread of salmonella, to assure people a healthy live and work environment.PCR technology is based on the molecular biology technique; it has had a wide range of applications and deeply exploration in a number of scientific fields. PCR in detecting foodborne pathogens area have a lot of successful examples. However, this method also faces an enormous technical challenge, it is because the bacterial DNA has high stability, even the cell died, and its DNA can still be used as a template for PCR amplification. PCR method can detect whether DNA contains the target fragment, but the living state of the target cannot be clearly known. This will result in a non-selective amplification which can cause false-positive results. Therefore, the use of PCR technology was somehow limited in the detection of food-borne pathogens; it cannot be uesd as a major method. If you get a positive result by using PCR method, we still need traditional methods to test and verify.In this experiment, we use Samonella enteritidis strain (ATCC13076) as experimental subjects, use DNA dye EMA (Edthidium Monoazide Bromide azide ethidium bromide) and PMA (Propidium Monoazide Bromide)as the pretreatment of bacteria, combined with real-time quantitative PCR technology,to inhibit theDNA amplification of dead cells, selectively make the DNA amplification of living bacteria work. Therefore, in order to achieve the purpose of distinguishing live/dead bacteria. EMA and PMA are nucleic acid dyes, a lot of domestic and international experts’ researches showed that the2dyes have the characteristics of penetrating the damaged cells and then binds to DNA, while the viable cells whose cell wall is intact can be amplified successfully, by this way to overcome the shortcoming of PCR technique that cannot distinguish between live/dead bacteria and to reduce false-positive rate.In this study,we treat Salmonella enteritidis at the concentration of5×107CFU/mL with EMA. After a series experiments with pure culture bacteria suspension and the real samples,we get the following conclusion:In the saline, the detection limit of salmonella enteritidis was104CFU/mL. Continue to incubate for6h, the salmonella enteritidis can be detected at an initial concentration of101CFU/mL; after a24h enrichment culture,the initial concentration of the suspension that can be detected was improved to100CFU/mL.In the pure Salmonella suspension, the suspension treated with the EMA at a concentration of10μg/mL, were placed in the dark environment for10min to allow the EMA to penetrate the heat-killed dead cells and to bind to the DNA. The suspension was exposed to light from a halogen bulb (500w) at a distance of14cm to activate and photolyse the EMA. This experiment condition can fully suppress the amplification effect of the DNA derived from dead cells (Ct value>40), and EMA had a slight impact on the PCR amplification of viable cells, no significant difference with the control group (p>0.05), which will not affect the final testing result of the qualitative detection.Under the same experiment condition with PMA, the result was not satisfactory; PMA cannot completely inhibit PCR amplification from dead cells by adjusting the concentration of dye and exposure time. The results showed that in the case of Salmonella enteritidis, the work condition and effect of EMA and PMA were not identical. EMA is better than PMA in inhibiting the amplification of dead cells.We test the effect of EMA-qPCR method in3kinds of food samples:frozen mutton, frozen poultry (chicken) and fresh eggs (egg liquid). Results showed that this experiment system: 10μg/mL of EMA,10min incubation in dark and15min exposure under500w halogen lamp can be used to differentiate the dead from the viable in all three samples, which was consistent with the results in previous tests on the pure bacteria suspension. |
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