What Is Pseudomonas Fluorescens?

P. Fluorescens is a kind of environmental pollution bacteria. It is a rare opportunistic pathogen for humans. Pseudomonas fluorescens belongs to the genus Pseudomonas and is a gram-negative heterotrophic gram-negative bacterium. It is rod-shaped and has flagella. It can secrete yellow-green fluorescent pigments and emit fluorescence. It can produce antibiotics, hydrolase and other metabolites. It can grow in a neutral environment in the range of 4 -37 , and has significant physiological and biochemical characteristics. Microbes ^[1] .

It can be separated from wounds, sputum, pleural fluid, urine and blood, as well as from blood banks. Can reproduce in blood and blood products stored in the refrigerator, and release endotoxin after autolysis. The phospholipid portion of its endotoxin can cause irreversible shock after blood transfusion.

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[Beijing Administration for Industry and Commerce said

At present, the number of psychrophiles in dairy products in China is measured by the plate counting method in the national standard NYT 1331-2007. There is no special national standard for Pseudomonas fluorescens, but there are abundant detection technologies for Pseudomonas fluorescens at home and abroad. the study.

Pseudomonas fluorescens plate counting method

The International Dairy Federation (IDF) uses IDF Standard 101A and IDF Standard 132A. The former uses 6.5 ° C for 10 days, and the latter counts at 21 ° C for 25h. The 132A has short stability and good bacteria count. The plate counting method dilutes the raw milk, and then uses the coating method, pouring method, 3M total bacteria test paper and other methods to count. The pouring method has lower accuracy due to higher temperature, and the 3M test paper has higher accuracy and efficiency. The plate counting method is a traditional detection method. The counting is accurate, but it takes too long, and 25h is far from the requirement of rapid detection in industrial production.

Direct fluorescent filtration of Pseudomonas fluorescens

The direct fluorescence method DEFT (Direct Epifluorescent Filter Technique) is a method for counting colonies of food samples using ionizing radiation. It is easy to operate and has high throughput. After passing the sample through the filter, staining with acridine orange, under the microscope, live cells can be observed in orange and yellow, while dead cells are stained in green, and the total number of all colonies in the sample can be counted. The raw milk was stained with a filtration membrane, and the correlation coefficient of Pseudomonas fluorescens was 0.91, and the detection was completed within 25 minutes. Compared with the traditional plate counting method, DEFT greatly shortens the detection time. Using its principle, it can realize the rapid detection of the number of colonies in various foods. However, the sensitivity of DEFT is not high, and it can only be used to detect the number of colonies in a certain range. When the number of bacteria in the food is small, there is no good linear relationship, and the experimental equipment is relatively expensive, which is not suitable for industrial production.

Pseudomonas fluorescens flow cytometry

FCM (Flow Cytometry Method) can detect single cells labeled with fluorescent signals in the bacterial solution, and achieve a certain amount of analysis of the strain. The number of bacteria detected by flow cytometry was 3.8 times that of plate counting, because the total number of colonies could be counted. Using this method to detect Pseudomonas in milk, the detection can be completed within 4 hours, and the sensitivity is greatly improved when the number of colonies is small. This method can quickly and accurately detect the content of Pseudomonas fluorescens in raw milk, but the flow cytometry procedure is more complicated, and it is easy to be affected by various factors.

PCR Pseudomonas fluorescens PCR

Primers are designed for the 16S rRNA conserved sequence. PCR amplification can amplify the 147bp DNA fragment of psychrophilic bacteria in milk. After labeling, it can be detected by ELISA to obtain the OD450 and bacteria concentration equation of Pseudomonas fluorescens AH-70. The analysis showed that the correlation coefficient between this method and the plate counting method reached 0.94, and the concentration range of detectable bacteria was 103-107CFU / mL. PCR detection has high sensitivity and strong specificity. It has been reported in the literature that when the concentration of E. coli in raw milk reaches 10 CFU / mL, it can be detected. The results of PCR detection of colony number will also be affected by the number of dead strains. In actual production, it is difficult to extract DNA that can be PCR amplified from food, which is easily affected by factors in the food system.

Pseudomonas fluorescens aminopeptidase method

The aminopeptidase method detects the activity of aminopeptidase by reacting the aminopeptidase on the cell wall of Gram-negative bacteria with a specific compound. Lu Yuan et al. Used the aminopeptidase method to quickly detect three dominant psychrotrophic bacteria, Pseudomonas fluorescens, Enterobacter sakazakii, and Acinetobacter ruber in raw milk in Hangzhou. The results showed that there was a significant correlation between the concentration of the three bacteria and the aminopeptidase activity, and there was no significant difference from the results of the traditional plate counting method, which can greatly shorten the detection time. In the detection of cold bacteria in refrigerated meat, it was reported that the detection range of the aminopeptidase method was 104-108 CFU / mL, the detection time was 2.5 h, and the correlation with the plate counting method result was 0.88. Obviously, the aminopeptidase method can achieve rapid detection. The aminopeptidase method is suitable for qualitative detection. It can be observed with the naked eye according to the reaction color, but the detection sensitivity is worse than other methods. The biggest problem is the Gram-positive bacteria in the raw material sample Unable to be detected, leading to deviations in experimental results.

ELISA Pseudomonas fluorescens ELISA

The ELISA method detects the number of colonies through an antibody-specific reaction, has high sensitivity, and is easily combined with other methods. PicardC et al. Calculated the content of psychrotrophs in raw milk by measuring casein glycopeptides. Using monoclonal antibodies, psychrotrophs were detected in the finished milk samples, and the correlation with plate counting method was 0.96. Six types of Salmonella were detected from skimmed milk powder, the detection line was 10 CFU / mL, and the detection time was 3 hours. CrociL et al. Compared the ELISA method with the PCR method and plate count in the detection of pork samples, and concluded that the former two are more sensitive. The minimum detection line is 1-10 / 25g, and the test results meet the ISO regulations.

Pseudomonas fluorescens immunomagnetic beads method

Immune magnetic bead technology can quickly enrich low-concentration bacteria in dairy products. By combining methods such as PCR and ELISA, fast and accurate detection can be achieved. Lu Qi et al. Designed primers for the conserved sequence genes of 4 bacteria and established a multiplex PCR system. In 7-8h detection time, the detection sensitivity reached 102CFU / mL, which is specific. Immunomagnetic bead technology detection has certain advantages in all aspects, and improved conserved sequences of psychrotrophs can show high throughput. There are few reports on the detection of bacterial concentration in dairy products by immunomagnetic bead technology, but the studies applied to detect harmful compounds are relatively mature and widely used ^[2] .