Rapid detection of total number of colonies in food by ATP luminescence

【Summary】
Objective: To explore the feasibility of ATP luminescence technology for rapid detection of total number of colonies. Methods The total number of bacteria was detected by comparing ATP bioluminescence values ​​with plate count method.
RESULTS: The correlation analysis of the test results showed that the two had significant correlation within a certain range.
Conclusion: ATP bioluminescence method is easy to operate and has a wide range of applications. It can be applied to the rapid detection of the total number of colonies in food.

The total number of colonies is an important indicator in the detection of food microbial national standards, mainly as a marker for determining the degree of bacterial contamination of food. The food hygiene standards issued by the state and the health indicators of many foods in the import trade have a limit on the total number of colonies. The conventional method for detecting the total number of colonies (plate counting method) has problems such as cumbersome operation, long test period, and little significance in the quality control of actual production. ATP bioluminescence assay is one of the methods for rapid detection of microorganisms. In this paper, the correlation between ATP bioluminescence assay and plate counting method was studied and analyzed, and the feasibility and operability of ATP bioluminescence assay for hygienic testing were discussed.

1 Materials and methods

1.1 Instruments and reagents
ATP bioluminescence rapid detection system supporting instruments and reagents (produced by Shenyang Zhongke Yuma Bioengineering Co., Ltd.), including: Hamamatsu 9505 microphotometer, special cuvette, luminescent reagent (LLR), bacterial cell ATP release reagent ( XRA). Vortex mixer. Diluted into a 9 ml tube of phosphate buffered saline (PBS) and dispensed into 10 tubes (autoclaved at 121 ° C). Solid nutrient agar medium (autoclaved at 121 ° C). PE gloves. Escherichia coli (ATCC 25922-3, purchased from the China National Institute for Pharmaceutical and Biological Products). Constant temperature shaker. Benchtop centrifuge.

1.2 method

1.2.1 Experimental preparation <br> One day before the counting, a loop of the E. coli slant culture was scraped with an inoculating loop and inoculated into a 100 ml Erlenmeyer flask containing 40 ml of LB, and cultured overnight at 37 ° C with shaking. The culture was centrifuged (10000 r/min, 1 min), and the supernatant was decanted. The pellet was washed once with an equal volume of PBS, and suspended in the same volume of PBS, that is, the culture stock solution. Dilution: (assuming a culture concentration of approximately 1 x 10 ⁶ cells/m1) The culture was serially diluted 10-fold with PBS.

1.2.2 plate count <br>determine 3 dilution gradients for plate counting, ensure that the count of at least 1 gradient is in the range of 30-300, draw 1ml and add to the glass sterilization empty culture dish, each The gradient was inverted parallel to the two plates while making a blank control. The cells were cultured in a 37 ° C incubator for 48 hours. The release gradients in which the colony counts were uniformly dispersed in the range of 30 to 300 were selected for plate count and the average value was taken.

1.2.3 ATP metering <br>Open the microphotometer screen in advance to show that the RLU is “0”, and use the sterilized pipette tip to draw 50 bacteria to be tested and add it to the special cuvette. Place the cuvette in the microphotometer drawer, hold the XRA reagent bottle, and add two full XRA drops vertically to the bottom of the cuvette. Using a sterile 100 μl pipette tip, 50 μl of LLR was added to the cup, and gently sucked 3 times, and the reaction mixture was mixed. Keep the pumping time and number of times consistent. And strive to consistent the operation time of all samples. Close the microphotometer drawer. At this time, the microphotometer automatically performs the fluorescence value measurement. After the microphotometer shows the fluorescence value, the fluorescence value is recorded.

2 results

2.1 ATP assay and plate count test results <br> Two methods for determining the total number of bacteria in parallel test results are shown in Table 1, Table 2.

2.2 Correlation between ATP measurement method and plate counting method <br>Based on the data of Table 1 and Table 2, the logarithm of the average value of the light value is the Y-axis, and the logarithm of the average value of the plate count is the x-axis for x and Y dispersion. The dot plot depicts the relationship curve, Y = 0.8834x - 1.6807, R ² = 0.9778. It can be seen from the data and scatter points that the values ​​of the 10 ^-5 and 10 ^-6 gradients of the first test and the first test are linearly inferior to other values, outside the detection limit. It can be seen that plate counts are significantly correlated with bioluminescence values ​​in the range of bacteria above 10 cfu/ml.

3 Discussion

The sensitivity of ATP bioluminescence detection is high. It is found that the color, operation and non-bacterial ATP of the test solution have an effect on the luminescence intensity, but the effect on the overall luminescence intensity is <0.9%. From the preparation of the sample to be tested to the extraction of bacterial ATP, until the detection of ATP luminescence intensity, the whole process can be completed within 1h, which has the characteristics of fast and simple, and can meet the requirements of general rapid detection. The detection limit of ATP luminescence technology can reach l0 ^-15 molATP, the corresponding total number of bacteria should be above 10 ³ cfu/ml, and the sample hygiene index standard above 10 ³ cfu/ml can instantly determine the total number of bacteria in the sample. Bad. The microbiological test health index is below 10 ³ cfu/ml, and the appropriate and simple enrichment technology can be used to make the total number of sample colonies reach the effective detection range, and this technology is also the key to the application of ATP luminescence technology to sanitary inspection. In recent years, luciferase has been produced through genetic engineering, and the price has been greatly reduced. With the miniaturization of related instruments, ATP bioluminescence technology will be rapidly popularized in related industries in China.

(Marnie Zhao Hong, China Health Engineering)