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Method for fast detecting salmonella in fresh milk

A technology for salmonella and fresh milk, which is applied in the field of microbial detection, can solve problems such as time-consuming, false negative results, and low detection rate, and achieve the effects of shortening the detection time, increasing the detection rate, and solving detection problems

Active Publication Date: 2015-06-24
HENAN BUSINESS SCI RES INST
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  • Application Information

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Problems solved by technology

The traditional salmonella detection method needs to go through pre-enrichment, enrichment, isolation and culture, biochemical test confirmation and other test steps. It takes at least 4-7 days to get a clear diagnostic result. The operation steps are cumbersome and time-consuming, and only 25g / ml can be detected The detection rate of Salmonella surviving in samples is lower than that of Salmonella in actual samples, and false negative results are prone to occur, endangering the safety of consumers

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  • Method for fast detecting salmonella in fresh milk
  • Method for fast detecting salmonella in fresh milk
  • Method for fast detecting salmonella in fresh milk

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Screening test

[0028] 1.1 Selection of fresh milk sample buffer

[0029] Take 4 copies of 25mL fresh milk Salmonella positive samples and add them to homogeneous bags containing 225mL buffer respectively. The buffers in the homogeneous bags are: ddH 2 O, 0.01mol / L PBS buffer (pH7.4, the same below), carbonate buffer, buffered peptone water (BPW). Then, according to the traditional salmonella detection method, the above-mentioned fresh milk mixture was enriched, streaked and separated, and biochemical tests were carried out. The test results are shown in Table 1.

[0030]

[0031] It can be seen from Table 1 that both BPW and 0.01mol / L PBS can effectively protect Salmonella in samples, but the cost of BPW is high, so 0.01mol / L PBS was selected as the pretreatment buffer.

[0032] 1.2 Comparison of the detection rate between the double filter method and the national standard method

[0033] Take 25mL of positive fresh milk samples, put them into ...

Embodiment 2

[0048] A kind of rapid detection method of Salmonella in fresh milk, comprises the following steps:

[0049] 1) Aseptically take 25 mL of fresh milk sample, add it into a sterile homogeneous bag containing 25 mL of phosphate-buffered saline solution containing 2 g of cellulose resin, and prepare a 50 mL fresh milk mixed solution. Homogenizer beating for 2min;

[0050]2) Rinse the mixed solution of fresh milk with 200mL PBS and quickly filter it into the container with filter paper, centrifuge the filtrate at a centrifugation speed of 6000r / min and a centrifugation temperature of 4°C for 10min, and filter the supernatant with a 0.22μm microporous membrane. Concentrate microorganisms on microporous membranes;

[0051] 3) Cut the microporous membrane into pieces with sterile scissors, rinse and soak for 2 minutes with a mixture of 5ml TTB enrichment solution and 5ml PBS buffer solution to obtain a concentrated bacterial suspension;

[0052] 4) Mix 100 μL Salmonella immunomagnet...

Embodiment 3

[0056] Embodiment 3 comparative test

[0057] Set the concentration to 1 x 10 9 The CFU / mL Salmonella bacterial liquid was diluted according to a certain gradient, and 1 mL of the diluted solution was added to 25 mL of fresh milk samples to prepare fresh milk positive samples. According to the method described in Example 2, and at the same time, the "GB 4789.4-2010" national standard method was used for comparison, and the fresh milk positive sample and the fresh milk sample were detected. The comparison test results are shown in Table 5.

[0058]

[0059] It can be seen from Table 5 that, compared with the national standard method, the detection method of the present invention has higher sensitivity and greatly improves the detection rate of Salmonella in fresh milk samples. In addition, for this group of tests, the national standard method takes 4-7 days to detect a sample, and the method of the present invention takes 50 minutes to detect a sample, and the latter grea...

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Abstract

The invention relates to a method for fast detecting salmonella in fresh milk and belongs to the technical field of microbiological detection. The method includes: adding a fresh milk sample into buffer solution to obtain fresh milk fixture, wherein the buffer solution contains reagent for removing protein and fat; filtering the fresh milk mixture with filter paper, and using a micro-pore filter membrane to perform extraction filtration on filtrate; cutting the micro-pore filter membrane into pieces after filtration, and adding tetrathionate broth and phosphate buffer solution for washing and soaking to obtain bacterium suspension; adding salmonella immunomagnetic beads into the bacterium suspension, mixing, oscillating, standing on a magnetic rack for layering, and abandoning supernatant; taking lower-layer solution, adding phosphate buffer solution for washing, standing on the magnetic rack for layering, and abandoning the supernatant to obtain concentrated to-be-detected solution; using an Elisa detecting method to detect the to-be-detected solution. By the method, the total salmonella detecting time is 50 minutes, and detecting time is shortened greatly as compared with a national standard method.

Description

technical field [0001] The invention belongs to the technical field of microbial detection, and in particular relates to a method for rapidly detecting Salmonella in fresh milk. Background technique [0002] Food safety is related to the national economy and people's livelihood. Foodborne diseases and food contamination are still important factors that endanger public health and are the most concerned public safety issues in the world today. According to statistics, food poisoning caused by Salmonella is often listed among bacterial food poisoning in various countries in the world. top of the list. Statistical analysis of foodborne disease outbreaks in China from 2001 to 2010 showed that foodborne diseases caused by microbial contamination accounted for 56.39% of the total, and foodborne diseases caused by Salmonella accounted for 10% of the total. Salmonella (Salmonella) is a group of Gram-negative bacilli with similar antigenic structure and biochemical properties. The po...

Claims

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Application Information

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IPC IPC(8): C12Q1/10G01N33/569C12R1/42
Inventor 王永王法云朱海华王慧周莉章建军邱红玲关炳峰谭静平洋
Owner HENAN BUSINESS SCI RES INST
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