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Quantitative detection method of escherichia coli in soil and assay kit thereof

A technology for quantitative detection of Escherichia coli, which is applied in the direction of microbial-based methods, microbial measurement/inspection, biochemical equipment and methods, etc., can solve problems such as difficult operation, insufficient quantitative detection, time-consuming and labor-intensive troubles, etc., and achieve good results. Economic and social benefits, improving detection efficiency, and reducing cumbersome steps

Inactive Publication Date: 2012-02-01
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

Among them, the traditional detection method is highly authoritative, but it has the disadvantages of time-consuming, labor-intensive and troublesome operations, and the quantitative detection of pathogenic microorganisms in soil has certain complexity and operational difficulty; with the modern molecular biology With the development of scientific methods, PCR technology is widely used in the qualitative detection of pathogenic microorganisms in food because of its rapidity and accuracy, but the PCR method cannot distinguish dead bacteria from live bacteria well, and it is also insufficient in quantitative detection. lead to inaccurate results

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  • Quantitative detection method of escherichia coli in soil and assay kit thereof
  • Quantitative detection method of escherichia coli in soil and assay kit thereof
  • Quantitative detection method of escherichia coli in soil and assay kit thereof

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Effect test

Embodiment 1

[0033] Using representative Escherichia coli DH5α strain as the experimental strain, prepare a series of concentration gradient Escherichia coli and add them to the sterilized soil for correlation analysis of plate colony counting method and MPN counting method counting results. Escherichia colonies were inoculated into 100ml LB liquid medium, cultured at 30°C with shaking at 200r / min for 8h. Then, serial dilutions of different concentrations were prepared by ten-fold dilution and added to the sterilized soil, and the number of Escherichia coli was counted by plate colony counting method and MPN counting method, respectively.

[0034] Plate colony count method refers to the literature [Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiments [M]. Beijing: Higher Education Press, 1999, 92-94. ], firstly carry out gradient dilution on the soil bacterial suspension prepared above, take 0.1ml of the bacterial suspension with appropriate dilution and spread it on the LB mediu...

Embodiment 2

[0048] The specificity of the PCR detection method mainly depends on the specificity of the primers, so the selection of the target gene should have species or genus specificity in order to avoid the occurrence of false positives. According to the phoA gene sequence of Escherichia coli, reference [Kong RY, DungW F, Vrijmoed L L, et al. Marine Pollution Bulletin, 1995, 31(4): 317-3241. 】Primer design software Primer 5.0 was used to design primers. The amplified fragment of the primers was 684bp. The above two pairs of primers were synthesized by Shanghai Yingjun Company. The specific sequence is as follows: phoA-1: 5'-TACAGGTGACTGCGGGCTTATC-3', phoA-2: 5'-CTTACCGGGCAATACACTCACTA-3'.

[0049] Using phoA-specific primers of Escherichia coli for Bacillus subtilis 168, Pseudomonas putida KT2440, Agrobacterium, Brevibacterium, Microbacterium cellulosus, Microbacterium, Pseudomonas, Rhodococcus, Salmonella, Escherichia coli DH5α, etc. 10 The strains were subjected to conventional b...

Embodiment 3

[0051] There are a lot of humic acid and metal ions in the soil, and their existence will seriously affect the activity of Taq enzyme in the PCR reaction, which may lead to the failure of the PCR reaction. In order to improve the efficiency of PCR detection and obtain a clean DNA template that can amplify the target band, usually the DNA template is obtained by phenol / chloroform extraction. It is toxic and is not suitable for DNA preparation of a large number of samples. According to past experience, even if DNA can be extracted, the target band must be amplified after recovery and purification of DNA samples due to the large amount of impurities in the soil. In order to simplify the procedure of template preparation, this method was compared with other commonly used simple methods.

[0052] Escherichia coli proliferation method, PCR system, conditions and electrophoresis method are all the same as in Example 4.

[0053] Method 1: Direct amplification of the enrichment soluti...

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Abstract

The invention discloses a quantiative detection method of escherichia coli in soil and an assay kit thereof. On the basis of taking related researches as reference, the method performs quantitative detection on pathogenic microorganism in the soil by adopting a method of combining MPN (most probable number) with PCR (polymerase chain reaction). By utilizing advantages of quickness and accuracy ofthe PCR method, biochemical identification, serological identification and other steps of positive results in the traditional MPN method are substituted, so that complex steps in a national standard method are reduced, the detection efficiency is improved, and a pre-enrichment step in the national standard method is adopted at the same time to improve the accuracy of detection targeting pathogenic bacteria; and due to the complexity of soil, the soil contains massive microorganisms, as well as various pathogenic microorganisms of different items, so that the targeting pathogenic bacteria is selectively cultured to improve the detection efficiency in order to quantitatively detect the targeting pathogenic bacteria better and more accurately. By virtue of creative effort of the inventor andscientific test verification, the sensitivity of the escherichia coli quantitative detection method in an experiment of adding pure bacteria in the soil is 25 per gram of soil.

Description

technical field [0001] The invention belongs to the technical field of microorganism quantitative detection, and in particular relates to a method for quantitatively detecting Escherichia coli in soil and a detection kit thereof. Background technique [0002] With the acceleration of urbanization in our country, cities migrate and expand to the edge. The urban-rural fringe is at the junction of urban development and rural construction areas. Soil health and quality issues in the urban-rural fringe are too important to ignore. At present, experts and scholars mainly focus on the indicators of pesticide residues, heavy metal pollution and organic pollution in the research of soil quality, and pay little attention to the biological indicators of pathogenic microorganisms in soil. Pathogenic microorganisms from poultry manure and sewage irrigation soil can remain latent in the soil for a long period of time, and can enter vegetables, eventually affecting human health. In parti...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06C12R1/19
Inventor 曹慧董丽宁崔中利
Owner NANJING AGRICULTURAL UNIVERSITY
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