EMA-PCR (Ethidium Monoazide-Polymerase Chain Reaction) detection method of viable pathogen in farm environment

A detection method and farm technology are applied in the field of EMA-PCR detection of live bacteria, which can solve the problems of false positive detection results, inability to distinguish dead bacteria from live bacteria, and interference with the authenticity of detection, achieving simple operation and suitable for promotion. , the effect of short time

Inactive Publication Date: 2012-07-25
SICHUAN ANIMAL SCI ACAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PCR technology has been widely used in the detection of pathogenic bacteria in farms due to its strong specificity, high sensitivity, accuracy and speed. However, traditional PCR technology cannot distinguish between dead bacteria and live bacteria in samples. Can amplify target gene
Although the dead bacteria are no longer harmful, their DNA has existed for a long time and has also been detected. On the one hand, the number of live bacteria is overestimated, and on the other hand, the detection results are false positive when there are no live bacteria. Interfering with the authenticity of the detection

Method used

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  • EMA-PCR (Ethidium Monoazide-Polymerase Chain Reaction) detection method of viable pathogen in farm environment
  • EMA-PCR (Ethidium Monoazide-Polymerase Chain Reaction) detection method of viable pathogen in farm environment
  • EMA-PCR (Ethidium Monoazide-Polymerase Chain Reaction) detection method of viable pathogen in farm environment

Examples

Experimental program
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Effect test

Embodiment 1

[0038] This embodiment mainly consists of the following steps:

[0039] (1) The bacterial liquid is obtained after the sample is pretreated;

[0040] (a1) Take 1.5g wet weight stool sample;

[0041] (a2) Add 30ml of sterile PBS buffer, vortex to suspend, centrifuge at 500×g for 4min, and take the supernatant;

[0042] (a3) Repeat step (a2) for the precipitate formed after centrifugation; centrifuge the obtained supernatant at 9000×g for 5 minutes at high speed;

[0043] (a4) Collect the precipitate, suspend it with 30ml of 0.1M PBS buffer, and centrifuge at 9000×g for 5min;

[0044] (a5) After repeating step (a4), the bacterial cells were obtained, and the bacterial cells were suspended in 3ml of TE buffer solution, shaken and mixed evenly, and the bacterial liquid was prepared.

[0045] (2) Add 100ug of ethidium azide bromide to 1ml of the bacterial solution, place it in the dark for 5 minutes, and treat it with a 650W halogen light source for 1 minute. In this embodiment...

Embodiment 2

[0058] The difference between this embodiment and Example 1 is that the bacterial species detected are different. The bacterial species detected in this embodiment is enterohaemorrhagic Escherichia coli O157:H7. Taking the rfbE specific amplification primer as an example, the primer sequence is: F : 5'-AGATTGCGCTGAAGCCTTTG-3'; R: 5'-ATTGGCATCGTGTGGACAG-3', the size of the amplified target band is 495bp, and the detection result is as follows Figure 5 .

[0059] Figure 5 1 to 3 are the detection results of enterohemorrhagic Escherichia coli O157:H7, and 4 to 12 are the detection results of the control strain.

[0060] The control experiment of the present embodiment is: the EMA-PCR amplification of the mixed bacterial suspension of different viable cell ratios, and detection result is as follows Figure 6 .

Embodiment 3

[0062] The difference between this embodiment and Example 1 is that the strains detected are different. The strains detected in this embodiment are Staphylococcus aureus. Taking the nuc-specific amplification primer as an example, the primer sequence is: F:5'- TTAGCCAAGCCTTGACGAAC-3′; R: 5′-AGGGCAATACGCAAAGAGGT-3′, the size of the amplified target band is 480bp, and the detection result is as follows Figure 7 .

[0063] Figure 7 1 to 4 are the test results of Staphylococcus aureus, and 5 to 12 are the test results of the control strain.

[0064] The control experiment of the present embodiment is: the EMA-PCR amplification of the mixed bacterial suspension of different living cell ratio, detection result is as follows Figure 8 .

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Abstract

The invention discloses an EMA-PCR (Ethidium Monoazide-Polymerase Chain Reaction) detection method of viable pathogen in a farm environment. The EMA-PCR detection method mainly comprises the following steps of: (1) pre-processing a sample to obtain bacterial liquid; (2) adding 50-100 micrograms of azided ethidium bromide in 1 ml of the bacterial liquid, standing the bacterial liquid for 5-10 min in a dark condition, and carrying out illumination treatment for 1-3 min by using a 650 W halogen light source; (3) extracting a template DNA (Deoxyribonucleic Acid) from the treated bacterial liquid; and (4) carrying out PCR detection by using the template DNA. The EMA-PCR detection method provided by the invention has the advantages of effectively removing interference of dead cell DNA, greatly improving the detection accuracy, rapidly and effectively detecting the normal viable pathogen in the sample, obtaining more accurate detection result and the like.

Description

technical field [0001] The invention relates to a method for detecting live pathogenic bacteria in a farm environment, in particular to an EMA-PCR detection method for live bacteria. Background technique [0002] With the development of animal husbandry, my country's breeding structure is gradually changing from traditional breeding to modern breeding and large-scale breeding, which is also an important symbol of modern animal husbandry. However, large-scale farming also has many disadvantages. Among them, large-scale farms overuse antibiotics in the breeding process, resulting in a large number of antibiotics remaining in animals for a long time, seriously affecting the quality of livestock and poultry products. Pathogens are not monitored at any time, resulting in the abuse or overuse of a large number of antibiotics. On the one hand, it leads to the emergence of drug resistance. Therefore, it is very important to monitor the pathogenic bacteria in the farm environment. O...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/19C12R1/42C12R1/445
Inventor 赵素君曹冶廖党金谢晶李江凌王秋实罗丹丹叶勇刚
Owner SICHUAN ANIMAL SCI ACAD
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