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Multiple-PCR detection method for pathogenetic bacteria in soil

A detection method and technology for pathogenic bacteria, which are applied in the directions of microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc., can solve problems such as time-consuming, labor-intensive, and complicated operation

Inactive Publication Date: 2009-11-11
INST OF SOIL SCI CHINESE ACAD OF SCI
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Problems solved by technology

[0004] Technical problem: The purpose of the present invention is to overcome the cumbersome operation of conventional pathogenic bacteria detection technology, time-consuming and labor-intensive and other deficiencies, give full play to the application of multiple PCR technology in the detection of soil pathogenic bacteria, increase the types of pathogenic bacteria and the logarithm of primers as much as possible, and improve the specificity of detection and sensitivity

Method used

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  • Multiple-PCR detection method for pathogenetic bacteria in soil
  • Multiple-PCR detection method for pathogenetic bacteria in soil

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Embodiment 1

[0067] Soil sample collection and pretreatment: Take the soil 1-5cm away from the surface of the soil layer and put it into a sterilized sealed plastic bag. Remove large particles such as plant residues and sand and stones in the sample, grind and sieve (20 mesh sieve), weigh 100g soil sample and store it at -80°C for later use.

[0068] Extraction of bacterial DNA from soil: using FastDNA SPIN Kit for Soil and FastPrep TM FP120 Nucleic Acid Extractor (Bio 101, Carlsbad, CA, USA) was used to extract and purify total DNA from soil samples. For specific methods, refer to the manufacturer's instructions. The main process is to weigh 500 mg of fresh soil sample into a lysis tube containing ceramic and silica particles, then add 978 μL of sodium phosphate buffer and 122 μL of microtube buffer, and put the tube into FastPrep TM FP120 Nucleic Acid Extractor, set the speed at 6.0 and run for 40 seconds, and finally filter and elute the total DNA with a silica gel column for purifi...

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Abstract

The invention discloses a multiple-PCR detection method for pathogenetic bacteria in soil, which is characterized in that: multiple PCR is adopted to simultaneously amplify specific genes of enteropathogenic E. Coli, salmonella, Staphylococcus aureus, pseudomonas aeruginosa and Shigella flexneri for one time, namely phoA, ttrBCA, vicK, ecfX, ipaH and virA, and then PCR amplification products are detected by agarose gel electrophoresis. The multiple-PCR detection method overcomes defects of complicated operation, time and labor consumption and the like of the conventional pathogenetic bacteria detection technology, fully applies the multiple-PCR detection technology in detecting the pathogenetic bacteria in the soil, increases the types of the pathogenetic bacteria and number of pairs of primers as possible, and improves the specificity and sensitivity of the detection.

Description

1. Technical field [0001] The present invention relates to a kind of multiplex PCR amplification method that is used for the detection of pathogenic bacteria in soil, be specifically related to the detection method of multiplex PCR to be used in Escherichia coli (Escherichia coli), Salmonella (salmonella), Staphylococcus aureus (Staphylococcus aureus), Shiga Application in the analysis of soil samples contaminated by Shigella and Pseudomonas aeruginos. 2. Background technology [0002] Rapid detection and identification of pathogenic bacteria in the soil environment is the prerequisite for timely and effective control and prevention of pathogenic bacteria transmission, which is of great significance to human health. At present, the detection method of pathogenic bacteria mainly relies on the conventional bacteriological culture method, which generally takes 4-7 days, and the operation is cumbersome, time-consuming and labor-intensive; the latex agglutination method, immunoma...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N27/447C12R1/19C12R1/42C12R1/445C12R1/385C12R1/01
Inventor 钟文辉尹睿刘燕
Owner INST OF SOIL SCI CHINESE ACAD OF SCI
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