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Fast quantitative detection method of shigella contained in sludge

A quantitative detection method, shigella technology, applied in the field of pathogenic bacteria detection in biotechnology, can solve the problems of various reagents, sensitivity interference, and complex sludge composition, so as to improve sensitivity and specificity, avoid interference, and operate steps and The effect of labor intensity reduction

Inactive Publication Date: 2011-03-30
JIANGNAN UNIV
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Problems solved by technology

However, when this method is applied to the detection of sludge samples, on the one hand, due to the complex composition of sludge and a large number of microorganisms, it will interfere with the detection sensitivity; Various disadvantages
There are also other patents reporting Shigella detection methods, such as a Shigella detection primer and detection method invented by South China Agricultural University (CN101845493A) and a detection kit for Salmonella and Shigella in feed invented by Cao Jijuan and its detection method (CN101624624A), but none of them can meet the requirements of rapid quantitative detection of Shigella in sludge samples

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  • Fast quantitative detection method of shigella contained in sludge
  • Fast quantitative detection method of shigella contained in sludge
  • Fast quantitative detection method of shigella contained in sludge

Examples

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Embodiment 1

[0024] Example 1 Determination of the number of Shigella in the residual sludge before and after dehydration in the secondary sedimentation tank of a sewage treatment plant in Wuxi, Jiangsu

[0025] 1. Gradient dilution of samples and pre-enrichment culture: Weigh 3.00g of the remaining sludge samples before and after dehydration in the secondary sedimentation tank of a sewage treatment plant into a 50mL centrifuge tube filled with 27mL GN enrichment solution, vortex Shake for 5-10 minutes to make the mixture even. Take 0.5mL GN enrichment solution and add the mixed mud-water mixture into sterilized 4.5mL GN enrichment solution to obtain 6 dilution gradients, each dilution set three replicates, and incubate at 37°C for 18h.

[0026] 2. Selective enrichment culture includes: add 0.5mL of the suspension in each test tube of the enriched bacterial solution into the Shigella enrichment broth, and incubate at 37°C for 24 hours.

[0027] 3. The DNA extraction process includes: take...

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Abstract

The invention relates to a fast quantitative detection method of shigella contained in sludge, belonging to the technical field of pathogenicbacteria detection in biotechnologies. The fast quantitative detection method of the shigella contained in the sludge comprises the following steps of: (1) carrying out gradient dilution and front enriched culture on a sludge sample; (2) carrying out selectively enriched culture; (3) carrying out DNA extraction; (4) carrying out PCR (Polymerase Chain Reaction) amplification and amplified product detection; and (5) calculating to obtain a result by utilizing MPN (Most Probable Number) counting software. The invention realizes the fast quantitative detection of the shigella contained in the sludge by adopting a method combining an MPN method with specific PCR detection and can prevent the disturbance of other microbes through selective enrichment and specific PCR, thereby enhancing the sensitivity and the specificity of detection; and in addition, the invention also has the advantages of shortening the detection time, enhancing the detection efficiency and increasing the quantity of detection samples.

Description

technical field [0001] The invention discloses a rapid quantitative detection method for Shigella in sludge environment, and belongs to the technical field of pathogenic bacteria detection in biotechnology. Background technique [0002] Shigella ( Shigella ) bacteria, commonly known as Shigella, rank first in the proportion of the original bacteria that cause infectious diarrhea in my country. They are highly infectious and serious Gram-negative enteropathogenic bacteria. Every year, 160 million people around the world suffer from bacterial infections, and about 1.1 million people die, most of whom are children under 5 years old. Since domestic sewage contains many pathogenic microorganisms, such as bacteria, viruses, protozoa, parasites, etc. from the intestinal tract, most of these microorganisms will enter the residual sludge with sewage treatment. During the agricultural land use of sludge, It diffuses into the environment through various ways such as polluting surface...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12Q1/06
Inventor 刘和符波王燕陈燕
Owner JIANGNAN UNIV
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