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Spiroplasma pathogenic microorganism PCR fast checking technique

A pathogenic microorganism and detection technology, which is applied in the field of PCR rapid detection technology for spiroplasma pathogenic microorganisms, can solve the problems of large sample volume, inability to quantify, unsuitable for live blood detection, etc., and achieve the effect of low cost

Inactive Publication Date: 2006-02-15
NANJING NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, in the conventional polymerase chain reaction (PCR) detection process, the DNA extraction of the tested sample is usually carried out by the phenol method or a kit. These methods are cumbersome and time-consuming. The phenol method requires a large amount of samples and a lot of DNA loss, which is not suitable Trace detection and live blood detection of animals; the cost of the kit is relatively high, and the scope of use is relatively limited. Usually, a kit can only extract templates for one or two samples from different sources
In addition, the existing PCR detection of pathogenic microorganisms can only be qualitative, but not quantitative

Method used

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  • Spiroplasma pathogenic microorganism PCR fast checking technique
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: Detection of environmental sediment

[0016] In July 2004, a small amount of sediment samples were collected from a pond where crab shivering disease broke out in Quanjiao, Anhui Province, and brought back for testing. The specific operation is as follows:

[0017] a) Pretreatment: Dissolve about two medicine spoons of the mud sample to be tested in 50mL water for several hours, stir several times during the period to mix thoroughly → filter with quantitative filter paper to remove mud sample residue → put the filtrate in a 50mL centrifuge tube, 12000rpm , 4°C, centrifuge for 1 hour → carefully remove the supernatant, transfer the bottom liquid to a 1.5mL Eppendorf tube → 8000rpm 4°C, centrifuge for 1 hour → carefully remove the upper layer of liquid, leaving about 0.1mL in the tube;

[0018] b) Template extraction: (1) Add 150-200 μL of 5% Chelex-100 to the tube, shake vigorously for 10 seconds; (2) bathe in water at 56°C for 20 minutes, and mix several ...

Embodiment 2

[0021] Embodiment 2: detect the pathogenic bacteria of isolation culture

[0022] Take 1mL of the culture of spiroplasma pathogenic bacteria purely cultivated in the laboratory and dilute it to a spiroplasma concentration of 10 6 Individual / mL, 13000rpm, 4 ℃, centrifugal 30 minutes, remove supernatant; Template extraction, PCR reaction are the same as embodiment 1, 2% agarose electrophoresis as figure 2 Shown in track 6. figure 2 Middle M, Marker, from bottom to top are 100bp, 200bp, 300bp, 400bp, etc. The spiroplasma-specific band appears between 200bp and 300bp, which is 271bp.

[0023] According to the above method, the spiroplasma concentration was detected to be 10 4 pcs / mL, 10 2 samples / mL, the results are as follows figure 2 Tracks 7 and 8 are shown.

Embodiment 3

[0024] Example 3: Detection of host blood

[0025] During the high temperature in the summer of 2004, a few crabs suffering from shivering disease were collected.

[0026] a) Pretreatment: about 10 μL of blood was drawn from the living body, mixed in a small amount of PBS (pH=7.0), mixed evenly, and transferred into a 1.5 mL Eppendorf tube.

[0027] b) Template extraction: (1) Add 150-200 μL of 5% Chelex-100 to the tube containing the sample, and mix well with proteinase K (final concentration is 100 μg / mL); (2) 1.5-2 hours in a 56°C water bath, Mix several times during the period; (3) take out the vigorous shaking for 10 seconds, 99 ° C water bath for 8 minutes (strict control); (4) take out the vigorous shaking for 10 seconds, 13000 rpm, 4 ° C, centrifuge for 5 minutes; (5) transfer the supernatant ( DNA template) to another clean Eppendorf tube, stored at -20°C, and amplified as soon as possible.

[0028] c) PCR reaction is the same as in Example 1,

[0029] d) 2% agaros...

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Abstract

Disclosed is a PCR fast detection technique for Spiroplasma pathogen microorganisms comprising the following steps: (1) extracting template DNA in the detected sample with Chelex-100 method, (2) proceeding polymerase chain reaction with the 16S rDNA specific primer of the Spiroplasma, (3) subjecting the PCR product to electrophoresis detection, wherein the existence of Spiroplasma can be determined by the emergence of destination electrophoresis strip. The destination electrophoresis strip is at the position of 271bp, it can product certain diagnosis within 2-4 hours, and can carry out further semi-quantitative determination.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection of pathogenic microorganisms, in particular to a PCR rapid detection technology of spiroplasma pathogenic microorganisms. Background technique [0002] Spiroplasma is a pathogenic microorganism that parasitizes plants and insects discovered in the 1970s. In recent years, we have found this pathogenic microorganism in river crabs and crayfish. Using specific primers for the 16S RNA gene of Spiroplasma in diseased crabs and shrimps A 271bp band was amplified in vivo, and after sequencing and comparison in GenBank, it was confirmed that they were all spiroplasma microorganisms. [0003] At present, in the conventional polymerase chain reaction (PCR) detection process, the DNA extraction of the tested sample is usually carried out by the phenol method or a kit. These methods are cumbersome and time-consuming. The phenol method requires a large amount of samples and a lot of DNA los...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N27/447
Inventor 王文丁正峰顾伟
Owner NANJING NORMAL UNIVERSITY
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