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Method and kit for detecting multiple high-pathogenicity pathogenic bacteria

A highly pathogenic and pathogenic bacteria technology, applied in the field of molecular biology and nucleic acid detection, can solve the problems of lack of highly pathogenic pathogens, low sensitivity, cumbersome process, etc.

Active Publication Date: 2012-11-28
SHANGHAI TELLGEN LIFE SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The second is to detect and identify the DNA of the specific gene of the bacteria. This method does not require cultivation, which greatly shortens the detection time, and generally only takes a few hours to complete; but this method has a disadvantage, that is, each detection It can only judge whether it is a certain germ or not, and then conduct another reaction to judge whether it is another germ. Therefore, this method has a corresponding detection range, that is, it can only detect the germs within its detection range. If you want to detect Ten kinds of germs need to carry out ten reactions. Although these reactions can be carried out sequentially or in parallel, it is generally cumbersome.
However, like other solid-state chip detection, due to the defects of the solid-state chip itself, this method requires multiple washings, the process is still cumbersome, and the sensitivity is generally not high. More importantly, the repeatability is poor and the detection quality is unstable.
[0009] In summary, there is still a lack of satisfactory technology in the field that can simultaneously detect multiple highly pathogenic pathogenic bacteria

Method used

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  • Method and kit for detecting multiple high-pathogenicity pathogenic bacteria
  • Method and kit for detecting multiple high-pathogenicity pathogenic bacteria
  • Method and kit for detecting multiple high-pathogenicity pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Example 1 Detection of highly pathogenic pathogens

[0121] 1. Probe microsphere coating:

[0122] 1. Follow the list below to synthesize the probe

[0123]

[0124] Note: The spacer arm is a poly(T) composed of 10 Ts 10 -, so the probe Ban-P1 is equal to poly(T) 10 +SEQ ID NO.: 3, probe Ype-P1 is equal to poly(T) 10 +SEQ ID NO.: 4, and so on.

[0125] 2. Select 11 kinds of fluorescent coded microspheres No. 22, 24, 26, 28, 31, 32, 33, 34, 35, 36, and 42 [Luminex Company], and perform probe analysis corresponding to each probe in the table above. Coating, the method is as follows:

[0126] (1) Put all kinds of microspheres and a portion of EDC powder stored at -20°C to equilibrate at room temperature for 30 minutes;

[0127] (2) Take each corresponding probe and dissolve it with double distilled water, the concentration is 0.01mM (10pmol / μL);

[0128] (3) Mix the microspheres uniformly with an oscillator;

[0129] (4) Take 50ul each (6.0×10 5 ) The microspheres are placed in a clea...

Embodiment 2

[0195] 1. Probe microsphere coating:

[0196] 1. Follow the list below to synthesize the probe

[0197]

[0198] Note: The spacer arm is -HEG-.

[0199] 2. Select 11 kinds of fluorescent coded microspheres No. 22, 24, 26, 28, 31, 32, 33, 34, 35, 36, and 42 [Luminex Company], and perform probe analysis corresponding to each probe in the table above. Coating, the method is as follows:

[0200] (1) Put all kinds of microspheres and a portion of EDC powder stored at -20°C to equilibrate at room temperature for 30 minutes;

[0201] (2) Take each corresponding probe and dissolve it with double distilled water, the concentration is 0.01mM (10pmol / μL);

[0202] (3) Mix the microspheres uniformly with an oscillator;

[0203] (4) Take 50μl each (6.0×10 5 ) The microspheres are placed in a clean 1.5ml centrifuge tube with a pre-marked number;

[0204] (5) Centrifuge at 8000g centrifugal force for 5 minutes to precipitate the microspheres, carefully discard the supernatant;

[0205] (6) Add 100μl of d...

Embodiment 3

[0265] Example 3 Detection of highly pathogenic pathogens

[0266] Repeat Example 2 with the difference that samples 16-27 are replaced with samples Nos. 28 and 29. Among them, the No. 1 and No. 16 samples were combined to form the No. 28 sample. Combine No. 3 and No. 25 samples as No. 29 sample.

[0267] The test results showed that sample No. 28 contained Clostridium botulinum and Streptococcus suis; sample No. 29 contained Tulaversa and Legionella pneumophila. The test result is consistent with the actual situation of the sample.

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Abstract

The invention provides a method and kit for detecting multiple high-pathogenicity pathogenic bacteria, particularly a method for simultaneously detecting multiple high-pathogenicity pathogenic bacteria, which comprises the following steps: in a polymerase reaction system, carrying out PCR (polymerase chain reaction) on high-pathogenicity pathogenic bacteria by using a specific primer set, thereby obtaining an amplification product; and detecting with a specific probe or probe microsphere. The invention also provides a corresponding kit. The invention can sensitively and conveniently detect and identify multiple high-pathogenicity pathogenic bacteria, including Bacillus anthracis, Yersinia pestis, Clostridium botulinum, Brucella, Streptococcus suis, Vibrio cholerae, Francisella tularensis, Pseudomonas mallei (or Pseudomonas pseudomallei), Coxiella burnetii and Legionella pneumophila.

Description

Technical field [0001] The invention relates to the technical fields of molecular biology and nucleic acid detection. Specifically, the present invention relates to methods and kits for detecting multiple highly pathogenic pathogenic bacteria. Background technique [0002] Pathogens that cause sudden infectious diseases can cause severe epidemics in peacetime, and can be used as biological weapons in wartime. These pathogenic microorganisms are mainly divided into six categories: viruses, bacteria, rickettsiae, chlamydia, mycoplasma and fungi, and there are more than 40 species. Bacillus anthracis (Bacillus anthracis), Yersinia pestis, Clostridium botulinum, Brucella mallei, Brucella abortus, Streptococcus suis 2 suis II), Vibrio cholerae, Francisella tularensis, Burkholderia mallei, Burkholderia pseudomallei, Coxiella Burnetii), Legionella pneumophila (Legionella pneumophila) are all highly pathogenic pathogenic bacteria. [0003] At present, the main bacterial pathogens that ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12Q1/04C12N15/11
CPCY02A50/30
Inventor 谭畅王勤熙张秀斐姚见儿
Owner SHANGHAI TELLGEN LIFE SCI CO LTD
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