PCR detection kit and method for Klebsiella pneumonia infecting Chinese giant salamanders

A Klebsiella and detection kit technology, applied in the field of biomedicine, can solve the problems of lack of rapid, accurate and sensitive detection methods

Active Publication Date: 2015-12-09
CHONGQING UNIV OF ARTS & SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The diagnosis of traditional pathogenic bacteria requires microscopic observation, and there is a lack of rapid, accurate and sensitive detection methods

Method used

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  • PCR detection kit and method for Klebsiella pneumonia infecting Chinese giant salamanders
  • PCR detection kit and method for Klebsiella pneumonia infecting Chinese giant salamanders
  • PCR detection kit and method for Klebsiella pneumonia infecting Chinese giant salamanders

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The PCR rapid detection kit of the Chinese giant salamander infected with Klebsiella pneumoniae of embodiment 1

[0055] All the chemical reagents and primers in the PCR rapid detection kit for Chinese giant salamander infected with Klebsiella pneumoniae described in this example were purchased from professional reagent companies, or they could be prepared by themselves, and the upstream and downstream primers could also be synthesized by themselves.

[0056] The kit consists of the following parts (9 samples):

[0057] (1) A total of 1.0 mL of 2-fold reaction mixing buffer (ReactionMixBuffer), including the following components:

[0058]

[0059] (2) A total of 200 μL of detection primers: upstream primer K-F: 5’-CAGCGATTATGACAGCAAAG-3’, downstream primer K-R: 5’-TCCAGACCATCCACCAGA-3’, the concentration is 10 μM / L, and the upstream and downstream primers are mixed.

[0060] (3) Taq enzyme 5U / μL.

[0061] (4) Sterilized ddH 2 O1.0mL.

[0062] (5) Positive control...

Embodiment 2

[0071] Example 2 The PCR detection method of Chinese giant salamander infection Klebsiella pneumoniae

[0072] (1) Using the test kit described in Example 1, proceed as follows:

[0073] ①Take the lung, liver or foot skin tissue of the diseased giant salamander, cut them into pieces, put them in a homogenizer, add the homogenate solution, grind them in an ice bath, grind them and put them in a centrifuge tube;

[0074] ②Add 1%-3% β-mercaptoethanol solution, mix well and then bathe in water at 60-70℃ for 0.5-1h, take it out and shake well every 3-5 minutes;

[0075] ③ Centrifuge at 10000-15000rpm for 3-7min. After centrifugation, remove the supernatant into another sterile centrifuge tube and discard the precipitate;

[0076] ④Add an equal volume of chloroform:isoamyl alcohol mixed solvent with a volume ratio of 20-30:1 to the supernatant, mix well, centrifuge at 8000-12000rpm for 5-15min, and take the supernatant to another sterile centrifuge tube middle;

[0077] ⑤Add an e...

Embodiment 3

[0085]Example 3 Specificity experiment of Chinese giant salamander infection Klebsiella pneumoniae PCR rapid detection kit

[0086] Using the kit described in Example 1, proceed as follows:

[0087] (1) Using the extracted DNA of Chinese giant salamander as a template, carry out PCR detection.

[0088] (2) Take 10 μL of 2-fold reaction mixture buffer, 0.5 μL each of upstream and downstream primers (K-F, K-R), 1 μL of TaqDNA polymerase, ddH 2 O1 μL, template 1.0 μL. After mixing evenly, centrifuge for a few seconds, and place on a PCR reaction instrument for amplification reaction.

[0089] (3) Carry out PCR amplification reaction according to the following conditions: pre-denaturation at 95°C for 5 minutes, 1 cycle; then denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 30 seconds, a total of 30 cycles; extension at 72°C 7 minutes, and finally stored at 4°C.

[0090] (4) After the reaction is over, take 10 μL and add it to 2 μL br...

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Abstract

The invention discloses a PCR detection kit and method for Klebsiella pneumonia infecting Chinese giant salamanders. The detection kit comprises 1.0mL of 2-fold reaction mixed buffer solution, upstream and downstream primers, Taq DNA polymerase, positive control liquid, negative control liquid and ddH2O. The detection method for clinically diagnosing the Klebsiella pneumonia infecting the Chinese giant salamanders is practicable, fast, accurate and specific, capable of satisfying the requirements of clinical diagnosing, and capable of bringing convenience to the detection of the Klebsiella pneumonia infecting the Chinese giant salamanders.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a bioengineering technology and a molecular biology method, in particular to a detection reagent and a detection method for Klebsiella pneumoniae infecting Chinese giant salamanders, which are suitable for Klebsiella pneumoniae infecting Chinese giant salamanders Bacteria detection. Background technique [0002] The Chinese giant salamander is a second-class protected animal in the country. Due to its high scientific research, nutritional and medicinal value, people's demand for it is gradually increasing. However, in recent years, due to habitat loss, environmental pollution, and human overfishing, this species has become extremely endangered. In order to effectively protect species diversity, many places in my country have begun large-scale and industrialized artificial breeding and breeding of giant salamanders. . However, with the continuous increase in the number of breeding, variou...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/22
CPCC12Q1/689
Inventor 樊汶樵孙翰昌吴宝红姜玉松徐敬明
Owner CHONGQING UNIV OF ARTS & SCI
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