PCR detection kit and detection method of rana boulengeri-infected shewanella putrefaciens

A technology of Shewanella spoilage and detection kit, which is applied in the fields of bioengineering technology and biomedicine, and can solve problems such as cumbersome detection methods

Inactive 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

[0006] The above detection methods are cumbersome, especially for spiny-bellied frogs, which lack specificity and sensitivity for detecting whether spiny-bellied frogs are infected by Shewanella putrefaciens. For spiny-bellied frogs, it is necessary to study a simple, fast, specific and Sensitive detection kit for detection

Method used

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  • PCR detection kit and detection method of rana boulengeri-infected shewanella putrefaciens
  • PCR detection kit and detection method of rana boulengeri-infected shewanella putrefaciens
  • PCR detection kit and detection method of rana boulengeri-infected shewanella putrefaciens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 detects the rapid detection kit of PCR that the spiny-bellied frog is infected with Shewanella putrefaciens

[0049] The chemical reagents and primers used in the PCR detection kit for detecting Shewanella putrefaciens of the present invention are all purchased from professional reagent companies, and relevant reagents and synthetic relevant primers can also be prepared by themselves.

[0050] The kit consists of the following parts (9 samples):

[0051] (1) 1.0 mL of 2-fold reaction mixing buffer (ReactionMixBuffer), containing the following components:

[0052]

[0053] (2) Detection primers: upstream primer S-F: 5'-CAGCGATTATGACAGCAAAG-3', downstream primer S-R: 5'-TCCAGACCATCCACCAGA-3', the concentration is 10 μM / L, the upstream and downstream primers are mixed, 200 μL.

[0054] (3) Taq enzyme 5U / μL.

[0055] (4) Sterilized ddH 2 O1.0mL.

[0056] (5) Positive control solution: genomic DNA of Rana spinosa.

[0057] (6) Negative control solution: ...

Embodiment 2

[0067] The PCR detection method of the Shewanella putrefaciens of embodiment 2 spiny-bellied frogs

[0068] (1) Using the kit described in Example 1, proceed as follows:

[0069] ①Take the oral cavity, esophagus, stomach or intestinal tissue of the diseased frog, cut it into pieces, put it in a homogenizer, add the homogenate solution, grind it in an ice bath, and put it in a centrifuge tube after grinding;

[0070] 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;

[0071] ③Centrifuge at 10000-15000rpm for 3-7min, after centrifugation, remove the supernatant into another sterile centrifuge tube, and discard the precipitate;

[0072] 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 into another sterile centrifuge tube ;

[0073] ⑤Add an equal vo...

Embodiment 3

[0081] The Shewanella putrefaciens PCR detection kit specificity experiment of embodiment 3 spiny-bellied frog

[0082] Using the kit described in Example 1, proceed as follows:

[0083] (1) The DNA of Rana spinosa extracted according to the extraction method in Example 2 was used as a template for PCR detection.

[0084] (2) Take 10 μL of 2-fold reaction mixture buffer, 0.5 μL each of upstream and downstream primers (S-F, S-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.

[0085] (3) Perform PCR amplification under the following conditions: 95°C for 5 min, 1 cycle; 95°C for 30 s, 56°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 7 min, and finally store at 4°C.

[0086] (4) After the reaction is over, take 10 μL and add it to 2 μL bromophenol blue and mix well. After electrophoresis on 1% agarose gel for 20 minutes, observe it on a UV instrument. If a bright reaction band...

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PUM

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Abstract

The invention discloses a PCR detection kit and a detection method of rana boulengeri-infected shewanella putrefaciens, wherein the detection kit consists of 1.0mL of a double-reaction mixed buffer solution; forward and reverse primers according to an optimized design; Taq DNA polymerase; a positive control solution; a negative control solution; and ddH2O. The detection kit disclosed by the invention can overcome the shortcomings of an existing shewanella putrefaciens detection method; and the rana boulengeri shewanella putrefaciens detection kit disclosed by the invention has the advantages of being rapid, accurate and specific in clinical field, so as to facilitate the detection of rana boulengeri-infected shewanella putrefaciens.

Description

technical field [0001] The invention belongs to the fields of bioengineering technology and biomedicine, and specifically relates to a detection kit and a detection method for detecting Shewanella putrefaciens infection of spiny-bellied frogs, which are applicable to the detection of Shewanella putrefacilis infection of spiny-bellied frogs. Background technique [0002] The spiny-bellied frog is a large-scale edible frog with rich nutrition and medicinal value. The protein content of its fresh meat jerky sample is 16.25%, and the fat content is 1.26%. It has rich nourishing effects and medicinal value. With the continuous improvement of the level, the edible value of spiny-bellied frog is also gradually rising, and it has a good development prospect. At the same time, as a transitional animal in the evolution of vertebrates, it is an ideal experimental animal for the study of anatomy, genetics and embryonic development. Due to serious environmental pollution, destruction of...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q2545/113
Inventor 徐敬明姜玉松樊汶樵吴宝红李晓英
Owner CHONGQING UNIV OF ARTS & SCI
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