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Fish Rickettsia specific PCR detection kit and detection method

A Rickettsia-like and Rickettsia-like technology, applied in the field of breeding, can solve the problems of low accuracy, long time-consuming, poor sensitivity, etc., and achieve the effect of fast detection, high efficiency and accurate detection

Active Publication Date: 2018-03-16
GUANGZHOU JINSHUI ANIMAL HEALTH PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some of these methods have low accuracy, some take a long time, and some have poor sensitivity.

Method used

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  • Fish Rickettsia specific PCR detection kit and detection method
  • Fish Rickettsia specific PCR detection kit and detection method
  • Fish Rickettsia specific PCR detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: PCR rapid detection kit for fish rickettsiae

[0045] All the chemical reagents and primers of the fish Rickettsia PCR rapid detection kit described in this embodiment are purchased from professional reagent companies. However, the sources of the above-mentioned reagents and primers do not constitute any limitation to the present invention. The present invention can prepare relevant reagents and synthesize relevant primers by itself.

[0046] The kit consists of the following parts (9 samples):

[0047] (1) 2× 1.0 mL reaction mix buffer, containing the following ingredients:

[0048]

[0049] (2) Detection primers: upstream primer RLO-F: 5'-TAGGAGATGAGCCCGCGTTG-3', downstream primer RLO-R: 5'-ATTTCACATCCAACTTAATCT-3', the concentration is 10 μM, the upstream and downstream primers are mixed, 400 μL.

[0050] (3) Taq DNA polymerase 5U / μL.

[0051] (4) Sterilized ddH 2 O 1.0mL.

[0052] (5) Positive control solution: fish Rickettsia genomic DNA.

[0053] (6) Negative contr...

Embodiment 2

[0070] Example 2: PCR detection method for fish rickettsiae

[0071] Using the kit described in Example 1, proceed as follows:

[0072] (1) Take 50 mg of the sample to be tested, add 600 μL of sterilized double-distilled water, fully grind it with a glass homogenizer, place it in a refrigerator at -20°C, freeze-thaw for 3 times, centrifuge at 6000 rpm for 10 minutes, and take the supernatant Add 200μL of Tris-saturated phenol, shake and mix thoroughly, let stand for 5 minutes, then centrifuge at 12000rpm for 5 minutes, take the supernatant and add the same amount of phenol: chloroform: isoamyl alcohol for extraction twice, take the supernatant and add 2 times Volume of isopropanol, after mixing, let stand for 10 minutes, centrifuge at 12000rpm for 10 minutes, remove the supernatant, add 1mL of pre-cooled 75% ethanol, let stand for 5 minutes, centrifuge at 12000rpm for 10 minutes, remove the supernatant, and set After drying in a vacuum oven, use 100μL of sterilized ddH 2 O dissolv...

Embodiment 3

[0077] Example 3: Specific experiment of PCR rapid detection kit for fish Rickettsia

[0078] Using the kit described in Example 1, proceed as follows:

[0079] (1) The extracted rickettsiae, carp herpes virus type II, koi herpes virus, grass carp hemorrhagic disease virus, mandarin fish rhabdovirus, red sea bream iris virus, viral neuronecrosis virus, viral hemorrhage The nucleic acid of sepsis virus is used as a template for PCR detection.

[0080] (2) Take 10μL of 2× reaction mix buffer, 0.5μL of upstream and downstream primers (RLO-F, RLO-R), 0.5μL of TaqDNA polymerase, ddH 2 O 5.5μL, template 3.0μL. After mixing uniformly, centrifuge for a few seconds and place on PCR reaction machine.

[0081] (3) Carry out PCR amplification under the following conditions: 95°C for 5min, 1 cycle; 95°C for 30s, 55°C for 30s, 72°C for 30s, 30 cycles; 72°C for 10min, and finally stored at 4°C.

[0082] (4) After the reaction is over, take 10μL and add 2μL bromophenol blue to mix well, pass it on 1....

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Abstract

The invention relates to the field of breeding, in particular to a fish rickettsia-like organism specificity PCR detection kit and method. The fish rickettsia-like organism specificity PCR detection kit comprises a 2*reaction mixture buffer solution, preferably-designed upstream or downstream primers, Taq DNA polymerase, positive control fluid, negative control fluid and ddH2O. The novel and rapid PCR detection kit and method overcome defects of an existing fish rickettsia-like organism detection method. The fish rickettsia-like organism specificity PCR detection kit and method are practicable for clinic diagnosis on the fish rickettsia-like organism, have the advantages of being rapid, accurate and special, meet the requirement of clinic diagnosis, and provide convenience for fish rickettsia-like organism detection.

Description

Technical field [0001] The present invention relates to the field of aquaculture, in particular to a rapid detection kit and a detection method for specific PCR detection of Rickettsia like fish sarcoidosis, that is, a gene detection reagent for Rickettsia like fish sarcoidosis and The detection method is suitable for the epidemiological monitoring of fish-like rickettsiae, the detection of rickettsia-like body, and genetic engineering technology. Background technique [0002] For aquaculture animals, it has been clear that rickettsiae has pathogenic significance, but there is no clear description of a specific kind of rickettsia that causes aquaculture animal diseases, often rickettsia-like ( Rickettsia-likeorganisms (RLO) are described and reported. [0003] Rickettsia-like bodies are pleomorphic, but mainly spherical Gram-negative bacteria with a diameter of 0.5 to 1.5 μm. They are obligate intracellular proliferation and replicate in the host tissue cells with membrane-bound c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 雷燕卢刚肖洋张文文王娟马家好
Owner GUANGZHOU JINSHUI ANIMAL HEALTH PROD