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Reagent box and detection method

The technology of a kit and a detection kit, which is applied in the field of breeding, can solve the problems of nucleic acid and primer reporting that have not yet been invented, and achieve the effects of rapid detection, high efficiency, and high detection sensitivity

Inactive Publication Date: 2015-08-19
GUANGZHOU JINSHUI ANIMAL HEALTH PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] After searching the literature of the prior art, it is found that there are no reports related to the microsporidia detection method, nucleic acid and primer pair of Litopenaeus vannamei intestinal epithelial cells of the present invention.

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1: Microsporidia PCR Rapid Detection Kit for Litopenaeus vannamei Intestinal Epithelial Cells

[0047] All the chemical reagents and primers of the microsporidia rapid detection kit for Litopenaeus vannamei intestinal epithelial cells described in this example were purchased from professional reagent companies. However, the sources of the above reagents and primers do not constitute any limitation to the present invention, and the present invention can prepare relevant reagents and synthesize relevant primers by itself.

[0048] The kit consists of the following parts (9 samples):

[0049] (1) 1.0mL 2×Reaction Mix Buffer, containing the following components:

[0050]

[0051] (2) Detection primers: upstream primer ECZ-F: 5'-TGTGGGAGAAATCTTAGTTTT-3', downstream primer ECZ-R: 5'-ATTGCGCTTGCTGCCCAGGAT-3', the concentration is 10 μM, the upstream and downstream primers are mixed, 400 μL.

[0052] (3) Taq enzyme 5U / μL.

[0053] (4) Sterilized ddH 2 O 1.0 mL. ...

Embodiment 2

[0072] Example 2: PCR detection method for microsporidia in intestinal epithelial cells of Litopenaeus vannamei

[0073] Using the kit described in Example 1, proceed as follows:

[0074] (1) Take 50 mg of hepatopancreas and intestine samples of Litopenaeus vannamei to be tested, add 600 μL of sterilized double-distilled water, grind them thoroughly with a glass homogenizer, and place them in a -20°C refrigerator for three times of freezing and thawing. Centrifuge at low temperature at 6000rpm for 10 minutes, take the supernatant, add 200μL Tris-saturated phenol, shake and mix well, let stand for 5 minutes, then centrifuge at 12000rpm for 5 minutes, take the supernatant and add an equal amount of phenol: chloroform: isoamyl alcohol to extract Extract twice, take the supernatant and add 2 times the volume of isopropanol, mix well, let stand for 10 minutes, centrifuge at 12000rpm for 10 minutes, remove the supernatant, add 1mL pre-cooled 75% ethanol, let stand for 5 minutes, Ce...

Embodiment 3

[0079] Example 3: Specificity experiment of PCR rapid detection kit for microsporidia in intestinal epithelial cells of Litopenaeus vannamei

[0080] Using the kit described in Example 1, proceed as follows:

[0081] (1) The extracted intestinal epithelial microsporidia, white spot syndrome virus, infectious subcutaneous and hematopoietic organ necrosis virus, prawn baculovirus, hepatopancreatic parvovirus, taura virus, yellow head virus, infectious muscle Necrosis virus, Vibrio parahaemolyticus, and Vibrio alginolyticus were used as templates for PCR detection.

[0082] (2) Take 10 μL of 2× reaction mixture buffer, 0.5 μL of upstream and downstream primers (ECZ-F, ECZ-R), 0.5 μL of Taq DNA polymerase, ddH 2 O 5.5 μL, template 3.0 μL. After mixing evenly, centrifuge for a few seconds and place on a PCR reaction instrument.

[0083] (3) Perform PCR amplification under the following conditions: 95°C for 5 min, 1 cycle; 94°C for 30 s, 53°C for 30 s, 72°C for 40 s, 30 cycles; 7...

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Abstract

The invention belongs to the field of breeding and particularly discloses a reagent box and a detection method. The reagent box comprises a 2x reacting, mixing and buffering solution, preferably designed upstream and downstream primers, a Taq DNA polymerase, a positive control solution, a negative control solution and ddH2O. The invention overcomes the defects of the method for detecting microsporidia of intestinal epithelial cells in the prior art and provides the novel reagent box and the detection method for rapidly detecting PCR (Polymerase Chain Reaction). The reagent box and the detection method are practicable and feasible in clinical diagnosis of microsporidia of intestinal epithelial cells of litopenaeus vannamei, have the advantages of high speed, accuracy and specificity, meet the requirements of clinical diagnosis and provide convenience for detecting the microsporidia of intestinal epithelial cells of litopenaeus vannamei.

Description

technical field [0001] The invention relates to the field of breeding, in particular to a genetic detection reagent and a detection method for Microsporidia in intestinal epithelial cells of Litopenaeus vannamei, which are suitable for epidemiological monitoring of Microsporidia in intestinal epithelial cells of Litopenaeus vannamei, intestinal Detection of epithelial microsporidia and genetic engineering technology and other fields. Background technique [0002] Enterocytozoon hepatopenaei (EHP), short for Enterocytozoon hepatopenaei, is a very small sporozoite with a body length of less than 1 micron. Intestinal epithelial microsporidia were first discovered and reported in 2009 by Thai scientists in cultured Penaeus monodon. In 2014, Thai scientists found that microsporidia of intestinal epithelial cells were one of the main reasons for the slow growth of Litopenaeus vannamei in recent years. The Yellow Sea Fisheries Research Institute of the Chinese Academy of Fishery ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/90
CPCC12Q1/6893C12Q1/686C12Q2565/125C12Q2563/173
Inventor 雷燕肖洋张会军
Owner GUANGZHOU JINSHUI ANIMAL HEALTH PROD
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