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Primer set for simultaneously detecting three kinds of microsporidia and application of primer set

A technology of microsporidia and primer sets, which is applied in recombinant DNA technology, microbial determination/inspection, resistance to vector-borne diseases, etc., can solve the problems of complex microsporidia process, low sensitivity and low specificity, etc. Simplified steps, broad application prospects, good sensitivity and specificity

Active Publication Date: 2020-06-12
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For this reason, the present invention provides the primer group and its application for simultaneously detecting three kinds of microsporidia, to solve the microsporidia detection process complexity in the prior art, the problem of low sensitivity and specificity

Method used

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  • Primer set for simultaneously detecting three kinds of microsporidia and application of primer set
  • Primer set for simultaneously detecting three kinds of microsporidia and application of primer set
  • Primer set for simultaneously detecting three kinds of microsporidia and application of primer set

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1, Establishment of Triple PCR of Enterocystis prawn hepatica, Enterocystis artemia and Microsporidia river crab 1. Materials and methods

[0031] 1. Samples: The positive samples of Enterocystis prawns, Enterocystis artemia and Microsporidium river crabs were all collected from Yingkou and Panjin, Liaoning, and separated by the laboratory.

[0032] 2. Reagents: DNA extraction kit was purchased from Tiangen (Beijing) Biochemical Technology Co., Ltd.; PCR Mix and PCR product recovery kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0033] 3. DNA extraction of three microsporidia

[0034] (1) Cut no more than 30 mg of tissue materials containing three types of microspores, put them into centrifuge tubes containing 200 μl GA buffer solution, and vortex for 15 seconds.

[0035] (2) Add 20 μL of Proteinase K (20mg / ml) solution, vortex to mix, and briefly centrifuge to remove water droplets on the inner wall of the tube cap. Place at 56°C until the ...

Embodiment 2

[0062] Embodiment 2, the specificity experiment of above-mentioned primer set detection sample

[0063] 1. Single primer specificity experiment

[0064] The three microsporidian DNA templates were mixed in equal proportions, and a pair of primers were added for PCR amplification to test the specificity of the primers. The results showed that only the gene corresponding to the specific primers could be amplified. After mixing the DNA of the three microsporidia, PCR amplification was performed with a single primer, and the results were as follows: image 3 As shown, each pair of primers can amplify the corresponding target gene fragment, but cannot amplify other gene fragments.

[0065] 2, 3 pairs of primer specificity experiments

[0066] After mixing the DNA of the three microsporidia, three pairs of specific primers from the above primer group were added to the sample at the same time, and PCR amplification was performed at the same time. The results showed that the gene fr...

Embodiment 4

[0067] Embodiment 4, the sensitivity experiment of above-mentioned primer set detection sample

[0068] The concentration of the PCR products recovered by gel cutting in Example 1 was measured, and the copy number of each sample was calculated according to the DNA copy number calculation formula. The results are shown in Table 2.

[0069] Table 2

[0070]

[0071] Copy number = (6.02×10 14 ×concentration (ng / μL) / (DNA length×660)

[0072] The copy numbers of the three microsporidian DNA templates were diluted to approximately 5.7×10 8 , 5.7×10 7 , 5.7×10 6 , 5.7×10 5 , 5.7×10 4 , 5.7×10 3 , 5.7×10 2 , 5.7×10 1 , the three DNA templates with the same copy number are mixed in equal proportions. Take 2 μL from the mixture as a template for PCR amplification.

[0073] The mixed sample was proportionally diluted according to the above method and used in the multiple PCR method established above to test the sensitivity of the established method. The results are as follo...

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Abstract

The invention discloses a primer set for simultaneously detecting three kinds of microsporidia and application of the primer set. The primer set comprises a first specific primer pair for detecting enterocytozoon hepatopenaei, a second specific primer pair for detecting enterocytozoon artemia and a third specific primer pair for detecting enterocytozoon eriocheir. The first specific primer pair aims at a shrimp enterocytozoon hepatopenaei ptp2 gene, the second specific primer pair aims at an enterocytozoon artemia STPK gene, and the third specific primer pair aims at an enterocytozoon eriocheir swp7 gene. According to the invention, the infection conditions of enterocytozoon hepatopenaei, enterocytozoon artemia and enterocytozoon eriocheir in shrimps and crabs can be rapidly and accuratelydetected through multiple PCR reactions, and the sensitivity and specificity are better.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer set for detecting microsporidia and its application. Background technique [0002] Enterocytozoon hepatopenaei (EHP) can infect a variety of economical shrimps. After infection, the size of shrimps will be uneven, and the growth will slow down or stop, which will seriously reduce the economic value of farming. Artemia Enterocytospora (Enterocytospora Artemiae) is found in Artemia, a commonly used bait for aquatic products, and it can infect the economical shrimp, the Chinese short arm shrimp. The Chinese small long arm shrimp infected with Artemia Enterocytospora will have white turbid muscles, Phenomenon of hepatopancreatic erosion. Hepatospora eriocheir is a strictly intracellular parasitic sporozoite, mainly parasitic in the hepatopancreas of river crabs, which seriously threatens the healthy culture of river crabs. The above-mentioned three kinds of mi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6893C12N15/11
CPCC12Q1/6893C12Q2600/16Y02A50/30
Inventor 姜宏波宁梓健包杰陈启军
Owner SHENYANG AGRI UNIV
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