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Nucleic acid sequence combination, kit and detection method for LAMP-CRISPR isothermal detection of prawn enterocytozoon hepatopenaei

A technology of LAMP-CRISPR and hepatic enterocystosis, applied in the field of biotechnology detection, can solve the problem of low sensitivity

Pending Publication Date: 2020-10-23
HANGZHOU ALLSHENG INSTR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the problems of low sensitivity in the detection method of Enteroplasma hepatica in the prior art, the present invention provides a combination of nucleic acid sequences, a kit and a detection method for isothermal detection of Enteroplasma hepatica LAMP-CRISPR

Method used

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  • Nucleic acid sequence combination, kit and detection method for LAMP-CRISPR isothermal detection of prawn enterocytozoon hepatopenaei
  • Nucleic acid sequence combination, kit and detection method for LAMP-CRISPR isothermal detection of prawn enterocytozoon hepatopenaei
  • Nucleic acid sequence combination, kit and detection method for LAMP-CRISPR isothermal detection of prawn enterocytozoon hepatopenaei

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Experimental program
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Effect test

Embodiment 1

[0069] like Figure 1-13 As shown, a reaction tube for nucleic acid amplification and detection in a two-step method includes a tube body 1 for adding the first reaction solution and a cover body 2 that cooperates with the tube body 1 to seal. A liquid box 3 for temporarily storing the second reaction solution during a one-step reaction. The bottom surface of the liquid box 3 has a first opening 31 for the second reaction solution to flow into the tube body 1. The top surface of the liquid box 3 is provided with The second opening 32 for adding the second reaction solution, and the first opening 31 is sealed with a sealing film before adding the second reaction solution. The overall structure of the tube body 1 is similar to that of a conventional PCR reaction tube, and the bottom is used for adding reaction solution for reaction.

[0070] like Figure 5 and 7 As shown, the liquid box 3 has a plug portion 33 extending into the tube body 1 and plugged with the mouth of the t...

Embodiment 2

[0080] 1. Experimental materials

[0081] Actual sample 1: No. ND01, sampled from shrimp seedlings in the Ningde shrimp seedling breeding base in 2019;

[0082] Actual sample 2: No. HZ02, sampled in 2019 from juvenile shrimps in Hangzhou Shrimp Breeding Base;

[0083] Actual sample 3: No. HZ03, sampled in 2019 from the juvenile shrimp in the shrimp seedling breeding base in Hangzhou;

[0084] Actual sample 4: No. FZ04, sampled from juvenile shrimp in Fuzhou Shrimp Breeding Base in 2019;

[0085] Actual sample 5: No. FZ05, juvenile shrimp sampled at the Fuzhou Shrimp Breeding Base in 2019;

[0086] Actual sample 6: No. FZ06, sampled from juvenile shrimp in the Fuzhou Shrimp Breeding Base in 2019.

[0087] 2. Extraction of Shrimp Iridovirus DNA

[0088] (1) Weigh 30 mg of experimental samples (parotid glands for sample 1, whole shrimp seedlings for sample 2), add 500 μL of physiological saline, remove the liquid after mixing upside down, and repeat this step once;

[0089] ...

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Abstract

The invention discloses a nucleic acid sequence combination, a kit and a detection method for LAMP-CRISPR isothermal detection of prawn enterocytozoon hepatopenaei. The nucleic acid sequence combination comprises a primer group for LAMP detection, crRNA and a probe. The detection method uses the kit, and the detection method comprises the following steps: (1) extracting DNA of a to-be-detected sample; (2) taking the DNA extracted in the step (1) as a template, and carrying out LAMP isothermal amplification reaction by using the primer group in the claim 1; (3) carrying out CRISPR isothermal detection reaction by using the crRNA and the probe in the claim 1 by taking an LAMP isothermal amplification reaction product in the step (2) as a template; and (4) analyzing the CRISPR isothermal detection result obtained in the step (3) through an isothermal amplification instrument, and if an amplification curve occurs, the result is positive, otherwise, the result is negative. The detection method is rapid, efficient, simple and convenient to operate, high in specificity, high in sensitivity and simple and convenient to identify.

Description

technical field [0001] The invention relates to the technical field of biotechnology detection, in particular to a nucleic acid sequence combination, a kit and a detection method for the isothermal detection of Enterococcus hepatica LAMP-CRISPR. Background technique [0002] Enterocytozoon hepatopenaei (EHP) belongs to the family Microsporidiaceae and the genus Enterococcus, and was first discovered in 2009 in the slow-growing Penaeus monodon cultured in Thailand. The shrimps infected with Microsporidia have been reported in China to include Litopenaeus vannamei, Palaemoncarincauda and Fenneropenaeus chi-nensis. Generally, death does not occur immediately after being infected with EHP. Shrimp can eat normally, but the muscles turn white, the intestinal absorption function decreases, and the hepatopancreas atrophy occurs in severe cases. It is usually found that the growth is slow after 30-45 days of breeding. One of the main reasons for the slow growth of prawns has serious...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/6844C12N15/11C12M1/34C12M1/40C12M1/24
CPCC12Q1/6893C12Q1/6844B01L3/502B01L2300/044C12Q2531/119C12Q2521/327C12Q2563/107
Inventor 黄俊骆志成樊伟东张徐俞骆广进杨稳付媛媛郑晓叶俞彬荣郑利俊俞晓平
Owner HANGZHOU ALLSHENG INSTR
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