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Visual rapid detection kit for enterocytozoon hepatopenaei (EHP)

A technology of shrimp hepatic enterocystosis and detection kit, which is applied in the field of pathogen detection in aquaculture, and can solve the problems that hinder the popularization and application of LAMP detection technology, cumbersome and complicated operation, etc.

Inactive Publication Date: 2019-02-15
天津市水生动物疫病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, researchers have developed and established the LAMP detection technology of EHP. However, the traditional virus nucleic acid extraction requires professional laboratory equipment, and the operation is cumbersome and complicated, which hinders the promotion and application of LAMP detection technology in the breeding process.

Method used

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  • Visual rapid detection kit for enterocytozoon hepatopenaei (EHP)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] A rapid detection kit for shrimp hepatic enteroplasma visualization, comprising:

[0042] Reagent A: tissue lysate Ⅰ;

[0043] Reagent B: tissue lysate II;

[0044] Reagent C: isopropanol;

[0045] Reagent D: 70% ethanol water solution by volume;

[0046] Reagent E: sterile double distilled water;

[0047] Reagent F: 100mg nucleic acid extraction magnetic beads resuspended in 1ml double distilled water;

[0048] Reagent G: Shrimp Enterococcus positive plasmid;

[0049] Detection tube with reagent H: 2.5 μl of 10×Buffer, 2.5 μl of 10 mM deoxyribonucleotide, 100 mM MgSO in the detection tube 42 μl, 5 M betaine 1 μl, 100 μM primer EHP-FIP 0.4 μl, 100 μM primer EHP-BIP 0.4 μl, 100 μM primer EHP-LF 0.2 μl, 100 μM primer EHP-LB 0.2 μl, 100 μM primer EHP-F3 0.05 μl, 100 μM primer EHP-B3 0.05 μl, 8 U / μl Bst DNA polymerase 1 μl, calcein dye Reagent H mixed with 11.7 μl of sterile double distilled water;

[0050] Grinding rod, disposable straw, toothpick, 1.5ml sample tu...

Embodiment 2

[0052] A rapid detection kit for shrimp hepatic enteroplasma visualization, comprising:

[0053] It is the 70% ethanol water solution that the volume percentage concentration of reagent D of embodiment 1 is replaced by the volume percentage concentration of 65% ethanol water solution;

[0054] The 100 mg nucleic acid extraction magnetic beads in the reagent F of Example 1 were replaced with 90 mg nucleic acid extraction magnetic beads; the others were the same as in Example 1.

Embodiment 3

[0056] A rapid detection kit for shrimp hepatic enteroplasma visualization, comprising:

[0057] It is the 70% ethanol aqueous solution that the volume percent concentration of the reagent D of embodiment 1 is replaced by the 80% ethanol aqueous solution with the volume percent concentration;

[0058] The 100 mg nucleic acid extraction magnetic beads in the reagent F of Example 1 were replaced with 110 mg nucleic acid extraction magnetic beads; the others were the same as in Example 1.

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Abstract

The invention discloses a visual rapid detection kit for enterocytozoon hepatopenaei (EHP). The visual rapid detection kit comprises a reagent A, a reagent B, a reagent C, a reagent D, a reagent E, areagent F, a reagent G and a detection tube loaded with a reagent H. The kit provided by the invention is high in detection specificity and detection sensitivity, and magnetic beads are adopted for extracting nucleic acid, thereby adequately extracting the nucleic acid out of a sample to be tested, avoiding the use of large-scale instruments, simplifying the complicated operation process and greatly shortening the time spent in nucleic acid extraction. The use of dye calcein can enable a user to strictly follow the principle of not opening a lid during a loop-mediated isothermal amplification(LAMP) reaction, thereby greatly reducing the false positive rate. The kit provided by the invention is simple and convenient in the whole operation process, and the detection can be finished within 1.5-2 hours without adopting high-end instruments.

Description

technical field [0001] The invention relates to a detection technology for pathogens in aquaculture, in particular to a detection kit for Enterocytozoon hepatopenaei (EHP). Background technique [0002] Enterocytozoon hepatopenaei (EHP), the full name of intestinal epithelial microsporidia, belongs to Microsporidiaceae, Enterocystis genus, and was first discovered and isolated from the slow-growing Penaeus monodon in Thailand in 2009. In 2013, EHP was discovered for the first time in China. EHP-infected prawns show normal food intake, but their body color is whitish, intestinal absorption function decreases, and hepatopancreas atrophy occurs in severe cases. Generally, there will be no large-scale death phenomenon, but it will cause slow growth of prawns, resulting in feed loss and low yield. Thereby causing greater economic losses to fishermen. At present, there is no effective treatment for EHP. Early detection and necessary measures to prevent the spread of pathogens ar...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888
CPCC12Q1/6888
Inventor 薛淑霞孙妍
Owner 天津市水生动物疫病预防控制中心
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