Shrimp health system visual rapid detection kit based on LAMP technology

A detection kit and shrimp technology, applied in the development of aquatic pathogen detection reagents, can solve the problems of high detection cost for users, unsuitable for promotion and use by grassroots farmers, and inability to visualize display, etc., and achieve the effect of simple operation

Active Publication Date: 2020-12-08
FISHERIES RES INST OF FUJIAN
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few studies on the simultaneous detection of multiple pathogens; moreover, the amplification process of most existing LAMP detections is carried out in a reactor similar to a PCR instrument, the detection cost is high for users, and the detection results are not intuitive enough to be displayed visually. The measurement method is complicated
For example, Chinese patent application 201910090452.6 discloses a high-throughput quantitative detection kit for aquaculture animal viruses, which includes a LAMP detection primer set for 14 kinds of aquaculture animal viruses. The detection of 14 kinds of aquaculture animal viruses within 3 hours has the advantages of high throughput and rapid detection; however, this method does not get rid of the dependence on expensive instruments, and is not suitable for customs quarantine and the promotion and use of grassroots fa

Method used

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  • Shrimp health system visual rapid detection kit based on LAMP technology
  • Shrimp health system visual rapid detection kit based on LAMP technology
  • Shrimp health system visual rapid detection kit based on LAMP technology

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Experimental program
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Effect test

Embodiment 1

[0047] The gene sequence of 18S ribosomal RNA of shrimp, crab, fish and ginseng was used for multiple alignment with the 18S ribosomal RNA gene sequence of Litopenaeus vannamei shown in SEQ ID No: 1; When there are too many alignment sequences, sequences with highly conserved regions cannot be obtained; therefore, after screening, Macrobrachium rosenbergii18S (GQ131934), Penaeus indicus 18S (MH400902), Penaeus semisulcatus 18S (DQ079766), Procambarus clarkii 18S (AF436001), Scylla Multiple alignment of paramamosain 18S (KC902763) with the sequence shown in SEQ ID No: 1; the highly conserved region of the sequence shown in SEQ ID No: 1 was obtained by screening; PrimerExplorer V4 software was used to design the internal reference quality control LAMP primer set according to the conserved region; The invention designs a total of four sets of primers, which are respectively recorded as NCZK-1, NCZK-2, NCZK-3, and NCZK-4, and each set of primers respectively includes two inner prim...

Embodiment 2 4

[0052] Example 2 Amplification analysis of four sets of internal reference quality control primer sets

[0053] Template preparation: Extraction of Crayfish, Australian freshwater lobster, blue crab, large yellow croaker, double-spotted pufferfish, oblique grouper, gentian grouper, vannamei prawn, rough sea cucumber, flower sea cucumber, The genomic DNA of 11 species such as Apostichopus japonicus was used for the amplification analysis of four sets of primer sets of NCZK-1, NCZK-2, NCZK-3 and NCZK-4.

[0054] 1. The NCZK-1, NCZK-2, NCZK-3, and NCZK-4 primer combinations are respectively based on the genomic DNA of Crayfish, Australian freshwater lobster, blue crab, large yellow croaker, and Penaeus vannamei as templates, and Using water as a negative control, the LAMP reaction was performed at 63° C. for 60 minutes. After the reaction, the amplification results were analyzed by observing the amplification curve.

[0055] The LAMP reaction system (25 μL) is:

[0056]

[0...

Embodiment 3

[0088] Shrimp health system visualization rapid detection kit based on LAMP technology, including: internal reference quality control primer set and IHHNV virus primer set, white spot syndrome virus primer set, hepatic enterococcus primer set, shrimp iridescent virus primer set, shrimp acute hepatopancreas At least one of the Necroptosis-V. parahaemolyticus primer sets; also contains reaction buffer, Bst DNA polymerase, dNTPs, ddH 2 O (RNase-free) and a chromogenic dye; wherein, the chromogenic dye is any one of calcein, HNB, and phenol red reagent.

[0089] Described internal reference quality control primer set is any group in NCZK-1, NCZK-2, NCZK-3, NCZK-4 described in embodiment 1;

[0090] The IHHNV virus primer set is a group in IHHNV-1, IHHNV-2:

[0091]

[0092]

[0093] The white spot syndrome virus primer set is any one of WSSV-1, WSSV-2, WSSV-3:

[0094]

[0095] The EHP primer set is any one of EHP-1, EHP-2, EHP-3:

[0096]

[0097] The shrimp iridov...

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Abstract

The invention discloses a shrimp health system visual rapid detection kit based on an LAMP technology, and belongs to the technical field of aquatic pathogen detection reagent development. The kit comprises an internal reference quality control primer group and a to-be-detected pathogen primer group. The kit is designed for common shrimp pathogenic microorganisms of IHHNV virus, white spot syndrome virus, enterocytozoon hepatopenaei, shrimp hemocyte iridescent virus, shrimp acute hepatopancreatic necrosis disease-vibrio parahaemolyticus and the like; the kit can be used for simultaneously detecting various pathogens; by adding the internal reference quality control primer group, the influence of failure of a reaction reagent and nucleic acid extraction quality of an amplification templateon a detection result can be eliminated; the accuracy of the detection result is ensured; and the kit is suitable for screening and purchasing young shrimps and parent shrimps by grass-roots farmers and customs rapid quarantine customs clearance.

Description

technical field [0001] The invention belongs to the technical field of aquatic pathogen detection reagent development, and in particular relates to a visual rapid detection kit for shrimp health system based on LAMP technology. Background technique [0002] China is the largest shrimp producer in the world; China's shrimp production accounts for about 40% of the world's total shrimp production; and the amount of shrimp farming is increasing year by year. However, shrimp farming diseases are also constantly occurring. At present, the most serious pathogens in shrimp aquaculture are: white spot syndrome virus (WSSV) of shrimp; Infectious hypodermal and hematopoietic necrosis virus (IHHNV); Shrimp hemocyte iridescent virus, SHIV); acute hepatopancreatic necrosis disease (AHPND); shrimp Enterocytozoon hepatopenaei (EHP). [0003] In addition, my country's broodstock is heavily dependent on imports; my country's shrimp seed industry introduces about 20% of the first-generation ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6893C12Q1/689C12Q1/6844C12N15/11C12R1/63C12R1/93
CPCC12Q1/701C12Q1/6893C12Q1/689C12Q1/6844C12Q2600/166C12Q2531/119Y02A50/30
Inventor 葛辉周宸林琪吴建绍
Owner FISHERIES RES INST OF FUJIAN
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