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LAMP (Lysosomal Associated Membrane Protein) detection kit for cryptocaryon irritans of large yellow croaker and detection method of LAMP detection kit

A technology for stimulating Cryptocaryonia and detection kits, which is applied in the field of LAMP detection kits for stimulating Cryptocaryonis by large yellow croakers, can solve the problems of lack of sensitivity and specificity, inability to meet on-site and grassroots inspections, etc., and achieve high sensitivity and results Easy to observe, highly specific effects

Inactive Publication Date: 2012-05-09
NINGBO UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional detection methods such as histopathological observation and microscopic examination cannot strictly distinguish Cryptocaryonia stimuli from other parasites with similar infection symptoms, lacking sensitivity and specificity
Although the polymerase chain reaction (PCR) has extremely high sensitivity and specificity, it has been used in the diagnosis practice of various parasitic diseases, but it needs the supporting use of many precision instruments such as PCR machines, which makes it unable to meet the requirements of on-site and The need for basic inspection

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Large yellow croaker stimulates Cryptocaryonia LAMP detection kit, including 10×ThermoPol reaction buffer 250μl, concentration of 100mM MgSO 4 50 μl solution, 200 μl dNTP solution with a concentration of 10 mM, 100 μl Bst DNA polymerase solution with a concentration of 8 U / μL, 50 μl FIP / BIP primer solution with a primer concentration of 1.6-2.4 μM, and a primer solution with a concentration of 0.4-0.6 μM F3 / B3 primer solution 50 μl and double distilled water 2×300ml, the nucleotide sequence of FIP primer is ACTCGAAATC GGTAGGAGCG TGTACTGATT ACGTCCCTG, the nucleotide sequence of BIP primer is GATCCGGTGA ACCTTCTGGA TCCTTCCTCT AAGTGAAGTG, the nucleotide sequence of F3 primer is AAGTGCAAGT CATCAGCT The nucleotide sequence of the B3 primer is CGGAAACCTT GTTACGACT, and the FIP primer, BIP primer, F3 primer and B3 primer are GENBANK, the conserved 18S-ITS2 gene-specific fragment with the accession number JN636814.1, which is automatically selected and analyzed by primer explorer...

Embodiment 2

[0018]Take 50-150 μg of parasitic large yellow croaker worms or about 1 g of large yellow croaker tissues (muscle, gills, mucus, etc.) / L) wash and centrifuge once, discard the supernatant, add 450 μl of TE buffer solution, 3 μl of proteinase K solution with a concentration of 20 mg / mL, and 20 μl of SDS solution with a concentration of 20% by mass, mix well, and put After the first warm bath at 37°C for 30 minutes, after the first warm bath, add 100 μl of NaCl solution with a concentration of 5 mol / L, and 80 μl of cetyltrimethylammonium bromide-sodium chloride mixture (CTAB concentration 10%, NaCl concentration 0.7 mol / L) , mix well, and incubate again at 65°C for 10 minutes to obtain worm body fluid; add chloroform-isoamyl alcohol mixture (volume ratio 24:1) equal to the volume of the worm body fluid, mix well, and centrifuge at 10,000r / min for 5min , retain the supernatant, mix the supernatant with a phenol-chloroform-isoamyl alcohol mixture (volume ratio 25:24:1) equal to t...

Embodiment 3

[0020] Large yellow croakers were taken to stimulate Cryptocaryonia, and at the same time, LAMP was tested with samples of common bacteria Vibrio harveyi, Vibrio alginolyticus, Vibrio parahaemolyticus and freshwater melon worms, valve body worms and Benedenia as samples The specificity of reaction is extracted and detected with the same extraction and detection method as in Example 2. Simultaneously, the DNA concentration of the stimulated Cryptocaryon spp. extracted by the large yellow croaker is measured with an ultraviolet spectrophotometer, and the For the stock solution, the DNA concentration was increased to 10 -1 ~10 -8 ng / μl was serially diluted, and 1 μl of each concentration was used as a template for LAMP amplification. LAMP test results: the stimulated Cryptocaryon spp. sample was positive, and the other samples were all negative, indicating that the kit of the present invention had no cross-reaction with other common bacteria and parasites in mariculture. Inse...

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Abstract

The invention discloses an LAMP (Lysosomal Associated Membrane Protein) detection kit and a detection method of the LAMP detection kit. The LAMP detection kit comprises a reaction buffer, a MgSO4 solution, a dNTP (deoxy-Ribonucleoside Triphosphate) solution, BstDNA (BstDeoxyribonucleic Acid) polymerase solution, an FIP (Forward Inner Primer) / BIP (Backward Inner Primer) solution, an F3 / B3 primer solution and double distilled water. The LAMP detection kit has high sensitivity and high specificity for the cryptocaryon irritans of the large yellow croaker and is suitable for quick field detection for cryptocaryon irritans disease of the large yellow croaker in a marine park; and the LAMP detection kit for detecting the cryptocaryon irritans of the large yellow croaker has the advantages of simpleness in operation, simpleness for requirement on instruments, easy observation for result and the like and provides technical support for early control over the cryptocaryon irritans disease.

Description

technical field [0001] The invention relates to a detection technology for stimulating Cryptocaryonia, in particular to a LAMP detection kit and a detection method for stimulating Cryptocaryon by large yellow croaker. Background technique [0002] Stimulate Cryptocaryon ( Cryptocaryon irritans ) is a ciliate that parasitizes the skin and gill epithelium of seawater bony fishes, causing fish abnormalities, epithelial hyperplasia, breathing difficulties, and mechanical damage, which in turn leads to secondary infection of pathogens. It has been listed as a new version of the "List of Types I, II, and III Animal Diseases" issued by the Ministry of Agriculture in December 2008. The "white spot disease" and secondary bacterial infections caused by it (white gill disease, bacterial infection) Ulcers, etc.) have brought huge losses to my country's mariculture. For example, in 2009, large-scale large yellow croaker-stimulated cryptocystosis broke out in the entire Ningde City. The i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/90
Inventor 王国良周旻曦张继挺
Owner NINGBO UNIV
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