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LAMP visual rapid detection kit of silkworm pathogenic micro spore worms and detection method thereof

A detection kit and technology of microsporidia, applied in the biological field, can solve the problems of time-consuming and laborious, low specificity, unsuitable field detection, etc., and achieve the effect of short detection time, easy results and guaranteed specificity

Active Publication Date: 2014-09-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above-mentioned detection methods all have disadvantages such as time-consuming and labor-intensive, low specificity, and not suitable for field detection, which are not conducive to widespread promotion and use in actual production.
At present, there are no relevant technical reports on the application of LAMP technology to the detection of various silkworm pathogenic microsporidia

Method used

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  • LAMP visual rapid detection kit of silkworm pathogenic micro spore worms and detection method thereof
  • LAMP visual rapid detection kit of silkworm pathogenic micro spore worms and detection method thereof
  • LAMP visual rapid detection kit of silkworm pathogenic micro spore worms and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The preparation method of the LAMP visual rapid detection kit for the pathogen of Bombyx mori pathogen Microsporidia, the specific method steps are as follows:

[0044] S1. Retrieving from the gene database the 16SrDNA of the pathogenic microsporidia of the silkworm: Nosema bombycis, Nosema rapae, Nospora litura, Microspores of Spodoptera litura, Microspores of locusts, Microsporidia of corn borer, etc. Sequence, homology analysis was carried out by BLAST software, and the conservative sequence of the 16S rDNA of Nosema bombycis was obtained as the target gene;

[0045] S2. Use online software: Primer ExplorerV4 (http: / / primerexplorer.jp / elamp4.0.0 / index.html) to design and synthesize four specific primer sets for LAMP detection; the primer set includes a pair of inner primers NFI1 / NBI1 and a pair The outer primer NF1 / NB1, the four LAMP primer sequences are: NF1: 5'-GGGGATAGTATGATCGCAAG-3', the primer sequence is shown in SEQ ID NO:1; NB1: 5'-CCTCTCCTTCATATGTATCACTAC-3', the...

Embodiment 2

[0055] The detection was carried out using the LAMP visualized rapid detection kit of Nosema bombycis, which was prepared by referring to Example 1, and the specific steps are as follows:

[0056] S1. Preparation of nucleic acid to be tested:

[0057] Adjust the temperature in an electric blast drying oven to 110℃, and heat; take 200 μL of spore liquid or moth moth grinding liquid sterile 1.5mL centrifuge tube; 12000 r / min, centrifuge for 5 minutes, discard the supernatant; DNA lysis solution ( 10 mM Tris-Cl, pH 8.0; 0.1 M EDTA, pH 8.0; 0.5% SDS) incubate in a 110 ℃ electric blast drying oven for 10-15 min; add 100 μL of lysis solution to the spore precipitation After mixing, boil for 10-15 min; centrifuge the mixture at 12000 r / min for 10 min, take the supernatant, which is the DNA solution, and store it in the refrigerator at -20℃ for later use.

[0058] S2. Configure the reaction system: configure in a 200μL reaction tube and use an optimized 25μL reaction system, as described in...

Embodiment 3

[0065] The LAMP visualized rapid detection kit of Nosema Bombyx mori pathogen prepared in Reference Example 1 was used to detect various Nosema Bombyx mori pathogens.

[0066] S1. Experimental materials and methods

[0067] S11. Experimental biological materials

[0068] Nosema Bombyx mori ( Nosema bombycis , Laboratory preservation); Microsporidia mulberry ( Microsporidium spp. Collected from the natural host Mulberry Phthonandria atrilineataButler ), Plutella xylostella (collected from the natural host Plutella xylostella Pluella xylostella ), Pieris rapae (Collected from the natural host Pieris rapae Pieristrapae linne ), Spodoptera litura (collected from the natural host Spodoptera litura) Prodenia litura ), Microsporidia ( Nosema antheraeae , Referred to N.a , Isolated from tussah moth), etc. are all collected and preserved by the subtropical silkworm disease prevention and control post expert laboratory of South China Agricultural University; corn borer microsporidia ( ...

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Abstract

The invention relates to the technical field of biology, and discloses a LAMP visual rapid detection kit of silkworm pathogenic micro spore worms and a detection method of the LAMP visual rapid detection kit of the silkworm pathogenic micro spore worms. The kit comprises four LAMP primers, deoxyribonucleic acid (DNA) lysate liquid, LAMP reaction liquid, positive reference products, negative reference products and color development liquid to form a detection reaction system. Under the constant temperature of 60 DEG C to 65 DEG C, formwork DNA is rapidly amplified, developed dye is added to the formwork DNA, and identification results are observed through naked eyes under natural light. If the color of reaction products changes into green, the fact that samples to be tested contain the silkworm pathogenic micro spore worms is confirmed, and if the color of reaction products changes into orange, the fact that the samples to be tested do not contain the silkworm pathogenic micro spore worms is confirmed. The kit can detect current various silkworm pathogenic micro spore worms fast and flexibly, is easy to operate, and low in cost, and needs no special or complex instruments, and reaction results are easy to judge, and specificity is strong. The kit can meet the urgent needs of disease surveillance, on-site emergency situation and the detection of production samples, and can be widely popularized and applied easily.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and more specifically, to a visualized rapid detection kit for the microsporidian LAMP of the silkworm pathogen and a detection method thereof. Background technique [0002] Microsporidia is a single-celled eukaryote that parasitizes obligate cells. The bombyx mori particle disease caused by the microsporidian parasitic silkworm is an infectious disease that is seriously harmful to mulberry production and cannot be cured for a long time, and it seriously restricts the healthy and stable development of the silk industry. The spread of the disease in the middle of the 19th century brought devastating damage to the silk industry in Europe. In the beginning of the last century, the silkworm particle disease also caused great harm to sericulture countries such as China, Japan, and India. Since Nageli first discovered Nosema Bombyx mori, many different species of Nosema Bombyx have been isolated from B...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/90
Inventor 刘吉平杨吉龙
Owner SOUTH CHINA AGRI UNIV
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