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Nucleic acid molecular hybridization detection method for silkworm nosema disease

A technology of nucleic acid molecular hybridization and silkworm microparticles, which is applied in the measurement/testing of microorganisms, biochemical equipment and methods, etc., can solve the problems of poor accuracy, low sensitivity, and long time consumption, and achieve the effect of good quality and high content

Inactive Publication Date: 2003-07-09
CHENGDU TIANCHUANG BIO TECH
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Problems solved by technology

These three methods all have the following disadvantages: time-consuming, low sensitivity, poor accuracy, and inability to achieve early detection
At the same time, the existence of multiple variants of pathogenic protosporidium also increases the difficulty of the traditional detection method of microspores and microspores, further reducing the accuracy of detection

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  • Nucleic acid molecular hybridization detection method for silkworm nosema disease
  • Nucleic acid molecular hybridization detection method for silkworm nosema disease
  • Nucleic acid molecular hybridization detection method for silkworm nosema disease

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Embodiment Construction

[0016] The specific operation steps of the present invention are as follows:

[0017] DNA extraction of Nosema bombycis → polymerase linked reaction (PCR) → PCR product cloning and recombinant plasmid identification → recombinant plasmid sequence analysis → DIG-labeled probe for specific identification of NB spores → DIG-labeled probe for detection of NB spore DNA Identification→Application of DIG-labeled probe in sericulture production.

[0018] 1. Extraction of Nosema bombycis DNA: Use proteinase K phenol chloroform extraction method to extract. To crush and purify the Bombyx mori, which is fed with microsporidia, is carried out as follows:

[0019] Take 400 microliters of centrifugation and remove the supernatant + equal volume of 0.2 mol / L K 2 CO 3

[0020] ↓30℃, 1 hour, remove impurities

[0021] Transfer to PBS buffer (pH 7.8)

[0022] ↓30℃, 2 hours, stimulate spore germination

[0023] 6000 rpm, 5 minutes, remove the supernatant, add...

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Abstract

The present invention discloses a nucleic acid molecular hybridization detection method of domestic silkworm nosema disease, including the following steps: using nucleic acid molecular hybridizatino technology to extract DNA of domestic silkworm microsporidian, utilizing PCR to clone the PCR product into recombinant vector plasmid, then using N.P.PCR product and DIG marker to make molecular probe, and adopting DNA Dot blot to detect the pathogenic spore of silwkorm nosame disease. The various microsporidian genomes extracted by said invention are good in quality, high in content, and the designed primers are specific for N.B spora, and said primer and template DNA specificity are highly matched, and its detection accuracy is greater than 98%.

Description

Technical field: [0001] The present invention relates to a method for detecting biological diseases, in particular to a method for detecting insect diseases, and more particularly to a method for detecting the hybridization of nucleic acid molecules of the silkworm microscopic disease. Background technique: [0002] Pebrine disease (Pebrine disease) is a silkworm disease caused by the parasitism of nosema bombycis (N.B.). It is a devastating chronic infectious disease of the silkworm. It is a serious harm to silkworm production and cannot be cured for a long time. The disease has a wide range of epidemics and difficult diagnosis and treatment. Historically, it has dealt a devastating blow to the sericulture production in the world's major sericulture countries such as Europe, Japan, and China. France, Italy and other countries have been devastated due to the outbreak of silkworm microparticle disease. Therefore, the prevention and control of microparticle disease is a worldwide p...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 白燕川秦启联赵力宾
Owner CHENGDU TIANCHUANG BIO TECH
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