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Diseased eggs of nosemosis in product eggs of domestic silkworm, and method for detecting rate of diseased eggs

A technology for microparticle disease and detection method, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, measuring devices, etc., can solve the problems of inaccurate poisoning rate of finished eggs, long inspection period, etc., and achieves short detection period, The effect of improving the yield and promoting the germination of microsporidia

Inactive Publication Date: 2005-10-26
SUZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this process usually takes about 10 days, and the inspection cycle is long, sometimes it is difficult to meet the actual production needs; at the same time, microscopic inspection of ant silkworms can usually only detect microsporidia in the state of spores, and special staining methods must be used , in order to detect microsporidia in the merozoite state, so the toxin-carrying rate of finished eggs obtained in actual operation is not accurate

Method used

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  • Diseased eggs of nosemosis in product eggs of domestic silkworm, and method for detecting rate of diseased eggs
  • Diseased eggs of nosemosis in product eggs of domestic silkworm, and method for detecting rate of diseased eggs
  • Diseased eggs of nosemosis in product eggs of domestic silkworm, and method for detecting rate of diseased eggs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Detection of microparticle disease diseased eggs in finished silkworm eggs.

[0030] (1) Take 50 finished silkworm eggs to be inspected, treat them with 0.3 ml of 25% potassium hydroxide (KOH) for 20 minutes at room temperature, wash them twice with water, and neutralize them with 0.3 ml of 1 mol / liter hydrochloric acid (HCl) Treat for 5 minutes, then wash with water twice;

[0031] (2) Extraction of DNA, comprising, adding 0.3 milliliters of a mixture of potassium hydroxide of 0.01 mol / liter and potassium chloride of 0.16 mol / liter to the above-mentioned treated silkworm ovum, as microsporidia germination inducing liquid, uniform After slurrying, induce at room temperature for 0.5 hours, then use TEK buffer with a pH of 8.0 as the germination buffer for 40 minutes, discard the supernatant, and use 0.3 ml of proteinase K (final concentration of 50 μg / ml) for precipitation at 50°C Digest protein for 30 minutes, extract and separate with saturated phenol, degr...

Embodiment 2

[0036] Example 2: Detection of the diseased egg rate of microparticle disease in finished silkworm eggs.

[0037] (1) Pretreatment of silkworm eggs: take 100 finished silkworm eggs to be inspected as an inspection group, treat them with 0.5 ml of 30% potassium hydroxide (KOH) for 30 minutes at 25°C, wash them twice with double distilled water, and After discarding the water, neutralize it with 0.5 ml of 1 mol / L hydrochloric acid (HCl) for 5 minutes, then wash it twice with double distilled water, 1.2 ml each time;

[0038] (2) Extraction of DNA, comprising, adding 0.1 mol / liter of potassium carbonate (K 2 CO 3 ) with 0.1 mol / L potassium bicarbonate (KHCO 3 0.5 milliliters of microsporidia germination inducing liquid formed by the mixture of ), mash the silkworm eggs with sterilized toothpicks, induce at room temperature for 1 hour, centrifuge at 3000 rpm for 5 minutes, remove the supernatant, add 0.5 milliliters of 0.01 mol / min One liter of phosphate buffer (pH 7.0), placed...

Embodiment 3

[0051] Example 3: Detection of Microparticle Disease Egg Rate in Silkworm Finished Eggs

[0052] (1) pretreatment of silkworm eggs: with embodiment two.

[0053] (2) Extraction of DNA: same as Example 2.

[0054] (3) According to the 16S rRNA gene sequence of Microsporidium silkworm published on GenBank (accession number is AY616662), a pair of primers were designed, respectively, Nb165: 5'-CGAGTGCCAGCAGCCGCGG-3'; and Nb163: 5'-GCAACCATGTTACGACTTATATCAGA- 3', the interval between the two primers is 0.8kb;

[0055] components

concentration

KCl

50mmol / L

Tris-HCl

10mmol / L

MgCl2

2.00mmol / L

bovine serum albumin

100μg / ml

Primer

Each 0.25μmol / L

4dNTPs

Each 200μmol / L

Taq enzyme

2.5U

[0056] After mixing the components, perform PCR amplification according to the conventional method. The PCR amplification conditions are 3.5 minutes of pre-denaturation at 94°C, 1 minute at 94°C, 1 minute...

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Abstract

This invention publishes a kind of method of detecting pebrine eggs in the domestic silkworm finished eggs. Firstly, the whole DNA of the silkworm finished eggs to be checked be taken out. Secondly we design and composite a pair of the specific primer according to the specific sequence of the gene of the silkworm mini sporozoon. The distance between the two primer is 0.5-2.0 kb. Thirdly we set the whole DNA as the form board to make PCR amplification and then we do the electrophoretic detection for the amplified product. When we detect out the specific gene fragment, we can determine the egg is sick. As we check the silkworm finished eggs in large groups, we can get the sick eggs' ratio. To sum up, this invention can directly check the silkworms' eggs and the eggs don't need to be catalyzed to incubation. As a result, the checking time is short and the sensitivity is high, so it can satisfy the need of the departments.

Description

technical field [0001] The invention relates to a method for detecting biological diseases, in particular to a method for detecting microparticle disease in silkworms, in particular to a molecular biological detection method for the rate of diseased eggs with microparticle disease in finished eggs of silkworms. Background technique [0002] Bombyx mori microparticle disease is an infectious silkworm disease caused by microsporidia infection. The disease is infectious to embryos and is the most serious infectious silkworm disease in silkworm egg production. [0003] In order to produce non-toxic silkworm eggs and reduce the risk of embryo seed infection, in the prior art, the female moth is usually detected. In 1870, Pasteur proposed the female moth microscope inspection method for the first time. Each female moth was inspected separately, and the embryo seed infection was eliminated by eliminating the silkworm eggs produced by the poisonous female mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447
Inventor 贡成良潘中华郑小坚沈卫德曹广力薛仁宇陶维华李掌林陶鸣潘丽芬
Owner SUZHOU UNIV
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