Detection method of number level of nosema bombycis in silkworm finished-product eggs

A microsporidia and detection method technology, which is applied in the measurement/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve problems such as the difficulty in inferring the number and impact of microsporidia in finished eggs

Inactive Publication Date: 2013-08-14
JIANGSU GRAINE
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Problems solved by technology

However, the current detection method can only infer the rate of microsporidia in finished silkworm eggs at best, but it is difficult to infer the number of m

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0030] Based on Microsporidium silkworm ( Nosema bombycis ) 16S rRNA gene copy number to judge the number level of Bombyx mori N. sporidia in finished eggs. Pretreatment of silkworm eggs: take 100 silkworm eggs, treat them with 30% KOH 0.5 ml at 25°C for 30 minutes, wash twice with double distilled water, 1.2ml each time, discard the water, and then use 0.5ml 1mol / L HCl Treat for 5 minutes, wash twice with double distilled water, 1.2ml each time. Microsporidia DNA extraction from silkworm eggs: silkworm eggs plus microspore germination induction solution (0.1mol / L K2CO3, 0.1mol / L KHCO 3) 0.5ml, mash silkworm eggs with a sterilized toothpick, induce at room temperature for 1 hour, centrifuge at 3000 rpm for 5 minutes, discard the supernatant, add 0.5m of 0.01mol / L phosphate buffer (pH 7.0) to Place at 25°C for 60 minutes, centrifuge at 5000 rpm for 10 minutes, discard the supernatant, digest the pellet with 0.5ml proteinase K (50μg / ml) at 37°C for 60 minutes, then extract wit...

Embodiment 2

[0036] Based on Microsporidium silkworm ( Nosema bombycis ) The copy number of the 5SrRNA gene region was used to determine the number level of N. spp. in finished eggs. Pretreatment of silkworm eggs: Take 100 silkworm eggs, treat them with 30% KOH 0.5 ml at 25°C for 30 minutes, wash twice with double distilled water, 1.2ml each time, discard the water, and then use 0.5ml 1mol / L HCl Treat for 5 minutes, wash twice with double distilled water, 1.2ml each time. Microsporidia DNA extraction from silkworm eggs: silkworm eggs plus microspore germination induction solution (0.1mol / L K2CO3, 0.1mol / L KHCO 3 ) 0.5ml, mash silkworm eggs with a sterilized toothpick, induce at room temperature for 1 hour, centrifuge at 3000 rpm for 5 minutes, discard the supernatant, add 0.5m of 0.01mol / L phosphate buffer (pH 7.0) to Place at 25°C for 60 minutes, centrifuge at 5000 rpm for 10 minutes, discard the supernatant, digest the pellet with 0.5ml proteinase K (50μg / ml) at 37°C for 60 minutes, th...

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Abstract

The invention provides a detection method of the number level of nosema bombycis in silkworm finished- product eggs. The detection method comprises the following steps of: designing a specific primer according to a DNA (deoxyribonucleic acid) sequence of nosema bombycis of silkworms, and with total DNA in the silkworm eggs as a template, carrying out fluorogenic quantitative polymerase chain reaction (Q-PCR) amplification to obtain a Ct value of a detected sample; meanwhile, with different copy numbers of DNA of recombinant nosema bombycis of the silkworms as a template, carrying out Q-PCR amplification, drawing a standard curve by taking the Ct value as a vertical coordinate and a log value of the copy number of the initial plasmid DNA as a horizontal coordinate, and solving an regression equation; and calculating the initial copy number of the nosema bombycis DNA in the silkworms in the detected sample according to the regression equation and the Ct value of the detected sample, thereby estimating the quantity level of the nosema bombycis of the silkworms in the sample. The detection method can be used for directly judging the number level of the nosema bombycis according to the copy number of the nosema bombycis DNA of the silkworms in the detected sample. The estimation of the occurrence risk of the microparticle disease in subsequent breeding according to the number level of the nosema bombycis in the finished-product eggs is more scientific than the estimation of the diseased egg rate of the microparticle disease of the finished-product eggs.

Description

Technical field [0001] The present invention involves a detection method of the quantity level of micro -spores in the eggs of a silkworm, especially a method of using fluorescent quantitative PCR microboscopy. Background technique [0002] Family silkworm particle disease Nosema bombycis ) Infectious silkworm disease caused by infection. The disease is infectious and is the most serious infectious silkworm disease that threatens in the production of silkworm species.In order to create a toxic silkworm species and eliminate embryonic infection, the microscope testing method of the mother moth can be used to eliminate the method of eliminating the antibody infection by eliminating the cricket eggs produced by the toxic female moth.Due to the large and time tightness of the moth inspection, the single moth inspection was also tested as the group's moth test, and the female moth's poison rate was calculated based on the principle of probability.In 1979, the "Guidelines for Words Dis...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 许刚秦鸿斌
Owner JIANGSU GRAINE
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