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Fluorescence PCR detection method of pebrine disease and kit thereof

A technology of silkworm microparticles and detection methods, applied in the field of biology

Inactive Publication Date: 2011-02-09
中华人民共和国浙江出入境检验检疫局
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is innovative to a certain extent, but it is only an attempt, and there are still many places to be improved

Method used

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  • Fluorescence PCR detection method of pebrine disease and kit thereof
  • Fluorescence PCR detection method of pebrine disease and kit thereof
  • Fluorescence PCR detection method of pebrine disease and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Specimen Collection

[0044] Applicable specimen types include samples such as silkworms and silkworm eggs. Silkworms, take the suspected diseased silkworms and transfer them to a 5ml centrifuge tube, seal them and submit them for inspection. For silkworm eggs or excrement, about 1 g of silkworm eggs or excrement from suspected diseased silkworms should be taken, transferred to a 1.5ml centrifuge tube, sealed and sent for inspection.

Embodiment 2

[0045] Embodiment 2: the extraction of silk microsporidia DNA

[0046] Take the above specimen and add an appropriate amount of normal saline to grind, filter the homogenate with gauze, collect the filtrate, and use differential centrifugation (500rpm, centrifuge for 2 minutes, discard the precipitate, and then centrifuge the supernatant at 5000rpm for 10 minutes) to discard the supernatant. Suspend with an appropriate amount of physiological saline to obtain a crude extract of Nb spores. Take 0.5ml (10 8 individual / ml) Microsporidia suspension, 5000rmp, centrifuge for 5 minutes, remove the supernatant, keep the spores, add 0.2mol / LKOH 0.25ml at the same time, mix well, induce at 27℃ for 1 hour, centrifuge at 5000rpm for 5 minutes, remove the supernatant . Resuspend with 0.5ml of normal saline and place at 25°C for 30 minutes. Centrifuge at 5000rpm for 5 minutes, add 50μl of nucleic acid extraction solution to the precipitate and mix thoroughly, then bathe in boiling water ...

Embodiment 3

[0047] Embodiment 3: the amplification detection of silk microsporidia DNA

[0048] Take out the NB-PCR MIX and Taq enzyme system from the kit and wait for room temperature to melt, shake and mix well, then centrifuge at 10,000rpm for 10 seconds. Each test reaction system was prepared as follows, PCR MIX 24μl, Taq enzyme system 2ul, put into a clean 0.2ml PCR tube, mix well, and centrifuge at 10,000rpm for 10 seconds. Add 4 μl of processed sample (extracted DNA) or positive quality control or negative quality control to the set PCR reaction tube, and centrifuge briefly at 10,000 rpm for 10 seconds.

[0049] Put each reaction tube into the reaction tank of the quantitative PCR instrument, set the name of each detection, the type of fluorescent group (FAM is selected as the reporter group, TAMRA is selected as the quencher group) and amplification conditions: ABI PRISM 7700, ABI PRISM5700, The amplification conditions of ABI GeneAmp 7000, ABI PRISM7300 / 7500, MJ Opticon and othe...

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Abstract

The invention provides a method for detecting pebrine-causing Nosema bombycis (N.b) from samples, such as silkworm, silkworm egg and the like by fluorescence quantification PCR technology (a PCR-fluorescence probe method) and a kit thereof. The method comprises the following steps of: (1) collecting the samples; (2) extracting Nosema bombycis DNA; (3) detecting the samples by the fluorescence quantification PCR augmentation method; and (4) analyzing the corresponding samples according to the fluorescence intensity of the reaction after the augmentation reaction is ended so as to judge existence of the Nosema bombycis in the collected samples, accurately quantify the Nosema bombycis and realize real-time quick detection on the Nosema bombycis.

Description

1. Technical field [0001] The invention relates to a method for detecting Nosemabombycis (N.b), the pathogenic microorganism of silkworm microparticle disease (Pebrine), in particular to a method for rapidly and accurately detecting Nosemabombycis by using a fluorescent PCR method, and belongs to the field of biotechnology. The present invention further relates to a kit for detecting the disease. 2. Background technology [0002] Pebrine is a devastating disease of silkworm production, and the pathogenic microorganism causing the disease is Nosema bombycis (N.b for short). Because of its special mode of ovum infection, it causes serious harm to sericulture production, especially silkworm egg production and cannot be cured for a long time. France, Italy and other major European sericulture countries have suffered heavy losses in the silk industry due to the outbreak of the disease. In recent years, millions of original species of all levels have been eliminated due to partic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/90G01N21/64
Inventor 何永强鲁兴萌王华雄史成军徐贵峰刘健翊帅江冰张晓峰
Owner 中华人民共和国浙江出入境检验检疫局
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