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Application of septin1 gene to detection of nosema bombycis

A technology of microsporidia and silkworm, applied in the application field of septin1 gene in the detection of microsporidia of silkworm, can solve the problems of interference with effective amplification of PCR, etc., and achieve the effect of strong specificity, high specificity and high sensitivity

Active Publication Date: 2014-09-03
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantage of microscopic inspection is that it requires high skills and experience for operators, and it is difficult to distinguish different species of microsporidia and their analogues.

Method used

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  • Application of septin1 gene to detection of nosema bombycis
  • Application of septin1 gene to detection of nosema bombycis
  • Application of septin1 gene to detection of nosema bombycis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 septin1 Gene function research

[0038] The inventors are developing silkworm microsporidia N. bombycis (Guangdong strain) based on the transcriptomic research, the results of the transcriptome sequencing analysis were identified for the first time that may be related to the N. bombycis infection, reproduction Septins genes, named respectively septin1 (Accession: KF421133.1), septin2 (Accession: KJ451481.1), septin3 (Accession: KJ451482.1). In this example, the infection of silkworm Microsporidia N. bombycis The silkworm larvae, eggs and other developmental stages were carried out septin1 differential expression analysis.

[0039] The test method was as follows: silkworms from the 4th instar (Bombyx mori variety: Liangguang No. 2) were fed with Microsporidia of Bombyx mori, once in the morning and once in the evening, and then raised normally thereafter. After poisoning, the silkworms fed with poisoning were randomly selected every day (from ...

Embodiment 2

[0047] Example 2 Septin1 Application of genes as target genes for detecting whether silkworm eggs are infected with silkworm Microsporidia

[0048] (1) Extraction of total DNA from silkworm eggs

[0049] Genomic DNA was extracted using the DNeasy Plant mini kit produced by QIAGEN. The steps are as follows:

[0050] A. Take 20 silkworm eggs and put them in a mortar, fully grind with liquid nitrogen, and collect the ground powder into a 1.5mL centrifuge tube;

[0051] B. Add 400 μL AP1 and 4 μL RNase, and mix by vortexing (do not mix 400 μL APL and 4 μL RNase before use); after mixing, incubate the mixed solution at 65°C for 10 min (invert the tube upside down for 2-3 Second-rate);

[0052] C. Add 130 μL AP2, mix and cool on ice for 5 min; then centrifuge at 14 000 rpm for 5 min;

[0053] D. Pipette the supernatant into the collection tube of the QIA shredder spin column, centrifuge at 14 000rpm for 2min;

[0054] E. Transfer the supernatant from the ...

Embodiment 3 Embodiment 2

[0066] Example 3 Specific detection of primers described in Example 2

[0067]Take 30 "toxic" silkworm eggs, healthy silkworm eggs and purified silkworm microsporidia, respectively, extract total DNA according to the method of Example 2, then carry out PCR amplification, and use agarose gel electrophoresis to detect the results. Test results such as figure 1 Shown: DNA fragments of 699 bp were detected in "toxic" silkworm eggs and purified silkworm microsporidia, while no DNA fragments of 699 bp were detected in healthy silkworm eggs. Except for the N.b-specific target band of about 699 bp amplified by the septin1R primer, there are no other heterobands of different sizes from the target fragment. septin1 The gene is used as the detection target gene. The inhibitory substances in the silkworm egg extract will not interfere with the effective PCR amplification of the pathogenic gene DNA, and the detection results are more accurate and sensitive. It can quickly detect wheth...

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Abstract

The invention belongs to the technical field of molecular biotechnologies and particularly discloses an application of a septin1 gene to detection of nosema bombycis. Researches prove that the transcriptional expression of the septin1 gene is related to the cell division of the nosema bombycis and the development of a host. A specific primer is designed by taking the septin1 gene as a target gene, whether silkworm eggs are infected by the nosema bombycis is detected through PCR (Polymerase Chain Reaction) amplification, and the detected result proves that if the septin1gene is used as the detecting target gene, the interference effects of inhibiting substances in silkworm egg extracts to the effective PCR amplification of pathogenic gene DNA (Deoxyribose Nucleic Acid) are very low, the detected result is more accurate and sensitive, and more importantly, whether the silkworm eggs are infected by the nosema bombycis can be rapidly detected at the early stage that the silkworm eggs are infected. The guarantee is provided for the detection and safe egg production of poisonous silkworm eggs in silkworm egg production.

Description

technical field [0001] The present invention relates to the field of molecular biology technology, more specifically, to septin1 Application of genes in detecting Microsporidia of silkworm. Background technique [0002] Bombyx mori microsporidia is a pathogenic microsporidia ( Nosema bombycis , N.b) A devastating disease that infects silkworms through ingestion or embryo (fetus) infection. It is also an important epidemic disease that affects the sustainable and stable development of my country's silk industry. Every year, silkworm egg production in various places suffers from heavy economic losses due to the damage caused by particulate disease. At the same time, the wild insect Microsporidium can cross-infect silkworms, and can spread among silkworms and between different silkworms, resulting in the scrapping of a large number of silkworm eggs, which seriously restricts the trade of silkworm eggs and the sustainable development of sericulture. Sericulture authorities a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/90
CPCC12Q1/6893C12Q2600/158
Inventor 刘吉平晏育伟程伟
Owner SOUTH CHINA AGRI UNIV
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