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Application of EB1 gene to detection of nosema bombycis

A microsporidia and silkworm technology, applied in the application field of EB1 gene in the detection of silkworm microsporidia, can solve the problems of interference with effective amplification of PCR, etc., and achieve the effects of strong specificity, high sensitivity, convenient use and industrial production

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 EB1 gene to detection of nosema bombycis
  • Application of EB1 gene to detection of nosema bombycis
  • Application of EB1 gene to detection of nosema bombycis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 EB1 Gene function research

[0032] Microsporidium Bombyx mori N. bombycis (Guangdong strain) on the basis of transcriptomics research, identified for the first time that it may be related to N. bombycis related to infection and reproduction EB1 gene (Accession No. KF421134), and against infection with Microsporidium silkworm N. bombycis Bombyx mori tissues of different development stages such as silkworm larvae and eggs were carried out EB1 Analysis of expression differences.

[0033] The test method is: silkworms from the 4th instar (bombyx mori variety: Liangguang No. 2) are fed with No. spp. silkworm, once in the morning and once in the evening, and then normally reared. Every day after the poisoning (from the first day of the fourth instar to the fifth instar mature silkworms), the silkworms reared with the poisoning were randomly selected, and the silkworms were dissected with sterile vessels, sterile water and a new wax dish, and the midgut wa...

Embodiment 2

[0041] Example 2 EB1 Application of the gene as a target gene to detect whether silkworm eggs are infected with Microsporidium mori

[0042] 1. Extraction of total DNA from silkworm eggs

[0043] Genomic DNA was extracted using the DNeasy Plant mini kit produced by QIAGEN, and the steps were as follows:

[0044] (1) Take 20 silkworm eggs and put them into a mortar, grind them thoroughly with liquid nitrogen, and collect the ground powder into a 1.5mL centrifuge tube;

[0045] (2) Add 400 μL AP1 and 4 μL RNase, and vortex to mix (do not mix 400 μL APL and 4 μL RNase before use); after mixing, incubate at 65°C for 10 min (invert the test tube up and down for 2~ 3 times);

[0046] (3) Add 130 μL AP2, mix and place in ice bath for 5 minutes; then centrifuge at 14 000 rpm for 5 minutes;

[0047] (4) Pipette the supernatant into the collection tube in the QIA shredder spin column, centrifuge at 14 000rpm for 2min;

[0048] (5) Transfer the supernatant in the ce...

Embodiment 3 Embodiment 2

[0060] The specific detection of the primer described in embodiment 3 embodiment 2

[0061] Take 30 "poisonous" silkworm eggs, healthy silkworm eggs and purified Nosporum silkworm respectively, extract total DNA according to the method in 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 496bp were detected in "poisonous" silkworm eggs and purified No. Except for the N.b specific target band of about 496bp, there are no other bands with sizes different from the target fragment. Therefore, the EB1 As the target gene for detection, the inhibitory substances in the silkworm egg extract will not interfere with the effective PCR amplification of pathogenic gene DNA, and the detection results are more accurate and sensitive. It can quickly detect whether it is infected with No. spp. silkworm. The above results show that the primers described in Example 2 have good specif...

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Abstract

The invention belongs to the technical field of molecular biotechnologies and particularly discloses an application of an EB1 gene to detection of nosema bombycis. Researches prove that the transcriptional expression of the EB1 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 EB1 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 EB1 gene 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 most 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 EB1 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 and r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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