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LAMP detection primers suitable for bombyx mori egg microsporidia and quick detection method thereof

A silkworm egg microspore and detection method technology, applied in the direction of microbial measurement/inspection, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of large interference, long time-consuming, many operation steps, etc., and reduce time Wasteful, easy to judge, and simple to operate

Inactive Publication Date: 2016-06-15
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved in the present invention is to design a group of microspores suitable for silkworm eggs in view of defects such as many operating steps, long time-consuming, and large interference in the prior art for detecting whether silkworm eggs are infected with No. spp. The detection primer of the LAMP detection of worm, based on said primer, can successfully establish the LAMP detection method of Bombyx mori silkworm egg microsporidia

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  • LAMP detection primers suitable for bombyx mori egg microsporidia and quick detection method thereof
  • LAMP detection primers suitable for bombyx mori egg microsporidia and quick detection method thereof
  • LAMP detection primers suitable for bombyx mori egg microsporidia and quick detection method thereof

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Effect test

Embodiment 1

[0085] The design and screening of embodiment 1 primer

[0086] In the present invention, the No. silkworm Septin1 gene (Accession number: KF421133.1) sequence verified by the transcriptome sequencing method and the cloning sequencing method is used for homology analysis by BLAST software, and no similar sequence is found. Septin1F / R primers were designed for the target gene. figure 1 Shown is a schematic diagram of the positions of PCR primers and LAMP primer sets on the Septin1 gene. figure 2 Shown is the schematic diagram of the target gene sequence and position designed by four LAMP primers.

[0087] ① The four primer sequences of primer set 1 are:

[0088] Septin1-F3: 5'-AAGGTGAAAGGATTAATGACTT-3';

[0089] Septin1-B3: 5'-TCATATAATTCACTTGCTGTGTA-3';

[0090] Septin1-FIP (F1c+F2):

[0091] 5'-TCCCCATTTAAACTTCCTAACTCGATTCCATTTTTTCGTTGTTTCATCT-3';

[0092] Septin1-BIP (B1c+B2):

[0093] 5'-AAGTTGATAACACGGAACACTGTCAAAAACACTTAAATGTGTACCAA-3';

[0094] ② The four primer...

Embodiment 2

[0109] The preparation of the template DNA of embodiment 2 silkworm silkworm egg and silkworm silkworm egg microsporidia

[0110] Take 10-50 samples of healthy silkworm eggs or silkworm eggs produced by silkworms fed with No. 2 O resuspended; draw the resuspended spore liquid into a precooled mortar, and grind fully with liquid nitrogen (more than 3 times); put the ground spores into a 1.5ml centrifuge tube, add 400 μl AP1 (see the kit produced by Qiagen) DNeasyPlantMiniKit instructions, the following other reagents are the same) and 4μl RNase, vortex mixing); the mixed solution was incubated at 65°C for 10min (during which the tube was inverted up and down 2-3 times); add 130μlAP2, mix and ice-bath for 5min; then 14000rpm Centrifuge for 5min; draw the supernatant into the collection tube in the QIAshredderspincolumn, and centrifuge at 14000rpm for 2min; transfer the supernatant in the centrifuge tube to a new tube (do not stir the residue that appears), add 1.5 times the volu...

Embodiment 3

[0111] Embodiment 3 prepares positive control substance:

[0112] Use the upstream primer Septin1F and the downstream primer Septin1R as reaction primers, and carry out PCR amplification with the DNA of Bombyx mori Microsporidia as the template, and the amplified product is cloned into the pMD-19T (purchased from Takara Company) vector to obtain the Septin1DNA-pMD plasmid and used as the positive result of the detection template.

[0113] The sequences of Septin1F and downstream primer Septin1R are:

[0114] Septin1F: 5'-TCATCATTCTTTAATACACTC-3';

[0115] Septin1R: 5'-CTTCTCATATAATTCACTTGC-3';

[0116] Use primer set Septin1-F3 and Septin1-B3 as external primers, Septin1-FIP and Septin1-BIP as internal primers, reaction buffer 2×ReactionMix (20mM Tris-HCl, pH8.8; 10mMKCl; 2mMMgSO 4 ; 20mM (NH 4 ) 2 SO 4 ; 0.1% TritonX-100; 2.8mMdNTPs; 1M Betaine; 25mMMgCl 2 ); Septin1F3 / B3 (20~40pmol / μL); Septin1FIP / BIP (5pmol / μL); Positive control substance; BstDNApolymerase (8U / μL); S...

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Abstract

The invention discloses LAMP detection primers suitable for bombyx mori egg microsporidia and a quick detection method thereof. The detection primers are Septin1-F3, Septin1-B3, Septin1-FIP and Septin1-BIP, the four primers are represented as the SEQ ID No: 1-4. On the basis of the primers, detection steps and system are optimized, and the LAMP quick detection method for the bombyx mori egg microsporidia is disclosed. The method is simple in operation, is free of complex instruments and amplification program, is short in detection time and is low in interference on amplification due to impurities. A reaction result is easy to determine and has strong specificity. The invention provides important basic and technical basis for applying the detection on the bombyx mori egg microsporidia with the LAMP and ensuring health and toxic-free for silkworm eggs. The method can satisfy toxicity detection demands in academic institutions, silkworm egg producing departments and silkworm egg quality detection centers and is easy to promote in large scope.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically, relates to a LAMP detection primer for the microsporidia of the silkworm egg and an application thereof and a rapid detection method for LAMP of the microsporidium of the silkworm egg. Background technique [0002] Bombyx mori microparticle disease is a devastating disease caused by the pathogenic silkworm Nosemabombycis (N.b) infected by food or embryo (fetus), and it is also an important disease that affects the sustainable and stable development of my country's silk industry. Every year, the economic losses caused by the damage of particulate diseases are very heavy. 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. my count...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6893C12Q2531/119Y02A50/30
Inventor 刘吉平杨思佳程伟
Owner SOUTH CHINA AGRI UNIV
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