Nucleic acid probe of specific marker nosema bombycis and in-vivo fluorescence hybridization detection method of nucleic acid probe

A technology for nucleic acid probes and microsporidia, which is applied in the field of nucleic acid probes for specific labeling of microsporidia and its in situ fluorescence hybridization detection. Problems such as training and research foundation are weak, and the results are intuitive and reliable, easy to operate, and simple and convenient to operate.

Inactive Publication Date: 2017-07-28
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the weak preliminary research foundation of N. silkworm, the lack of sufficient molecular data, and its special structure, which cannot be cultured in vitro, the FISH labeling method of N. silkworm has not yet been invented.

Method used

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  • Nucleic acid probe of specific marker nosema bombycis and in-vivo fluorescence hybridization detection method of nucleic acid probe
  • Nucleic acid probe of specific marker nosema bombycis and in-vivo fluorescence hybridization detection method of nucleic acid probe
  • Nucleic acid probe of specific marker nosema bombycis and in-vivo fluorescence hybridization detection method of nucleic acid probe

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Experimental program
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Embodiment 1

[0029] Embodiment 1 probe design

[0030] According to the DNA sequence of N.b large ribosomal RNA subunit (LSU rRNA) and its secondary structure model, combined with FISH probes, the design and screening of FISH probes was carried out. The nucleic acid probe designed by the technical solution can specifically bind the ribosomal RNA outside the nucleus, while the general probe can only bind the DNA in the nucleus and cannot achieve the purpose of distinguishing the microsporidia of silkworms in different stages. The requirements for the FISH probe are: the length is about 18nt, and the Tm value is 48-60°C. If the Tm value is greater than 60°C or less than 48°C, the length of the probe can be adjusted appropriately. According to the above principles, two nucleic acid probes were designed, the sequences of which are as follows:

[0031] Table 1 Nucleic acid probes for specific labeling of N. silkworm

[0032] serial number name sequence SEQ ID NO: 1 NbLSU-...

Embodiment 2

[0037] The method and result of embodiment 2NbLSU-1-Cy3 in situ hybridization experiments

[0038] 1) Preparation of silkworm BmE cells infected with N.b

[0039] Normal silkworm embryo cells (BmE) were counted according to 1X10 6 Sub-power climbing sheet (diameter 20mm), meanwhile, under aseptic conditions, get the silkworm chrysalis blood that infects Nosema bombycis (Nosema bombycis, N.b), the N.b spore wherein is counted, after counting with N.b:cell=10: The ratio of 1 was used to infect BmE cells, and the medium was changed after 6 hours of infection.

[0040] 2) FISH probe labeling of silkworm BmE intracellular hybridization N.b

[0041] 45 hours after N.b infected BmE cells, the BmE cells were fixed with 4% paraformaldehyde, and the NbLSU-1-Cy3 probe was diluted with hybridization solution (900mM NaCl, 20mM Tris[pH 7.5], 0.01%SDS), The final concentration was 5ng / μL, and the hybridization was carried out at 46°C for 12 hours. After the hybridization, the non-specifi...

Embodiment 3

[0048] The method and result of embodiment 3NbLSU-V1-Cy3 in situ hybridization experiments

[0049] 1) Preparation of silkworm BmE cells infected with N.b

[0050] Normal silkworm embryo cells (BmE) were counted according to 1X10 6 Sub-power climbing sheet (diameter 20mm), meanwhile, under aseptic conditions, get the silkworm chrysalis blood that infects Nosema bombycis (Nosema bombycis, N.b), the N.b spore wherein is counted, after counting with N.b:cell=10: The ratio of 1 was used to infect BmE cells, and the medium was changed after 6 hours of infection.

[0051] 2) FISH probe labeling of silkworm BmE intracellular hybridization N.b

[0052] 45 hours after N.b infected BmE cells, BmE cells were fixed with 4% paraformaldehyde, and the NbLSU-V1-Cy3 probe was diluted with hybridization solution (900mM NaCl, 20mM Tris[pH 7.5], 0.01%SDS), The final concentration was 5ng / μL, and the hybridization was carried out at 46°C for 12 hours. After the hybridization, the non-specifica...

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Abstract

The invention relates to a nucleic acid probe of specific marker nosema bombycis and an in-vivo fluorescence hybridization detection method of the nucleic acid probe. The sequence of the nucleic acid probe of the specific marker nosema bombycis is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, a fluorescence probe is prepared at the 5' end or 3' end of the sequence by the aid of corresponding fluorescent dyes according to requirements, and cells infected by the nosema bombycis are detected by an in-vivo fluorescence hybridization method provided by the fluorescence probe. Combined sensitivity between the specific rDNA (ribosome deoxyribonucleic acid) probe of the nosema bombycis and the nosema bombycis is high, the fluorescence probe prepared by the aid of fluorescein has higher specificity as compared with a fluorescent marking method by the aid of antibodies, and the fluorescence probe is simple and convenient to operate and more intuitive and reliable in result and can mark pathogens at various developmental stages except for mature spores.

Description

technical field [0001] The invention belongs to the field of biomolecular detection, and relates to a nucleic acid probe for specifically marking Microsporidium silkworm and an in-situ fluorescence hybridization detection method thereof. Background technique [0002] Nosema bombycis (N.b) is the pathogen of silkworm microsporidia, which can not only spread horizontally, but also vertically spread through eggs and infect offspring silkworms. Because N.b is an obligate intracellular parasite, it cannot be cultured in vitro, and its spore structure is special, most conventional research methods cannot be used or have not yet been established. Important features such as the pathogenesis are still unclear. At present, the detection of silkworm eggs in production is mainly to examine the spores of N.b with refraction in the female moth and finished eggs under the microscope, while the N.b in the non-spore stage cannot be checked by microscope. [0003] The life cycle of N. silkw...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q1/6841C12Q2563/107
Inventor 李田许晨罗堿徐金智周泽扬
Owner SOUTHWEST UNIVERSITY
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