A set of lamp detection primers and kits for microsporidia in silkworm eggs

A technology of silkworm egg microspores and detection kits, applied in the biological field, can solve the problems of silkworm egg DNA detection interference, complicated separation and collection of microsporidia, and many operating steps

Active Publication Date: 2016-08-17
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the PCR primers designed using this gene have low detection sensitivity for Microsporidia silkworm, and there are many operating steps, which take a long time, are not suitable for field detection and other shortcomings, which are not conducive to widespread promotion and use in actual production.
A bigger problem is that the existing public detection primers all use the DNA of microsporidia as a template, but the isolation and collection of microsporidia are complicated and time-consuming, which is extremely unfavorable for mass detection or on-site detection
[0004] On the other hand, microsporidia parasitize in silkworm eggs, and the content of silkworm eggs is significantly higher than that of the microsporidia to be detected. In the extracted DNA samples, both DNAs exist at the same time, and the DNA of silkworm silkworm eggs seriously affects the detection. Interference, therefore, if you want to directly use silkworm egg DNA as a template for the detection of microsporidia, higher requirements are put forward for the detection

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  • A set of lamp detection primers and kits for microsporidia in silkworm eggs
  • A set of lamp detection primers and kits for microsporidia in silkworm eggs
  • A set of lamp detection primers and kits for microsporidia in silkworm eggs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1 Primer Design

[0092] 1. According to the sequence of the No. silkworm septin3 gene (Accession number: KJ451482.1) verified by the laboratory through the transcriptome sequencing method and the clone sequencing method, the homology analysis was carried out by BLAST software, and no similar sequence was found. The invention designed a set of PCR primers with this gene as the target gene: primer Sep3F / Sep3R.

[0093] The sequence of primer Sep3F (shown in SEQ ID NO:5) is:

[0094] 5' TTCGGAGTAATAGCCAGTG 3'

[0095] The sequence of primer Sep3R (shown in SEQ ID NO:6) is:

[0096] 5'-AATTTGTTACAGAAGGACTCC-3'

[0097] 2. Use the above-mentioned PCR primers to detect healthy silkworm eggs and silkworm eggs infected with N. figure 1 shown).

[0098] Therefore, based on the position of the amplified fragment of the PCR primer, using the septin3 gene as the target gene designed by the LAMP primer, use the online software Primer ExplorerV4 (http: / / primerexplorer.j...

Embodiment 2

[0118] Embodiment 2 kit preparation

[0119] 1. For the preparation of DNA template of No. spp. silkworm, QIAGEN Company's plant mini-extraction kit was used, and the method was carried out according to the instructions. Specifically include the following steps:

[0120] S1. Take 200 μL microsporidia sample, centrifuge at 12,000 rpm for 5 minutes, discard the supernatant; then add 50-100 μL ddH 2 O resuspension;

[0121] S2. Draw the resuspended spore liquid into the pre-cooled mortar, and grind it fully with liquid nitrogen (more than 3 times);

[0122] S3. Put the ground spores into a 1.5 mL centrifuge tube, add 400 μL lysis buffer AP1 and 4 μL RNase A, and vortex to mix (400 μL lysis buffer AP1 and 4 μL RNase A should not be mixed before use);

[0123] S4. Incubate the mixed solution at 65°C for 10 min (invert the test tube 2 to 3 times during the period);

[0124] S5. Add 130 μL buffer P3, mix and ice-bath for 5 minutes; then centrifuge at 14,000 rpm for 5 minutes;

...

Embodiment 3

[0142] Example 3 Kit detection method

[0143] 1. The LAMP reaction conditions of the kit are: constant temperature reaction at 63°C for 30-60 minutes; then inactivation at 95°C for 2 minutes.

[0144] 2. When staining or agarose gel electrophoresis is used to determine the test result, the reaction system of the kit is:

[0145] 2×Reaction Buffer 12.5μL

[0146] Primers Sep3F3 / Sep3B3 and Sep3FIP / Sep3BIP 1 μL

[0147] Bst DNA polymerase 8U / μL 1μL

[0148] Template DNA 2 μL

[0149] wxya 2 O 8.5 μL;

[0150] When the detection result is judged by the real-time fluorescence method, the reaction system is:

[0151] 2×Reaction Buffer 12.5μL

[0152] Primers Sep3F3 / Sep3B3 and Sep3FIP / Sep3BIP 1 μL

[0153] Bst DNA polymerase 8U / μL 1μL

[0154] Fluorescent indicator 0.5μL

[0155] Template DNA 2 μL

[0156] wxya 2 O 8 μL;

[0157] Wherein, the concentration of the primer Sep3F3 / Sep3B3 is 5 pmol / μL, and the concentration of the primer Sep3FIP / Sep3BIP is 20˜40 pmol / μL.

...

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Abstract

The invention discloses a LAMP detection primer set and a kit for the silkworm silkworm egg Microsporidia. The primer set includes outer primers Sep3F3 / Sep3B3 and inner primers Sep3FIP / Sep3BIP, and the sequences are shown in SEQ ID NOs: 1-4. The present invention utilizes the primers to establish a LAMP detection method and kit for Bombyx mori ovale microsporidia. The kit includes the above-mentioned primer set, 2× reaction buffer, positive control substance, negative control substance, chromogenic solution (or fluorescence staining solution), Bst DNA polymerase, sealing solution and sterile water. The detection result of this method can be judged by naked eye observation or agarose gel electrophoresis observation or real-time fluorescence curve observation under natural light. The method has the advantages of simple operation, short detection time, easy determination of results, and strong specificity, and can detect silkworm egg DNA produced by silkworms infected with Microsporidia at a concentration of 5.0×10-3 ng / μL.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to a set of LAMP detection primers and kits for Microsporidia ova of Bombyx mori. Background technique [0002] Bombyx mori microparticle disease is a devastating disease caused by pathogenic Nosema bombycis (N.b) infected by ingestion or embryo (embryo) infection of silkworm, and it is also currently affecting the sustainable and stable development of my country's silk industry. It is an important epidemic disease, and the economic losses caused by the damage of particulate disease are very heavy every year. 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 country has listed silkworm microparticle disease as the qu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/90
CPCC12Q1/6844C12Q2531/119C12N15/11C12Q1/04C12Q1/68C12N1/105C12R2001/90
Inventor 刘吉平程伟晏育伟宋小景杨思佳
Owner SOUTH CHINA AGRI UNIV
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