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Primer pair for rapidly detecting silkworm microsporidia and reagent kit and detection method thereof

A technology of microsporidia and kit, which is applied in the fields of molecular biology and immunology, can solve the problems of small number of microsporidia, dependence on sensitivity, and low amount of antigen, and achieves high sensitivity, avoids complicated processes, and facilitates detection process. fast effect

Active Publication Date: 2015-12-16
SOUTHWEST UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

In the detection of immunocolloidal gold test strips, the characteristics of antigen-antibody specific binding are often used to construct a complex of antigen-antibody-colloidal gold particles. When the complex is concentrated on the detection line of the test strip, the colloidal gold The particles can form a red precipitation line, but the sensitivity of this method depends on the acquisition of high-affinity and high-specificity antibodies and the antigen content in the sample. The number of microsporidia in the finished silkworm eggs is very small, and the corresponding antigen content is also corresponding Rarely, it also limits the application of this method in the quarantine of finished eggs

Method used

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  • Primer pair for rapidly detecting silkworm microsporidia and reagent kit and detection method thereof
  • Primer pair for rapidly detecting silkworm microsporidia and reagent kit and detection method thereof
  • Primer pair for rapidly detecting silkworm microsporidia and reagent kit and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1, primer screening

[0044] Extract Genomic DNA of Microsporidium silkworm, the specific method is to take silkworm eggs, add water and grind, centrifuge at 9000r / min for 5 minutes, collect the precipitate, add the sample treatment solution to be tested in the precipitate, mix well and place it at 18-25°C for 10min, then wash with water, wash with water at 9000r After 5 minutes of centrifugation at 1 / min, the precipitate was collected, then a lysate was added to the precipitate, and placed at 100°C for 1 hr, wherein the sample treatment solution to be tested was KOH with a mass fraction of 4%; the lysate contained a volume fraction of 1% NP-40 and 2% TritonX-100 by volume.

[0045] Genomic DNA of Serratia, nuclear polyhedrosis virus, Beauveria bassiana and Staphylococcus aureus were also extracted as controls.

[0046] Then, according to the ribosomal large subunit RNA (LargesubunitrRNA) coding sequence of Bombyx mori, the amplification primers were designe...

Embodiment 2

[0056] Embodiment 2, specificity experiment

[0057] According to the method of Example 1, detect the following templates with LSU323 upstream primers and downstream primers respectively: 1. positive plasmid constructed after amplification with unlabeled LSU323-F and LSU323-R primer pairs; 2. Bombyx mori DNA; 3. Blank Water control; ④ DNA of silkworm nuclear polyhedrosis virus; ⑤ DNA of Beauveria bassiana; ⑥ DNA of Serratia spp; ⑦ DNA of Staphylococcus aureus; Amplification was performed with an Applied Biosystems PCR instrument, and the reaction parameters were: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 sec, annealing at 64°C for 15 sec, extension at 68°C for 35 sec, and 30 cycles; final extension at 68°C for 3 min, and storage at 12°C. Take 10 μL of the amplified product for electrophoresis in 2% agarose gel electrophoresis, then stain with ethidium bromide, and observe under ultraviolet light to determine whether there are specific bands. The results a...

Embodiment 3

[0058] Embodiment 3, sensitivity test

[0059] In order to detect the sensitivity of the colloidal gold immunochromatographic test strip, the silkworm microsporidia were counted and diluted to 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 10 silkworm microsporidia were mixed with 100 normal silkworm finished eggs respectively, and the DNA was extracted according to the method in Example 1, and amplified by the established PCR system. After amplification, 10 μL was used for agarose gel electrophoresis. The conditions of gel electrophoresis are: 1×TAE buffer, voltage 200V, electrophoresis time 17min. At the same time, take 10 μL to test with a test strip, then add it to 90 μL of developing buffer for detection, observe the test results after 5 minutes, and the results are valid within 15 minutes, and the results are as follows: Figure 4 shown. The results showed that the agarose gel electrophoresis did not detect positive in the sample mixed with 10 silkworm microsporidia a...

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Abstract

The invention relates to a primer pair for rapidly detecting silkworm microsporidia and a reagent kit and a detection method thereof. The nucleotide sequence of the primer pair is shown as SEQ ID NO.7 and SEQ ID NO.8. By the utilization of the primer pair, different nucleic acid molecular markers are added into the upstream primer and the downstream primer respectively, PCR products of the marked primers can be directly determined through immuno-gold test strips, the operation steps are simplified, convenience is achieved, specificity is good, and sensitivity is high. Compared with a traditional gel electrophoresis method, the sensitiveness is increased by 10 times, and the primer pair can serve as an ideal detection means for silkworm microsporidia in silkworm finished product eggs and has good application prospects.

Description

technical field [0001] The invention belongs to the fields of molecular biology and immunology, and relates to a primer pair for rapidly detecting the microsporidia silkworm, and also relates to a primer pair kit for rapidly detecting the microsporidia silkworm and a detection method for detecting the microsporidia silkworm using the kit. Background technique [0002] In order to effectively control the silkworm microsporidia caused by N. bombyx mori in sericulture production, so as to prevent it from causing mass death of silkworms, it is necessary to develop a sensitive, convenient and accurate detection method for N. bombyx mori. Nosemabombycis (Nosemabombycis), also known as silkworm microsporidia, mainly parasitizes in the body of the economic insect silkworm, and the silkworm microsporidia caused by it is a devastating disease in the sericulture industry. The area is listed as the legal quarantine object in sericulture production, and the successful detection of N. sil...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/90
CPCC12Q1/6804C12Q1/6893C12Q2531/113C12Q2563/131C12Q2563/137
Inventor 潘国庆倪琪周泽扬孙滨宋跃
Owner SOUTHWEST UNIVERSITY
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