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Kit and method for detecting Microsporidium silkworm by pcr-elisa method

A technology of microsporidia and kits, applied in the field of detection, can solve the problems of high cost, high requirements, confusion, etc., and achieve the effect of high specificity, specificity and accuracy

Active Publication Date: 2020-04-17
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its operation is simple and convenient, and the results are intuitive and easy to understand. However, due to its high requirements for inspectors, the sample preparation of female moths is cumbersome, and it is easy to confuse No. The speed and accuracy of the detection work, so the establishment of a method for the direct detection of Microsporidia silkworm in finished eggs has become the focus of sericulture scientific research
[0004] With the advancement of technology and the updating of instruments and equipment, more and more new detection methods have been applied to the detection of Microsporidia silkworm, mostly based on nucleic acid detection, such as PCR method, LAMP method, fluorescent PCR, etc. Compared with traditional female moth microscopy, the sensitivity and accuracy are higher, but some methods have higher requirements for inspectors in operation, require professional training, and are more dependent on instruments and equipment, and the cost is higher. These methods are still in the stage of laboratory research

Method used

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  • Kit and method for detecting Microsporidium silkworm by pcr-elisa method
  • Kit and method for detecting Microsporidium silkworm by pcr-elisa method
  • Kit and method for detecting Microsporidium silkworm by pcr-elisa method

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Experimental program
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Effect test

Embodiment 1

[0047] Embodiment 1, primer screening

[0048] Genomic DNA of N. silkworm was extracted by CTAB method.

[0049] At the same time, the genomes of Nos. tussah, No. bee and No. naka proteus were extracted as controls.

[0050] Then according to the comparison between the genome of Microsporidium silkworm and several other Microsporidium genomes, the sequences with similarity less than 30% were screened out for primer design.

[0051] The specific primers are shown in Table 1 below.

[0052] Table 1. Primer pairs for microsporidia detection

[0053]

[0054]

[0055]

[0056] With the silkworm Microsporidia genomic DNA as a template, the sequences shown in Table 1 are primers for PCR amplification, and the PCR amplification system is as shown in Table 2 below:

[0057] Table 2. PCR amplification system

[0058]

[0059]

[0060] Amplify according to the following conditions, pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 sec, annealing at 64°C ...

Embodiment 2

[0061] Embodiment 2, specificity experiment

[0062] According to the method of Example 1, the six pairs of primers screened are used to detect the following templates respectively: 1. DNA of Bombyx mori No. spp.; 2. No. naka proteus DNA; The company's MightyAmp DNA polymerase Ver.3, the total reaction system is 25μL. Amplification was performed with an Applied Biosystems PCR instrument, and the reaction parameters were: pre-denaturation at 98°C for 2 minutes; 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 are as follows: figure 2 shown in . The results show that OP1-748, K245, M247, U245, and U246 five pairs of primers can specifically amplif...

Embodiment 3

[0063] Embodiment 3, false positive detection

[0064] In order to detect whether there are false positives in the labeled primers, DNA of Bombyx mori was used as a template for positives, and ddH was used for negatives 2 O was used as a template and amplified according to the established PCR system. After amplification, 10 μL was used for agarose gel electrophoresis. The conditions of gel electrophoresis are: 1×TAE buffer, voltage 180V, electrophoresis time 30min. At the same time, it was detected by ELISA, and the results were as follows: image 3 shown. The results showed that except for U245, which could not get positive results, OP1-748, K245, M247, U246, and LSU323 could all get positive results, and the OD of the negative control was close to that of the blank control, so the next step of the experiment could be carried out. For the specific sequence of LSU323, please refer to the literature (Ni Qi, Establishment of a lateral flow test strip detection method for the...

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Abstract

The invention relates to a kit for detecting silkworm eggs by using a PCR-ELISA method and a detecting method of the kit. The kit comprises a nucleic acid molecular marker labeled primary pair for detecting the silkworm eggs and an ELISA reagent which detects a nucleic acid molecular marker; suitable gene segments are amplified by labeled specific primers, then an amplified product is subjected to ELISA, screened primers have species specificity, the sensitivity is high, multi-sample high-flux detection can be realized, and therefore, the kit plays an important role in detection of silkworm eggs in silkworm eggs.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a PCR-ELISA detection kit for the microsporidia silkworm, and also relates to a detection method. Background technique [0002] Nosema bombycis (Nosema bombycis) is a common pathogenic fungus of silkworms. The silkworm microsporidia caused by it is a devastating disease in the sericulture industry. Therefore, Nosema bombycis has been listed as a legal disease in various sericulture countries and regions. Sericulture production quarantine object. In order to effectively control silkworm microsporidia in sericulture production, so as to avoid catastrophic death of silkworms, how to complete the detection of silkworm microsporidia in the finished egg stage has become one of the important tasks in sericulture scientific research. [0003] In the 1860s, Louis Pasteur (Louis Pasteur) discovered and utilized the characteristics of silkworm microparticle disease embryo infection, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6804C12Q1/6806C12Q1/6893C12Q1/04
CPCC12Q1/6804C12Q1/6806C12Q1/6893C12Q2563/131C12Q2527/125
Inventor 潘国庆王锐周泽扬宋跃何章帅刘璐璐
Owner SOUTHWEST UNIV
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