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Molecular detection technology of microsporidian on infected bumble bees

A microsporidia and molecular technology is applied in the field of animal inspection and quarantine, and the field of microsporidia molecular inspection on import and export bumblebees, which can solve the problems of high cost, incomplete enzymatic cleavage and reduced enzymatic cleavage efficiency, and achieves low cost, The effect of avoiding contamination and reducing the loss of DNA

Inactive Publication Date: 2009-12-16
BEE RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method needs to use three enzymes, which is expensive, and the efficiency of one of the enzymes will be reduced during double digestion, resulting in incomplete digestion.
Second, N. thomsoni, another microsporidia infecting bumble bees, could not be identified by this method.

Method used

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  • Molecular detection technology of microsporidian on infected bumble bees
  • Molecular detection technology of microsporidian on infected bumble bees
  • Molecular detection technology of microsporidian on infected bumble bees

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Examination and species identification of three kinds of microsporidia

[0024] Using the purified N.ceranae, N.bombi, N.thomsoni three kinds of microsporidia and their mixture in pairs as samples, the Chelex-100 method of the present invention was used to extract DNA respectively, and specific primers (SSUrRNA-f1: 5'- CACCAGGTTGATTCTGCCT-3', SSUrRNA-r1b: 5'-TGTTCGTCCAGTCAGGGTCGTCA-3') was amplified, the amplified product was digested with Afa I, and the digested product was detected by 2% agarose gel electrophoresis.

[0025] PCR reaction system:

[0026] Add 5 μl of 5×GoTaq Buffer and 1.5 μl of MgCl to the 25 μl reaction system 2 (25mM) (Promega), 2.0μl of dNTPs (200μM) (TaKaRa), 1μl of SSUrRNA-f1 and SSUrRNA-r1b primers (10μM), 0.625U / tube of GoTaq DNA polymerase, 5μl of template DNA (about 5ng / μl), 9.375 μl of ddH 2 O. After pre-denaturation at 95°C for 4 minutes, it enters the cycle: denaturation at 95°C for 1 minute, annealing at 64°C for 1 minute, extension ...

Embodiment 2

[0031] Examination and Species Identification of Microsporidia in Bumblebees at Home and Abroad

[0032]Adopt the method of the present invention, carry out the inspection of microsporidia and the type identification of microsporidia to 5 bumblebees Bombus terrestris from Switzerland and 1 bumblebee Bombusladakhensis, 3 Bombus festivus, 1 Bombus friseanus in China.

[0033] The PCR reaction system and PCR product enzyme digestion reaction system are the same as Example 1, and the PCR amplification products and enzyme digestion products are detected by 2% agarose gel electrophoresis, as shown in the accompanying drawings. Figure 4 Agarose gel electrophoresis bands of microsporidia PCR products from different bumblebee host sources.

[0034] The digested products were detected by 2% agarose gel electrophoresis and found that 5 bumblebees from Switzerland and 5 domestic bumblebees were all infected with microsporidia, and the bands produced by the electrophoresis of the digested...

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Abstract

The invention relates to molecular detection technology of microsporidian on infected bumble bees, which belongs to the technical field of animal inspection and quarantine and particularly relates to the technical field of molecular inspection for accurately and quickly detecting the microsporidian on imported and exported bumble bees. The molecular detection technology is characterized in that a Chelex-100 method is adopted to extract DNA; specific primers are used for carrying out PCR for one time; SSUrRNA genetic fragments of 540bp microsporidian are amplified; then, enzyme digestion of PCR products is carried out by restriction endonuclease Afa I; electrophoretic detection of enzyme digestion products are carried out by 2.0 percent agarose gel; and the type of the microsporidian parasitizing on the bumble bees can be detected directly through the length of fragments of the enzyme digestion products. The molecular detection technology can be applied to the inspection and quarantine of pathogen microsporidian on the imported and exported bumble bees.

Description

technical field [0001] The invention relates to the technical field of animal inspection and quarantine, in particular to the technical field of accurate and rapid detection of microsporidia molecules on imported and exported bumble bees. Background technique [0002] Microsporidia are obligate intracellular parasitic protozoan parasites that are widely distributed in nature and have a large population. They have a very wide range of hosts, including vertebrates and invertebrates. They are economic insects, fish, rabbits, hair-producing Pathogens that cause disease in animals, rodents and primates. Bumble bee is an important class of economically pollinating insects, and it is also harmed by microsporidia. Therefore, microsporidia have become one of the important pathogens of bumblebee import and export inspection and quarantine. [0003] Microsporidia detection in bumblebee usually adopts microscopic examination and conventional molecular examination. The microscope inspe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/90
Inventor 李继莲彭文君吴杰陈文锋安建东黄家兴罗术东
Owner BEE RES INST CHINESE ACAD OF AGRI SCI
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