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A kind of pcr detection method of bumble bee sporozoite

A technology of sporozoites and bumblebees, which is applied in the field of PCR detection of bumblebee sporozoites, can solve the problems of confusion and easy missed detection, and achieve the effect of strong specificity and good stability

Active Publication Date: 2018-11-30
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Microscopic observation is easy to miss, and it is easy to be confused with microsporidiosis, brevihymenosis, etc.

Method used

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  • A kind of pcr detection method of bumble bee sporozoite
  • A kind of pcr detection method of bumble bee sporozoite
  • A kind of pcr detection method of bumble bee sporozoite

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 The specificity test of Bombus sporozoites

[0023] By adopting the method of the invention, the bumblebee sporozoite is tested for the DNA samples and other parasitic diseases and pests whose amplified sequence is positive for the bumblebee sporozoite.

[0024] Specific primer pairs are:

[0025] Api-F: 5'-AGCGATGGATGTCTTGGG-3';

[0026] Api-R: 5'-TTCAGCGGGTAACCTTGT-3'.

[0027] The PCR reaction system included 4 μL of 5×GoTaq Buffer, 1.5 μL of MgCl 2 (25mM) (Promega), 2.0 μL of dNTPs (200 μM) (TaKaRa), 1 μL each of primer Api-F and primer Api-R (10 μM), 0.5 μL of GoTaq® DNA polymerase, 2 μL of template DNA , ddH 2 O to make up to 20 μL:

[0028] The reaction conditions were pre-denaturation at 95°C for 4 min, followed by 40 cycles of denaturation at 95°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 10 min.

[0029] Take 6 μL of the amplification product and detect it by 1.5% agarose gel electro...

Embodiment 2

[0030] Example 2 Sensitivity test of Bombus sporozoites

[0031] By adopting the method of the present invention, the sensitivity test is carried out on different dilution multiples of the DNA of the whole gene synthesis of the bumblebee sporozoite.

[0032] The PCR reaction method, system and conditions are the same as in Example 1, and the PCR amplification product is detected by 1.5% agarose gel electrophoresis. figure 2 Bombus sporozoite DNA10 synthesized for whole gene 0 -10 15 Agarose gel electrophoresis bands of PCR products after doubling dilution. The PCR product was detected by 1.5% agarose gel electrophoresis and found that 10 8 Bands were amplified from the 139 ng / μL template DNA diluted twice, and the sensitivity of the specific primers in the present invention to Api-F / Api-R was 1.39 fg / μL.

Embodiment 3

[0033] Example 3 Detection of clinical samples of Bombus sporozoites

[0034] Extract the genomic DNA from the abdominal tissue of the bumblebee to be tested

[0035] Cut the bumblebee abdominal tissue and place it in a mortar, add an appropriate amount of liquid nitrogen to quickly grind it, grind it several times to form a slurry, and use the CTAB method to extract the bumblebee abdominal tissue genomic DNA. The specific steps are as follows:

[0036] The bumblebee abdominal tissue was ground into a slurry and transferred to a 1.5 mL centrifuge tube, centrifuged at 12000 rpm for 2 min, the supernatant was discarded, and 50 µL of TE buffer was added to fully suspend the sample, then 60 µL of 10% SDS solution and 10 µL of 20 mg / mL proteinase K, mix well and incubate at 37°C for 1 h;

[0037] Add 100 µL of 5 mol / L NaCl solution, mix well by inverting up and down, add 80 µL CTAB / NaCl solution, and incubate at 65°C for 10 min after mixing;

[0038] Add 700 µL chloroform / isoamy...

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Abstract

The invention discloses a PCR (polymerase chain reaction) detection method of Apicystis bombi and belongs to the field of biotechnologies. A PCR system contains a pair of specific primers Api-F and Api-R, and a 316 bp segment can be specifically amplified from bumblebee DNA infected with the Apicystis bombi by the aid of the primers, so that PCR detection of the Apicystis bombi can be realized. With the adoption of the PCR detection method of the Apicystis bombi, the detection sensitivity of the total gene synthesized Apicystis bombi DNA can reach 1.39 fg / mu L, and the method has the characteristics of high specificity, good stability, quickness and accuracy, can be applied to detection of the Apicystis bombi in bodies of imported bumblebees and domestically cultured bumblebees and provides reliable theoretical and technical bases for prevention of introduction of the Apicystis bombi and control on the domestic Apicystis bombi disease.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a PCR detection method for bumble bee sporozoites. Background technique [0002] Bombus sporozoa ( Apicystis bombi ) belongs to the class Pyramides ( Conoidasida ), Clusteria ( Gregarinasina ), Neoclusteria ( Neogregarinorida ), Sporozoa ( Apicystis ). Bombus sporozoa is a protozoan parasite parasitic on Bombus genus and Apis mellifera. It is similar in shape to No. bombus, but larger. The spores are boat-shaped, about 11.4-14.4 μm long and 3.6-5.4 μm wide. Since its appearance in Finland and Italy, this parasite quickly spread to European countries. At present, there is no report of bumblebee spores in China, but this parasite is highly pathogenic. If it is not strictly prevented, it will be imported and exported with commercial bumble bee colonies The increase in trade exchanges will have a huge impact on the breeding and breeding of bumble bees in our country...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6893C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2565/125
Inventor 张体银刘蓓郑腾白泉阳王武军张志灯于师宇林素洁
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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