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Specific primer for detecting pig metastrongylus elongatus and application thereof

A specific, nematode-based technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem that the test results are greatly affected by the experience of the test personnel, the shape is difficult to distinguish types, and the detection accuracy is low. problems, to achieve the effect of long storage and quality assurance time, high operation consistency and high detection efficiency

Inactive Publication Date: 2017-06-20
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Accurate diagnosis of strongyloidiasis in swine is the prerequisite for effective prevention and control of the disease. Existing detection methods are mainly based on clinical symptoms, epidemiological conditions, pathological changes, preliminary diagnosis, and further combined with fecal examination of eggs for diagnosis. Although the existing detection methods are simple and easy to implement, and have certain accuracy for adults, it is difficult to distinguish species in terms of morphology.
In particular, various nematodes are difficult to distinguish in the stage of eggs and larvae, and the 3 species of Strongyloides suis longus, Porcine genus, and Suimi are also difficult to distinguish from each other. At the same time, the existing The detection efficiency and detection accuracy of the detection method are low, and the detection results are greatly affected by the experience of the detection personnel

Method used

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  • Specific primer for detecting pig metastrongylus elongatus and application thereof
  • Specific primer for detecting pig metastrongylus elongatus and application thereof
  • Specific primer for detecting pig metastrongylus elongatus and application thereof

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Embodiment 1

[0038] The present invention provides a specific primer that can be used to detect Strongylus longina in pigs, and the specific primer includes an upstream primer MeJB3F and a downstream primer MeJB4.5R,

[0039] The nucleotide sequence of MeJB3F is: 5'-TGAAAGAATTCATGAATTGAAAACG-3';

[0040] The nucleotide sequence of MeJB4.5R is: 5'-AGATTTCATATTGTATTTAATTTTTG-3'.

[0041] The invention provides a detection kit which can be used for detecting strongylus longina in pigs. The detection kit is mainly composed of DNA lysate, PCR reaction solution, positive control solution and primer mixture.

[0042] The DNA lysate was mainly composed of 70 μl of 100 mM NaCl, 30 μl of 10 mM Tris-Cl with a pH value of 8.0, 150 μl of 25 mM EDTA with a pH value of 8.0, 30 μl of 1% W / V sodium dodecyl sulfate and 200 μl of 1.7 μg / μL proteinase K.

[0043] PCR reaction solution includes PCR buffer, 25mM MgCl 2 solution and 2.0mM deoxyribonucleotide solution.

[0044]The positive control solution co...

Embodiment 2

[0058] Adopt the detection kit provided by the present invention and the using method of the detection kit provided by the present invention, to Ascaris suum, Trichocephala nematode, esophagus stoma, Schistosoma japonicum, S. Strongyloides samui was tested. The specific method is as follows:

[0059] (1) DNA extraction: Take DNA samples from Ascaris suum, Trichocephala nematode, Oesophagostoma nematode, Schistosoma japonicum, Strongylus longina, Strongylus fuyin, and Strongylus Samsonii that have been verified for DNA validity spare;

[0060] (2) PCR amplification: add sterilized ddH to the centrifuge tube sequentially according to 9 times of the appropriate PCR reaction system 2 O, buffer, MgCl 2 , deoxyribonucleotides, primer mixture, Taq enzyme, and vortex to mix evenly. After mixing, put them into several 0.2μL centrifuge tubes to make multiple aliquots. Place 24μL in each centrifuge tube, take Each sample DNA prepared in step (1) is added to an aliquot and labeled, mi...

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Abstract

The invention provides a specific primer for detecting pig metastrongylus elongates. The specific primer comprises an upstream primer MeJB3 F and a downstream primer MeJB4.5 R, wherein a nucleotide sequence of the MeJB3 F is 5'-TGAAAGAATTCATGAATTGAAAACG-3'; a nucleotide sequence of the MeJB4.5 R is 5'-AGATTTCATATTGTATTTAATTTTTG-3'. The invention also provides a detection kit for detecting the pig metastrongylus elongates. The detection kit comprise a DNA (Deoxyribonucleic Acid) splitting solution for splitting sample DNA, PCR (Polymerase Chain Reaction) reaction liquid for amplifying the sample DNA, and primer mixed liquid, wherein the primer mixed liquid comprises the specific primer. The invention also provides application of the specific primer in detecting the pig metastrongylus elongates. The specific primer and the detection kit provided by the invention have the characteristics of high specificity, high detection efficiency and long storage time.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a specific primer for detecting strongylus longus in pigs and an application thereof. Background technique [0002] Strongylus porcini belongs to the order Cylindrical order, Strongylidae, and the genus Strongyloides. It is the general name of the three species of Strongyloides, Strongyloides longina, and Strongylus samsii. It is a nematode disease in which nematodes parasitize the bronchi and bronchioles of pigs. Because the worms are filamentous and parasitize in the lungs, it is also called lung filariasis or lung nematode disease. [0003] Strongylus swine is susceptible to sheep, cattle, and pigs. It is occasionally seen in sheep, deer, cattle, and other ruminants, and occasionally in humans. It mainly occurs in young pigs, with a high mortality rate and great harm. Swine pulmonary filariasis is endemic and occurs frequently in valley areas and low heat a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/158
Inventor 刘伟刘雪松谭磊尹德明
Owner HUNAN AGRICULTURAL UNIV
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