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PCR (Polymerase Chain Reaction) method for detecting Baylisascaris schoederi eggs in panda excrement sample

A technology of Zhongxi's and giant pandas, which is applied in the field of detection of Ascaris schnitzel eggs by means of PCR amplification, which can solve the problems such as the influence of DNA extraction from eggs, improve detection accuracy and sensitivity, facilitate extraction, and reduce loss Effect

Inactive Publication Date: 2012-03-28
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For any PCR method, the quality of the DNA template is one of the key factors to determine whether the DNA amplification is successful. , dense protein shell and impermeable lipid layer, so it has a certain impact on the extraction of DNA from eggs

Method used

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  • PCR (Polymerase Chain Reaction) method for detecting Baylisascaris schoederi eggs in panda excrement sample
  • PCR (Polymerase Chain Reaction) method for detecting Baylisascaris schoederi eggs in panda excrement sample
  • PCR (Polymerase Chain Reaction) method for detecting Baylisascaris schoederi eggs in panda excrement sample

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Effect test

Embodiment 1

[0043] Take 50-60g of fresh feces samples from giant pandas.

[0044] a) Design primers

[0045]According to the complete sequence of the 12s gene in the mitochondrial genome of Ascaria schnitzele of giant panda, the screening of the target fragment and the design of primers were carried out, and the feasibility was analyzed by Primer5.0 analysis software, and the upstream and downstream primers were determined as follows:

[0046] Upstream primer (P1): 5'-TTTTACCTTGGCATTTTGTC-3',

[0047] Downstream primer (P2): 5'-CTCTCAATTACTACTCAACCTCC-3',

[0048] The target gene fragment is 291bp, and the gene sequence is shown in Table 1: 12s amplified target fragment sequence.

[0049] b) Extraction of DNA from the eggs of Ascaris schwai in giant pandas

[0050] Filter the feces sample with a fecal filter, take out 1ml of the filtrate in a centrifuge tube, centrifuge at 12000r / min for 5min, pour off the supernatant, then take 500μl from the feces sample in a centrifuge tube, centrif...

Embodiment 2

[0059] Take 50-60g of fresh feces samples from giant pandas.

[0060] a) Primer design

[0061] According to the complete sequence of the mitochondrial cytochrome oxidase 2 gene of the giant panda Ascaris schinii, the target fragment was screened, and a pair of specific primers were designed by using Primer Premier 5.0 software:

[0062] Upstream primer: 5'-GAGTAAGAAGGTTGAGTATCAGTTT-3',

[0063] Downstream primer: 5'-ATGAATAACATCCCCCAGAAGTA-3',

[0064] The length of the target fragment is: 331bp, and the gene sequence is shown in Table 2: the sequence of the target fragment amplified by cox2.

[0065] b) Fecal DNA extraction

[0066] Centrifuge the microcentrifuge tube containing a small amount of feces sediment at 10,000 r / min for 5 minutes, discard the supernatant, and add 50 μl of double distilled water to the microcentrifuge tube. The samples were fully frozen and thawed in boiling water and liquid nitrogen, and repeated ten times.

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) method for detecting Baylisascaris schoederi eggs in panda excrement sample, which comprises the following four steps: design of a forward primer and a reverse primer, extraction of egg DNA, PCR amplification and result examination. With the method, the egg is treated before DNA extraction, general grinding is replaced by adopting a manner of repeated freeze thawing of boiling water of 100 DEG C and liquid nitrogen, the fracture of the shell of egg protein can be observed under a microscope so that the DNA can be exposed to be convenient for extraction, and the loss in DNA extraction can be reduced as much as possible. Simultaneously, the invention provides two PCR amplification primers. Compared with the prior art, with the method, the detection accuracy and the sensitivity are greatly improved, the relevance ratio is higher than that of a conventional method (a floating method) by 32-47.83 percent, and the situation of ascariasis infection of panda can be judged more accurately; therefore, the method has significant meaning on diagnosing and preventing and controlling ascariasis of the panda.

Description

technical field [0001] The invention relates to a method for detecting eggs of Ascaris schwai, in particular to a method for detecting eggs of Ascaris schwai by means of PCR amplification. Background technique [0002] The giant panda is a national treasure of our country. Baylisascaris schroederi (Baylisascaris schroederi) is a relatively common intestinal parasite in wild and captive giant pandas. Adults usually parasitize in the small intestine of giant pandas and feed on mucosal surface substances and semi-digested food. Nutrition, and seriously affect the absorption of protein, fat, sugar and vitamins, causing malnutrition, anemia and other symptoms in giant pandas. The parasitism of a large number of worms will lead to intestinal obstruction, intestinal perforation or intussusception, and sometimes it can drill into the pancreatic duct and bile duct, causing obstruction and inflammation, and can easily lead to death in severe cases. Under the microscope, the fertiliz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 杨光友周旋王凝古小彬王淑贤彭雪蓉
Owner SICHUAN AGRI UNIV
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