Primer for detecting schistosoma japonicum Yunnan geographical strains, kit containing same and detection method

A schistosomiasis and kit technology, which is applied in the field of schistosomiasis inspection and detection, achieves the effects of programmatic operation, objective judgment of results, and optimization of reaction conditions

Inactive Publication Date: 2010-11-10
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a primer that can identify and detect the Yunnan geographical strain of Schistosoma japonicum according to the probl

Method used

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  • Primer for detecting schistosoma japonicum Yunnan geographical strains, kit containing same and detection method
  • Primer for detecting schistosoma japonicum Yunnan geographical strains, kit containing same and detection method
  • Primer for detecting schistosoma japonicum Yunnan geographical strains, kit containing same and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The composition of embodiment 1 kit

[0050] The kit contains 27.5mL of DNA lysate, including Nuclei lysis solution, 0.5M EDTA at pH 8.0, proteinase K (20mg / mL) and RNase A solution (4mg / mL); CAPS-PCR reaction solution for 100 reactions (25 μL / reaction), dATP, dTTP, dGTP, dCTP with a final concentration of 200 μM each, upstream primers and downstream primers with a final concentration of 0.2 pmol / μL, and 2 mM MgCl 2 , Taq enzyme 25μL (5U / μL); CAPS reaction solution 100 reactions (10μL / reaction), which contains Nde I restriction endonuclease 100μL (1U / μL), 10×H buffer 100μL; Schistosoma japonicum Yunnan geographical strain DNA One positive control and one negative control of Schistosoma japonicum in other regions.

Embodiment 2

[0051] Embodiment 2 Kit CAPS-PCR test

[0052] Use 1 μL each of DNA from the geographical strain of Schistosoma japonicum that has been verified for DNA validity and control samples from Sichuan, Hunan, Hubei, Anhui, Jiangsu, Jiangxi, and Zhejiang as templates, perform specific PCR amplification according to the reaction conditions of the kit, and set a blank at the same time. Control and kit negative and positive controls.

[0053] Table 1 PCR amplification system

[0054]

[0055]

[0056] The PCR amplification conditions are: pre-denaturation at 94°C for 5 minutes

[0057]

[0058] Extend at 72°C for 10 minutes

[0059] After the PCR products were electrophoresed in 1.0% TBE agarose gel, the results were observed under an ultraviolet transilluminator and photographed by a gel imaging system.

[0060] Results kit can amplify about 580bp band from all Schistosoma japonicum strain DNA ( figure 1 ).

Embodiment 3

[0061] The PCR sensitivity test of embodiment 3 kit

[0062] First, the DNA of Schistosoma japonicum was extracted, diluted, vortexed and mixed, and the total DNA content was detected according to the operating procedures of the Eppendorf Biophotometer Nucleic Acid Protein Analyzer. Dilute the DNA at 40, 20, 10, 5, 2.5, 1.25, 0.63, 0.32, 0.16, 0.1 and 0.08 ng / μL. The PCR amplification conditions are the same as above, and a blank control is set at the same time. PCR products were detected by 1.0wt% agarose gel electrophoresis to determine their sensitivity. The test results show that the PCR detection method has high sensitivity, and can detect 0.16ng DNA of Schistosoma japonicum ( figure 2 ).

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Abstract

The invention discloses a primer for detecting schistosoma japonicum Yunnan geographical strains, a kit containing the same and a detection method. The nucleotide sequence of the upstream primer for detecting the schistosoma japonicum regional strains is expressed as SEQ ID No: 1, and the nucleotide sequence of the downstream primer is expressed as SEQ ID No: 2. When the primer is used for detecting the schistosoma japonicum regional strains, a to-be-detected template DNA is subjected to CAPS-PCR amplification, an amplification product is subjected to CAPS enzyme digestion reaction, and an enzyme digestion product is subjected to agarose gel electrophoresis and observed under ultraviolet light, wherein the positive result presents single band which is not digested by enzyme, and the negative result presents double bands which is fully digested by enzyme. The invention establishes a quick, specific and sensitive CAPS method for identifying the schistosoma japonicum regional strains, and the method can accurately identify the schistosoma japonicum regional strains. The operation of the kit containing the primer is simple and programmed; and the method has the advantages of strong specificity, high sensitivity and objective result judgment, and is suitable for popularization and application.

Description

technical field [0001] The invention relates to the field of inspection and detection of schistosomiasis, in particular to a primer for detecting schistosoma japonicum Yunnan geographical strain, a kit containing the primer and a detection method. Background technique [0002] Schistosomiasis japonicum is one of the six major tropical diseases identified by the World Health Organization and one of the three major human diseases in my country, which has important public health and socioeconomic significance. In my country, the disease is currently prevalent in seven provinces including Jiangsu, Anhui, Jiangxi, Hubei, Hunan, Yunnan, and Sichuan. About 50 million people are threatened, and about 800,000 people and more than 100,000 cattle are infected. In recent years, due to the abandonment of some measures to control schistosomiasis and environmental changes caused by floods and south-to-north water diversion, the disease has risen again in some areas, bringing new challenges...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 李娟林瑞庆赵光辉邹丰才宋慧群袁子国朱兴全陈芳
Owner SOUTH CHINA AGRI UNIV
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