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
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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
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[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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