Specificity detection primer, detection kit and detection method for schistosoma japonicam sex

A technique for detecting primers and detection methods, which is applied in the field of gender identification of Schistosoma japonicum, can solve the problems of low detection rate of feces detection, easy missed diagnosis, unclear differentiation of female and male worms, etc., and achieves objective and strong specificity in result determination , the effect of programmatic operation

Inactive Publication Date: 2009-12-09
SOUTH CHINA AGRI UNIV
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Problems solved by technology

[0003] At present, the diagnosis of schistosomiasis mainly relies on fecal pathogen detection and immunological methods, but the detection rate of feces detection is low, and it is easy to miss the diagnosis, and the immunological methods established by each laboratory are carried out according to the local actual conditions. Its widespread promotion and comparability still need further research; the prevention and treatment of schistosomiasis mainly relies on drug control and v

Method used

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  • Specificity detection primer, detection kit and detection method for schistosoma japonicam sex
  • Specificity detection primer, detection kit and detection method for schistosoma japonicam sex
  • Specificity detection primer, detection kit and detection method for schistosoma japonicam sex

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Experimental program
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Embodiment 1

[0039] The composition of embodiment 1 kit

[0040] The kit contains 27.5 mL of DNA lysate, including Nuclei lysis solution, 0.5M EDTA at pH 8.0, proteinase K (20 mg / mL) and RNase A solution (4 mg / mL); PCR reaction solution for 100 reactions (25 μl / reaction), dATP, dTTP, dGTP, dCTP of each 200μM final concentration, the primer Tsexu of final concentration 0.4pmol / μl 2 and Tsexd2 , 2mM MgCl 2 , Taq enzyme 25μl (5U / μl); Schistosoma japonicum female DNA positive control and Schistosoma japonicum male negative control each one.

Embodiment 2

[0041] Embodiment 2 kit specificity test

[0042] Use 1 μl of DNA from each of six control samples, including male Schistosoma japonicum, Fasciola large, Fasciola hepatica, Clonorchis sinensis, and Schistosoma mansoni (female and male), which have been verified for DNA validity, as templates, and react according to the kit. Conditions for specific PCR amplification, while setting a blank control and kit negative and positive controls.

[0043] Table 1 PCR amplification system

[0044]

[0045] The PCR amplification conditions are: pre-denaturation at 94°C for 5 minutes

[0046]

[0047] Extend at 72°C for 5 minutes

[0048] 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.

[0049] Results The positive control of Schistosoma japonicum female DNA in the kit amplified a band of about 153bp, while the other six control parasites and ...

Embodiment 3

[0050] The sensitivity test of embodiment 3 kits

[0051] Firstly, the DNA was extracted from the female Schistosoma japonicum, 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 according to 6, 4.8, 3.6, 2.4, 1.2, 0.9, 0.6, 0.3, 0.24 and 0.12 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 1.0% agarose gel electrophoresis to determine their sensitivity. The test results show that the PCR detection method is highly sensitive, and can detect 0.3ng DNA of Schistosoma japonicum female ( figure 2 ).

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Abstract

The invention discloses a specificity detection primer, a detection kit and a detection method for schistosoma japonicam sex, an upstream primer Tsexu2 nucleotide sequence of the specificity primer is 5'-ACGTTAGATACTGCTGTTCA-3, a downstream primer Tsexd2 nucleotide sequence is 5-ATATTGTTCCAAGTACGCAT-3'. In the invention, PCR amplification is carried out on the mould DNA to be detected by the specificity detection primer, the agarose gel electrophoresis is carried out on the amplifying product which is then observed under the ultraviolet light, the amplification strip appears in the positive result, which does not appear in the negative result. In the invention, the specificity detection primer is designed according to female worm specific sequence data of the schistosoma japonicam to establish a rapid, specific, sensitive PCR method used for schistosoma japonicam epidemiology and sex identification. The kit of the invention has simple procedures, strong specificity of the method, high sensitivity and objective result determination, and can be used for sex specific diagnosis of schistosoma japonicam of human and animals.

Description

technical field [0001] The invention relates to the gender identification technology of Schistosoma japonicum. Background technique [0002] Schistosomiasis japonicum is a serious zoonotic parasitism caused by Schistosoma japonicum parasitizing in the small blood vessels of the portal vein system of humans and cattle, sheep, pigs, horses, dogs, cats, rabbits, rodents and various mammals Sexual flukes. The disease is characterized by acute or chronic enteritis, liver cirrhosis, severe diarrhea, anemia, and emaciation, which seriously affects human health and the development of animal husbandry economy. It is mainly distributed in some countries in the Western Pacific region such as China, Japan, the Philippines and Indonesia. Among them, Japan has formally applied to WHO in 1997, declaring it a non-endemic area for schistosomiasis. In my country, the endemic areas are located in the Yangtze River Basin and the areas to the south. Except for Shanghai, Zhejiang, Fujian, Guan...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 朱兴全赵光辉林瑞庆袁子国李娟宋慧群
Owner SOUTH CHINA AGRI UNIV
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