Method for detecting NOD1 gene single nucleotide polymorphism with HRM (High Resolution Melting) technology
A single nucleotide polymorphism and technology detection technology, applied in the field of HRM technology detection of NOD1 gene single nucleotide polymorphism, can solve the problems of high reaction conditions, complicated operation process, cumbersome test process, etc., and achieve high sensitivity And the effect of high specificity, good repeatability, and shortened detection time
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[0023] Example 1
[0024] The high-resolution melting curve (HRM) method is used to detect the SNP site rs2075820 of the NOD1 gene in the subject, including obtaining sample DNA and providing reagents required for sequencing. Refer to Example 2 for specific operation steps. The reagents include: whole blood genomic DNA extraction kit (QIAGEN), HRM detection kit FP210 (Tiangen Biochemical Technology Co., Ltd., Beijing, China), primers for detecting rs2075820 site, standard substances and negative control substances. The primers for detecting rs2075820 site are:
[0025] NOD1-F-HRM:GCTTCAAGGAAAGTGACAGGC;
[0026] NOD1-R-HRM: CAGGTCCAAGTCCGAGTGC.
[0027] Standards are GG genotype, GA genotype and AA genotype DNA specimens; negative control: solution without DNA.
[0028] The PCR reaction solution of the detection system includes:
[0029]
[0030] The PCR reaction program is:
[0031]
[0032] The present invention also uses a sequencing method to further verify the result of the HRM meth...
Example Embodiment
[0041] Example 2
[0042] The operation process of the blood / cell / tissue genomic DNA extraction kit (QIAGEN):
[0043] (1) Extraction of genomic DNA from whole blood
[0044] 1) Add 20μl QIAGEN Protease (or proteinase K) to the bottom of a 1.5ml centrifuge tube. .
[0045] 2) Add 200μl of plasma to the centrifuge tube.
[0046] 3) Add 200μl Buffer AL and shake for 15s. (Note: Do not add QIAGEN Protease or proteinaseK directly to Buffer AL. If the sample size is large, QIAGEN Protease and Buffer AL will increase proportionally.)
[0047] 4) Water bath at 56°C for 10 minutes, and then centrifuge briefly to remove the liquid on the inner edge of the centrifuge tube cap.
[0048] 5) Add 200μl of ethanol (96%-100%), shake for 15s, and centrifuge briefly to remove the liquid on the inner edge of the centrifuge tube cap.
[0049] 6) Carefully add the above-obtained mixture (including the precipitate) to the QIAamp Mini spin column (without wetting the rim), put the spin column into a 2ml collec...
Example Embodiment
[0096] Example 3 Detection of clinical samples
[0097] Take 40 clinical samples to be tested, extract the genome, prepare the reagents and test according to the methods and reagents described in Examples 1 and 2.
[0098] Add 1 μL of each sample to the PCR reaction solution of the detection system. Make a standard product at the same time. Detect with a fluorescent PCR machine for 120 minutes.
[0099] Such as figure 1 Shown are the HRM results of standard AA, standard GA and standard GG.
[0100] Such as figure 2 Shown are the HRM results of 40 clinical samples and standards.
[0101] According to the HRM results, the genotypes of the NOD1 gene rs2075820 locus of 40 screening samples were determined as shown in Table 1.
[0102] Table 1
[0103]
[0104]
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