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Primers, probes and kit for quantitative detection of SENP2 mRNA level
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A quantitative detection and kit technology, applied in the direction of DNA/RNA fragment, recombinant DNA technology, microbial determination/examination, etc., can solve the problems of not reflecting the actual situation of myocarditis patients, lack of positive correlation of myocarditis, etc., achieving high sensitivity, Good specificity, sensitive and accurate detection
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Problems solved by technology
However, it is clinically found that the detection of myocardial enzymes often lacks positive correlation with viral myocarditis, and sometimes cannot reflect the actual situation of patients with myocarditis.
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[0033] 1. Design and synthesis of primers and probes:
[0034] The conserved gene sequences of SENP2 and GAPDH were found from GenBank, and Primer Primer 5.0 software was used to design amplification primers and probe sequences to ensure that each pair of primers could specifically amplify SENP2 and GAPDH.
[0035] The nucleotide sequences of primer F1 and primer R1 for amplifying the SENP2 gene, and primer F2 and primer R2 for amplifying the reference gene GADPH are respectively:
[0036] F1: 5'-GGACACTGATGAAATACCAG-3'
[0037] R1: 5'-CCGTGTTCCATTACAAGCAG-3'
[0038] F2: 5'-CCTGCCAAGTATGATGACATCAAGA-3'
[0039] R2: 5'-GTAGCCCAGGATGCCCTTTAGT-3'.
[0040] The nucleotide sequences of the probe P1 for detecting the SENP2 gen...
[0052] Reaction solution preparation: 2×NASBA reaction solution is 10µL×n, 5×RT-PCR reaction enzyme mixture is 4µL×n, DEPC-H 2 O is 2 µL x n. Mix the components evenly, and distribute 16 µL per tube into eight tubes (n is the number of reaction tubes).
[0053] Gradient dilution of the standard: the standard solution was diluted to 1×10 by 10-fold gradient dilution. 7 copies / mL, 1×10 6 copies / mL, 1×10 5 copies / mL and 1×10 4 copies / mL.
[0054] Preparation of the standard curve: Take 4 µL of the standard dilutions of each concentration, add them to the reaction solution containing 16 µL, and carry out PCR amplification on the LightCycler480 quantitative PCR instrument of Roche Company. The PCR reaction conditions are: 50°C, 15min→95°C, 3min→(95°C, 15s→55°C, 30s, 72°C, 30s). A total of 40 cycles of PCR amplification steps were performed.
[0055] According to the fluorescent quantitative PCR detection results of the standa...
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Abstract
The invention relates to the technical field of biological detection, in particular to primers, probes and kit for quantitative detection of the SENP2 mRNA level. The primers comprise the primer F1 and the primer R1 which are used for amplifying an SENP2 gene and the primer F2 and the primer R2 which are used for amplifying a reference gene GADPH. The probes comprise the probe P1 used for detecting the SENP2 gene and the probe P2 used for detecting the reference gene GADPH. The kit comprises the primers and the probes. The primers and the probes are good in specificity and sensitive and accurate in detection. The kit uses double channels of FAM and VIC for accurately and quantitatively analyzing the expression of SENP2 mRNA, and has the advantages of being rapid, simple, convenient, high in sensitivity and the like.
Description
technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer, a probe and a kit for quantitatively detecting SENP2 mRNA levels. Background technique [0002] Viral myocarditis (VMC) is a localized or diffuse acute or chronic inflammatory lesion of the myocardium caused by a virus. In recent years, the incidence of viral myocarditis in my country is on the rise, and it has become one of the main causes of sudden death in children and adolescents. Some patients have a self-limiting course of disease. In severe cases, heart failure, cardiogenic shock, and sudden death may occur. Some patients may develop dilated cardiomyopathy, and the fatality rate is as high as 50%. Viral myocarditis has become a major public health problem that needs to be solved urgently in our country. The current clinical detection of viral myocarditis mainly focuses on the changes of myocardial enzymes CK and CK-MB released in the serum after my...
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