Detection kit and method for copy number of spinal muscular atrophy pathogenic gene SMN1 based on real-time fluorescence quantitative PCR (Polymerase Chain Reaction) technique
A technology of real-time fluorescence quantification and spinal muscular atrophy, which is applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., and can solve the problem that the specificity of amplification products or indicator probes cannot be guaranteed, the amount of products varies greatly, and the copy In order to achieve the results of accurate and reliable detection results, avoid non-specific amplification, and improve specificity and amplification efficiency
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[0044] 1) Sample DNA concentration dilution
[0045] 152 DNA samples verified by other methods were diluted to a final concentration of 10-30ng / μl. The samples included 39 SMN1 double-copy normal individuals, 73 single-copy carriers, and 40 homozygous deletion patients.
[0046] 2) Dilution of standard plasmid DNA concentration
[0047] Standards were diluted 1:10:100:1000 for the preparation of the standard curve.
[0048] 3) PCR reaction system configuration
[0049] The PCR reaction system is 20 μl, and each reaction component is as follows:
[0050]
[0051] DNA was replaced with ddH in the negative control control reaction 2 O.
[0052] Primers and probes are as follows:
[0053] Primer: SMN1-F:ATAAAGCTATCTATATATAGCTATCTAT / LNA_G / (SEQ ID NO.1);
[0054] Primer: SMN1-R:CCTTCTTTTTGATTTTGTCT / LNA_G / (SEQ ID NO.2);
[0055] Probe: SMN-P:FAM-ACCCTGTAAGGAAAATAAAGGAAGT-MGB (SEQ ID NO.3);
[0056] Blocking probe: SMN2-B:TTCTTTTTGATTTTGTC / LNA_T / / LNA_A / AAACCC-C3 Spacer (SE...
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