Diagnostic kit and method for detecting human C-Kit gene exon 17 mutations
A kit and exon technology, which is applied in the field of diagnostic kits for detecting mutations in human C-Kit gene exon 17, can solve the problem of inability to evaluate multiple mutation sites in exon 17, unfavorable clinical detection, and detection sites. Single point and other problems, to achieve the effect of good clinical application prospect, good specificity and high sensitivity
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Embodiment 1
[0034] Example 1 The present invention detects the kit and detection method of exon 17 mutation of C-Kit gene
[0035] One, the composition of kit of the present invention
[0036] PCR amplification reagent (1 serving):
[0037] water
14.85μl
10XPCR buffer
2.5μl
MgCl2 25mM
0.75μl
UTP PLUS
0.5μl
Primer solution
1μl
UNG
0.2μl
Taq
0.2μl
overall system
20μl
[0038] Wherein "Primer solution" is prepared as follows:
[0039] C17F (100nM / ml)
12.5μl
C17F2 (100nM / ml)
12.5μl
C17F3 (100nM / ml)
12.5μl
C17R (100nM / ml)
12.5μl
C17P (100nM / ml)
2.5μl
H2O
47.5μl
total
100μl
[0040] Amplification primers and detection probes for c-kit gene exon 17 mutation:
[0041]
[0042] External control primers and probes:
[0043]
[0044]
[0045] 2. Using the kit of the present invention to detect mutations in exon...
experiment example 1
[0082] Experimental example 1 The accuracy and specificity detection of kit of the present invention and method
[0083] 1. Sample to be tested
[0084] One case of clinical GIST paraffin tissue samples with known mutation types of c-kit gene exon 17 mutation (respectively D816V, D820H, N822I, N822K mutation) were selected (for the sequencing results of D816V mutation, see figure 1 ) and a known wild-type clinical GIST paraffin tissue sample to detect the mutation type of human c-kit gene.
[0085] 2. Detection method
[0086] The method of the present invention: adopt the kit of embodiment 1, detect according to the method of embodiment 1.
[0087] Sample DNA extraction method: extract with Qiagen kit:
[0088] 1) Use a scalpel to remove the useless paraffin at the tissue border;
[0089] 2) Cutting the paraffin-embedded tissue into 4 μm thick slices;
[0090] 3) Quickly take 2-6 pieces with sterilized tweezers and put them into DNase / RNase Free EP tubes (each spreading ...
experiment example 2
[0109] Experimental example 2 sensitivity analysis of kit of the present invention and method
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