Kit for detecting genotype of fragile X syndrome FMR1
A kit and syndrome technology, applied in the field of kits for detecting the FMR1 genotype of Fragile X Syndrome, can solve the problems of inability to judge carriers of Fragile X Syndrome, low detection specificity and sensitivity, and unsuitable calculation formulas, etc. Achieve the effect of less sample nucleic acid, short detection time and large throughput
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Embodiment 1
[0062] Example 1 Detection of the primer set for the fragile X syndrome FMR1 gene
[0063] In order to realize the simultaneous detection of AGG and CGG repeats, it is necessary to overcome the non-specific amplification that may occur due to the high similarity between AGG and CGG, and the difficulty of amplification caused by the high C / G content of the target region sequence. In order to solve the above problems, specific primers were designed using the method of three primers, of which 2 primers were used for the full-length amplification of the target sequence, and the other primer was designed inside the pair of primers as a "nested" primer for detection AGG inserts information; this primer design significantly increases sensitivity while maintaining high specificity. After a large number of tests for combination, screening, optimization, verification, the experimental results are as follows: figure 1 shown. Finally, a primer set with strong stability, high amplificati...
Embodiment 2
[0065] Example 2 A kit for detecting the FMR1 genotype of Fragile X Syndrome
[0066] According to the capillary electrophoresis detection platform, this embodiment exemplarily provides a detection kit for detecting the FMR1 genotype of fragile X syndrome. It should be noted that the detection kits for Fragile X Syndrome FMR1 gene developed based on other detection platforms using the primer set of the present invention should also be within the protection scope of this application, and will not be exemplified here.
[0067] 1. Composition
[0068] 1. Primers
[0069] AGG / CGG primers (60 μL / tube) for detecting AGG insertion information, its nucleotide sequence is shown in SEQ ID NO: 1,
[0070] F / R primers (60 μL / tube) for amplifying the CGG repeat region of the FMR1 gene, the nucleotide sequence is as shown in SEQ ID NO: 2-3, and the nucleotide sequence is 5 of the primer shown in SEQ ID NO: 3 ' end with FAM fluorophore.
[0071] SEQ ID NO: 1: 5'-AGCGTCTACTGTCTCGGCACTTGCC...
Embodiment 3
[0095] Example 3 Detection of samples of different FMR1 genotypes
[0096] 1. Experimental method
[0097] Collect 6 samples of known different genotypes, use the kit of Example 1 and The PCR / CEFMR1 Kit kit was used to detect the FMR1 genotype respectively.
[0098] 2. Experimental results
[0099] The result is as Figure 2 to Figure 4 , put the peak fragment size of the full-length amplification product into the calculation formula Y=0.3399X-78.554 to calculate the CGG repeat number of the sample, see Table 2 for the specific results. Insertion of sample AGG: figure 2 A male sample has 2 AGG insertions, figure 2 B female sample has 3 AGG insertions; image 3 A and Figure 4 A male positive sample has no AGG insertion, image 3 The normal alleles of B and 4B female positive samples have 2 AGG insertions, using the kit of Example 2 and The specific detection results of the PCR / CEFMR1Kit kit are shown in Table 1.
[0100] Table 2:
[0101]
[0102]It shows tha...
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