A Fragile X Syndrome Clinical Rapid PCR Detection Kit
A kit and syndrome technology, applied in the field of bioengineering, can solve the problems of long time, cumbersome steps and high consumption, and achieve the effect of reducing the incidence rate
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Embodiment 1
[0027] Example 1 Preparation of a PCR detection kit for detecting Fragile X syndrome
[0028] 1. Primer design
[0029] Design a set of specific primers in the -600-+733 region of the FRM-1 gene, including primers F and R for amplifying the target fragment (including the CGG repeat region); and primer F for amplifying the internal reference fragment , M.
[0030] The sequences of F, R and M in the primer group are selected from any one of groups 1-6 in Table 1, and the primer sequences are shown in Attached Table 1.
[0031] 2. Prepare PCR reaction solution
[0032] The components of the PCR reaction solution are as follows: 1) 10× buffer solution, the buffer solution includes a concentration of 500mM Tris-HCl (pH9.0) and 20mM MgCl 2 . 2) dNTPs at a concentration of 2.5mmol / L; 3) the upstream primer F of the target gene primer at a concentration of 10 μmol / L; 4) the downstream primer R of the target gene primer at a concentration of 10 μmol / L; 5) the concentration of 10 μm...
Embodiment 2
[0033] Embodiment 2 application kit carries out negative sample detection
[0034] 1) DNA extraction: Peripheral blood was collected with the consent of the subject or the knowledge of his guardian. A commercially available full-type gold blood DNA extraction kit was used to extract, and the concentration was calibrated to be 100 ng / μl. The sample was clinically negative.
[0035] 2) Prepare a PCR amplification system using the kit of Example 1;
[0036] PCR amplification system:
[0037]
[0038] in,
[0039] The sequence of the primer group is shown in Group 1 in Table 1,
[0040] Composition of PCR enhancer: 6 μl of 2.5mol / L betaine, 4 μl of DMSO.
[0041] (3) PCR amplification conditions: pre-denaturation at 95°C for 8 minutes, suspend adding 1ul of Taq enzyme; denaturation at 95°C for 45s, annealing at 60°C for 45s, extension at 72°C for 1.5min, and final extension for 10min after 30 cycles.
[0042] (4) After the reaction, 1.5% agarose gel electrophoresis was used...
Embodiment 3
[0043] Embodiment 3 application kit carries out positive sample detection
[0044] (1) DNA extraction: Peripheral blood was collected with the consent of the patient or the knowledge of the guardian. A commercially available full-type gold blood DNA extraction kit was used to extract, and the concentration was calibrated to be 100 ng / μl. The sample tested positive for Fragile X syndrome clinically.
[0045] (2) Prepare a PCR amplification system using the kit of Example 1;
[0046] PCR amplification system:
[0047]
[0048] Wherein, the sequence of the primer group is shown in Group 1 in Table 1,
[0049] Composition of PCR enhancer: 6 μl of 2.5mol / L betaine, 4 μl of DMSO.
[0050] (3) PCR amplification conditions: pre-denaturation at 95°C for 8 minutes, suspend adding 1 μl of Taq enzyme; denaturation at 95°C for 45 seconds, annealing at 60°C for 45 seconds, extension at 72°C for 1.5 minutes, and final extension for 10 minutes after 30 cycles.
[0051] (4) After the r...
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