Kit for detecting MYD88 L265P mutation based on AS-PCR and application thereof
A kit and mutation-type technology, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial determination/inspection, etc., can solve system instability, confusion between weak positive samples and negative samples, and the increasing requirements for MYD88L265P mutation detection Advanced problems, to achieve the effect of high sensitivity and easy operation
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Embodiment 1
[0044] Example 1: Optimization of the amplification system
[0045] Due to the characteristics of AS-PCR technology, its non-specific amplification is strong in high-concentration samples. In order to suppress non-specific amplification, blocking primers and probes were introduced into the system. Using different ratios of probes and blocking primers (1:1, 1:2, 1:4, molar ratio), adding MYD88 L265P mutation-negative samples for amplification, it was found that when the molar ratio of probes and blocking primers was 1:2, The non-specific amplification of the mutant system was reduced, and the amplification of the wild system was not affected ( Figure 1-Figure 3 ).
Embodiment 2
[0046] Example 2: Construction of mutant, wild plasmid standard
[0047] According to the designed upstream and downstream primers, select the amplified fragment and about 100 bp upstream and downstream of the amplified fragment to construct the mutant plasmid and the wild plasmid respectively. The sequences of the mutant plasmid and the wild plasmid are respectively shown in SEQ ID NO.10 and SEQ ID NO.11. The copy number was calculated according to the quality of the plasmid, and the mutant plasmid and the wild plasmid were respectively prepared as 5×10 6 copies / μl (S1), and then use process water to dilute S1 with a gradient of 1:9 to obtain S2, S3, and S4 with a concentration of 5×10 5 copies / μl, 5×10 4 copies / μl, 5×10 3 copies / μl, and use the optimized system to amplify the standard to verify its linearity, R 2 = 1, see Figure 4 and Figure 5 .
Embodiment 3
[0048] Embodiment 3: kit composition and method of use
[0049] One, the composition of kit of the present invention is as follows:
[0050] Table 1: Kit Components
[0051]
[0052] The sequences of the primers and probes in the MYD88 L265P mutant master mix and the MYD88 L265P wild master mix are as follows:
[0053] Table 2: Primer Probe Sequences
[0054]
[0055] 2. How to use:
[0056] 1. Test samples
[0057] 20 negative clinical samples and 20 positive clinical samples were tested by the reference method.
[0058] 2. Extraction of sample DNA
[0059] Use whole blood nucleic acid extraction kit (Qiagen) to extract human genomic DNA, detect nucleic acid concentration and purity by spectrophotometer (DNA concentration ≥ 10ng / μl, OD260 / OD280 ratio between 1.8-2.0), and then test Dilute to 75ng / μl with DNA template. details as follows:
[0060] (1) Take a sufficient amount of 1.5ml centrifuge tube and mark it well, add 20μl Protease (or Protease K) solution to...
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