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CN-115261470-B - Kit for detecting tumor driving gene TP 53R 248W

CN115261470BCN 115261470 BCN115261470 BCN 115261470BCN-115261470-B

Abstract

The invention provides a kit for detecting tumor driving gene TP53R248W, which uses crRNA with a nucleotide sequence shown as SEQ ID NO. 5, designs R248W mutant base at the 5' end of a spacing sequence of the crRNA, introduces new mismatched base C, and cannot form a complex when two base mismatches exist between the spacing sequence of the crRNA and wild RNA, so that Cas13a protein cannot be activated. The kit of the invention can detect the mixed plasmid with the mutation frequency of 0.01% at the lowest and can detect TP53R248W variant with the concentration of 10 4 copies/. Mu.L in plasma at the lowest. The kit provided by the invention has the advantages of good sensitivity, high specificity, simplicity and convenience in operation, rapidness and high efficiency.

Inventors

  • LI LINHAI
  • Kuang Zhenzhan
  • CHEN JIANYUN
  • SUN CHAOHUI
  • XIAO BIN
  • CHEN LISHA

Assignees

  • 中国人民解放军南部战区总医院

Dates

Publication Date
20260505
Application Date
20220624

Claims (7)

  1. 1. A CRRNA, primer and probe composition for detecting tumor driving gene TP 53R 248W based on CRISPR/Cas13a nucleic acid detection system is characterized in that the nucleotide sequence of the crRNA is shown as SEQ ID NO. 5, the nucleotide sequence of the primer is shown as SEQ ID NO. 2 and SEQ ID NO. 4, and the nucleotide sequence of the probe is shown as SEQ ID NO. 6.
  2. 2. The composition of claim 1, wherein the probe is labeled with a FAM group at the 5 'end and a BHQ1 group at the 3' end.
  3. 3. Application of the composition of any one of claims 1-2 in preparing a product for detecting tumor driving gene TP53R 248W.
  4. 4. A kit for detecting a tumor driving gene TP 53R 248W, wherein the kit comprises the composition of any one of claims 1-2.
  5. 5. The kit of claim 4, wherein the kit comprises a RAA amplification reagent comprising a RAA reaction solution, mgCl 2 , water, and a RAA dry powder comprising a recombinase, a single-stranded binding protein, and a DNA polymerase.
  6. 6. The kit of claim 4, wherein the kit contains a PCR amplification reagent comprising a DNA polymerase and water.
  7. 7. The kit of claim 4, wherein the kit contains a CRIPSR/Cas13a detection reagent comprising Cas13a protein, T7 transcriptase, rnase inhibitor, 5 x T7 buffer, NTP, and water.

Description

Kit for detecting tumor driving gene TP 53R 248W Technical Field The invention relates to the technical field of gene detection, in particular to a kit for detecting a tumor driving gene TP53R 248W. Background TP53 is a very important oncogene, is expressed low in normal cells, and is expressed high in malignant tumors. Inactivating mutations caused by missense mutation, insertion or deletion of the gene are very common. Clinical studies confirm that 95.1% of TP53 point mutations in tumors occur predominantly at the highly conserved 175, 245, 248, 249, 273 and 282 positions. Loss of function events in TP53 are common in cancer, and the R248W variant not only results in loss of tumor suppression, but also as a function gain mutation, can promote tumorigenesis in a mouse model. Such mutants are more responsive to doxorubicin treatment than the wild-type mutants. It has been shown that the R248W mutation results in poorer survival. The detection of the tumor driving gene TP 53R 248W is beneficial to clinical medication guidance and prognosis evaluation, and has important significance for improving the total survival rate of patients. Currently, the detection means of TP 53R 248W in tissues and plasma mainly comprise digital PCR, high-throughput sequencing, mutation amplification systems and the like. The TP53 gene R248W mutation is that the base at position 742 of the gene is mutated from C to T, and because of the mutation of only a single base, false positive phenomenon can occur if qPCR technology is used for detection, because the probe used by qPCR has inherent defects that the probe is easy to be combined with non-target genes in a non-specific way to form mismatch, and particularly, the non-target genes are different from the target genes by only one base. The digital PCR technology has high cost, high requirements on instruments and operators, high-throughput sequencing sensitivity, complex experimental flow, simple operation of a mutation amplification system and poor stability, so that a novel tumor driving gene detection method with high sensitivity, good stability and low cost is needed to be found. With the continuous development of gene detection technology, many gene detection projects are presented in the market, and a large number of emerging enterprises begin to struggle in the field, such as Ancestry, illumina, helix and 23an dMe abroad, and China's Huada genes, san Xiang organisms, ani me genes and the like. At present, the national life quality is improved, the health consciousness of consumers is gradually enhanced, more and more people begin to pay attention to deep demands such as self physical health, nutrition conditions and the like, particularly, the people who are the consumers are more acceptable for fresh things, the medical knowledge such as genetic inheritance and the like is better known, and the genetic detection means are more favorable for knowing the genetic inheritance and the physical health information, so that the consumer-level genetic detection market is continuously increased. In the future commercial development, as the gene detection industry is mature, the downstream service business will be greatly expanded, and the competition of the gene detection enterprises will be more vigorous, so that the test is more huge. Disclosure of Invention The invention aims to overcome the defects in the prior art and provides a kit for detecting a tumor driving gene TP 53R 248W. It is a first object of the present invention to provide a composition. A second object of the invention is to provide the application of the composition in preparing products for detecting tumor driving genes TP53R 248W. The third object of the present invention is to provide a kit for detecting tumor driving gene TP 53R 248W. In order to achieve the above object, the present invention is realized by the following means: a composition comprises crRNA, a primer and a probe, wherein the nucleotide sequence of the crRNA is shown as SEQ ID NO. 5. Preferably, the nucleotide sequences of the primers are shown as SEQ ID NO. 2 and SEQ ID NO. 4. Preferably, the nucleotide sequence of the probe is shown as SEQ ID NO. 6. More preferably, the probe is labeled with a FAM group at the 5 'end and a BHQ1 group at the 3' end. The application of the composition in preparing and detecting tumor driving gene TP 53R 248W products. A kit for detecting tumor driving gene TP 53R 248W, wherein the kit comprises the composition. Preferably, the kit contains RAA or PCR amplification reagents, and CRISPR/Cas 13a detection reagents, wherein the RAA or PCR amplification reagents contain the primers, and the CRISPR/Cas 13a detection reagents contain the crRNA and the probes. Preferably, the RAA amplification reagent in the kit further comprises a RAA reaction solution, mgCl 2, water and RAA dry powder, wherein the RAA dry powder tube contains recombinase, single-chain binding protein and DNA polymerase. More preferably, the RAA ampli