Group of probes, detection kit and detection method for detecting thalassemia gene point mutation based on liquid chip of locked nucleic acid sensibilization

A liquid-phase chip detection and thalassemia technology, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problem of high non-specific hybridization signals and cannot clearly distinguish wild-type or mutant DNA Fragments and other issues to achieve the effect of easy distinction

Active Publication Date: 2015-01-21
GUANGDONG WOMEN & CHILDREN HOSPITAL
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Problems solved by technology

If there is only one base difference between wild-type and mutant DNA, probes designed according to conventional methods will often

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  • Group of probes, detection kit and detection method for detecting thalassemia gene point mutation based on liquid chip of locked nucleic acid sensibilization
  • Group of probes, detection kit and detection method for detecting thalassemia gene point mutation based on liquid chip of locked nucleic acid sensibilization
  • Group of probes, detection kit and detection method for detecting thalassemia gene point mutation based on liquid chip of locked nucleic acid sensibilization

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Embodiment 1

[0053] 1. Probe coupling

[0054] When synthesizing the probes for detection of thalassemia gene point mutations based on locked nucleic acid sensitization liquid-phase chips listed in Table 1, NH2-C12 connecting arms are brought on the 5' end of the probes, and NH2 can be used with liquid-phase chips The -COOH group of the microbead undergoes a chemical coupling reaction, allowing the probe to attach to the Luminex MicroPlex microbeads. Beads with different codes are coupled with different probes, as shown in Table 3. The steps for coupling the probe to the microbeads are as follows: take out 40 μl (1×10 5 1) the desired microbeads, centrifuge at 12000rpm for 2min, discard the supernatant, add 5μl 0.1M MES pH4.5, and mix well. The probe used was diluted to 0.1 mM, and 1 μl was added to the coupling system. Add 2.5μl 10mg / ml EDC for the first time, mix well and place in the dark for 30min. Repeat adding EDC again, mix well and keep in the dark for 30min. Wash once each wi...

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Abstract

The invention discloses a group of probes, a detection kit and a detection method for detecting thalassemia gene point mutation based on a liquid chip of locked nucleic acid sensibilization. Due to the characteristics that locked nucleic acid can flexibly modify probes, probes for detecting thalassemia gene point mutation are designed; these probes are respectively coupled to different numbers of microspheres; a specific primer disclosed by the invention is designed to amplify mutation sites, so as to obtain a PCR product; the PCR product is hybridized with the microspheres coupled to the probes; fluorescence labeling is carried out by using a fluorescence labeling reagent; and finally detection on the mutation sites is carried out through a liquid chip detector. The probes based on LNA sensibilization are shortened in comparison with a conventional probe, and still can keep relatively high Tm values; so that the hybridization signal ratio of completely matched probes and mismatched probes is improved to over four times from below twice in the past, thus the probes are easy to distinguish.

Description

Technical field: [0001] The invention belongs to the field of molecular diagnosis, and in particular relates to a group of probes, detection kits and detection methods for detecting thalassemia gene point mutations based on locked nucleic acid-sensitized liquid-phase chips. Background technique: [0002] Thalassemia (Thalassemia) is a genetic hemolytic disease caused by globin synthesis disorders, divided into two types: α-thalassemia (abbreviated as α-thalassemia) and β-thalassemia (β-thalassemia). The disease is mainly found in countries along the Mediterranean Sea and Southeast Asian countries. It is the single gene disease with the widest distribution and the largest cumulative population in the world. There are about 90 million thalassemia gene mutation carriers in the world. It is more common in the south of my country, and it is one of the genetic diseases with the highest incidence and the greatest impact in the provinces south of the Yangtze River, especially in Gua...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6834C12Q2537/143C12Q2563/149C12Q2525/117
Inventor 张小庄尹爱华张亮叶宁刘畅杜丽
Owner GUANGDONG WOMEN & CHILDREN HOSPITAL
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