Multiplex Taqman fluorescence detection kit for diagnosis of mitochondria mutational deafness

A detection kit and mitochondrial technology, which are applied in the determination/testing of microorganisms, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of inability to obtain timely and effective diagnosis, limit the popularization of hereditary deafness diagnosis, and limit the prevention of hereditary deafness and treatment issues, to achieve high accuracy, fast detection, and low cost

Active Publication Date: 2016-04-20
石玉玲
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This greatly limits the popularization of hereditary deafness diagnosis, resulting in sporadic hereditary deafness in most parts of China cannot be diagnosed in a timely and effective manner, limiting the prevention and treatment of hereditary deafness

Method used

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  • Multiplex Taqman fluorescence detection kit for diagnosis of mitochondria mutational deafness
  • Multiplex Taqman fluorescence detection kit for diagnosis of mitochondria mutational deafness
  • Multiplex Taqman fluorescence detection kit for diagnosis of mitochondria mutational deafness

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: PCR primers and probes for detecting mitochondrial mutation deafness

[0024] In the early stage of the present invention, a large number of PCR primers for detecting DNA related to mitochondrial mutation deafness were designed through the principle of primer design and combined with the actual situation, and then through a large number of experimental studies to screen out highly sensitive and high specificity primer sequences and detection probes, and finally select the detection The nucleotide sequences of PCR primers and probes with the best effect on DNA related to mitochondrial mutant deafness are as follows:

[0025] Primers:

[0026] F: 5'-GTGCTTAGTTGAACAG-3' (SEQ ID NO: 1),

[0027] R: 5'-CACTTTCCAGTACACTTA-3' (SEQ ID NO: 2).

[0028] Probe:

[0029] P1: CY3-CCCGTCACCCTCCTCA-BHQ2 (SEQ ID NO: 3);

[0030] P2: TexasRed-CCCGTCACTCTCCTCCA-BHQ2 (SEQ ID NO: 4);

[0031] P3: FAM-TTATATAGAGGAGACAAGTCGTA-BHQ (SEQ ID NO: 5);

[0032] P4: CY5-TTATATAGAGGA...

Embodiment 2

[0033] Example 2: PCR detection kit for detecting mitochondrial mutation deafness

[0034] The PCR detection kit for detection of mitochondrial mutation deafness includes the following components:

[0035] 1) containing the primers described in Example 1;

[0036] 2) Contain the detection probe described in Example 1:

[0037] 3) PCR reaction solution: BIO-RADIQ TM MultiplexPowermix (catalog#172-5848) American Bole Company

[0038] 4) It also contains DNA polymerase and negative control substance (water).

Embodiment 3

[0039] Example 3: Detection method for detecting mitochondrial mutation deafness

[0040] Utilize the detection kit established in Example 2 to detect the sample to be detected, the steps are as follows:

[0041] 1) DNA extraction:

[0042] Take the whole blood sample to be tested (EDTA anticoagulation), use the QIAampDNABloodMiniKit extraction kit, follow the whole blood sample extraction steps in the manual, the elution volume is 70uL, and extract the DNA in each group of samples as the subsequent PCR template;

[0043] 2) PCR reaction system:

[0044] 10 μM primer F1 μL

[0045] 10 μM Primer R 1 μL

[0046] IQMultiplexPowermix2×10μL

[0047] DNA template 1 μL

[0048] 10 μM probe P10.28 μL

[0049] 10 μM probe P20.28 μL

[0050] 10 μM probe P30.28 μL

[0051] 10 μM Probe P4 0.28 μL

[0052] MilliH20 (deionized water) 5.88 μL

[0053] The total volume is 20 μL.

[0054]3) PCR reaction procedure:

[0055] Step 1: 95°C for 5 minutes; Step 2: 95°C for 30 seconds, 58...

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Abstract

The invention discloses a multiplex Taqman fluorescence detection kit for diagnosis of mitochondria mutational deafness. The kit contains primers as shown in SEQ ID NO:1-2, probes as shown in SEQ ID NO:3-6, DNA polymerase, negative reference substances and the like. At present, clinical diagnosis of deafness is mainly conducted by means of a Jingxin nine deafness gene detection chip produced by the CapitalBio Corporation, and the detection cost of each site is 100 yuan; the requirement for the chip storage condition is high, high-end equipment is adopted, the detection process is complicated, and time consumption is high. The multiplex Taqman fluorescence detection kit has the advantages of being simple, efficient, low in cost and high in accuracy; no high-end equipment is needed, and all reagents can be stored in a refrigerator which is at 4 DEG C; two sites can be detected at the same time through a single hole, a result can be obtained through one step of PCR, and the detection cost of two sites is about 40 yuan. Therefore, the kit and detection method make detection quick, accurate and low in cost, and popularization of deafness screening is facilitated.

Description

technical field [0001] The invention relates to multiple Taqman fluorescence detection kits for mitochondrial mutation deafness. Background technique [0002] Deafness, also known as sensorineural deafness, is a hearing defect that seriously affects the quality of life of individuals and the quality of the social population. According to the etiology, deafness can be divided into hereditary deafness and non-hereditary deafness. Among them, hereditary deafness accounts for 50%. Hereditary deafness is divided into autosomal inheritance, sex chromosome inheritance and mitochondrial inheritance according to the mode of inheritance. Among them, mitochondrial inheritance is also called maternal inheritance, and the probability that a female carrier will pass on the mutation to her next generation is 100%. Mitochondrial hereditary deafness also has a special clinical manifestation of "one-shot deafness". Mitochondrial hereditary deafness is not only related to congenital inheri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 石玉玲曹诚李林海张亚松熊敏王岳彤陈建芸廖扬
Owner 石玉玲
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