WO2008077329A1 - Sonde destinée à détecter la mutation a1555g de surdité génétique mitochondriale héritée de la mère et son utilisation - Google Patents

Sonde destinée à détecter la mutation a1555g de surdité génétique mitochondriale héritée de la mère et son utilisation Download PDF

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WO2008077329A1
WO2008077329A1 PCT/CN2007/071003 CN2007071003W WO2008077329A1 WO 2008077329 A1 WO2008077329 A1 WO 2008077329A1 CN 2007071003 W CN2007071003 W CN 2007071003W WO 2008077329 A1 WO2008077329 A1 WO 2008077329A1
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probe
real
seq
fluorescent
mutation
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PCT/CN2007/071003
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English (en)
Chinese (zh)
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Zhengce Jin
Pu Dai
Yongyi Yuan
Dongyi Han
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Dongguan Shi Aomaier Gene Technological Co., Ltd
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Publication of WO2008077329A1 publication Critical patent/WO2008077329A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of genetic engineering technology, and particularly to a probe for diagnosing maternal mitochondrial genetic deafness and its use, and more particularly to a real-time fluorescent quantitative probe for detecting a maternal genetic mitochondrial deafness gene A1555G mutation and its use.
  • the invention further relates to the use of said probes and related products in the manufacture of kits or similar products for the diagnosis of maternal genetic mitochondrial deafness.
  • Aminoglycoside antibiotics are widely used clinically for their broad-spectrum and high-efficiency antibacterial effects and low price. Controls Gram-negative and positive bacterial infections, but such antibiotics have severe ototoxic side effects that can cause irreversible hearing loss in patients. In the past ten years, some aminoglycoside antibiotics have a family history of maternal inheritance, and the 1555 A ⁇ G (hereinafter referred to as A1555G mutation) with the mitochondrial DNA 12S rRNA gene (hereinafter referred to as mitochondrial gene, or mtDNA).
  • Kit Patent No.: ZL03156762.2
  • HSU03156762.2 which introduces a new restriction enzyme site including 1555 in the vicinity of 1555 mitochondrial gene, and then digested with Hae III restriction endonuclease (Daipu) Et al., Chinese Journal of Hearing and Speech Rehabilitation, 2004, 6: 21-23), although it has a significant cost advantage over BsmAI (Alw26I), it still requires tedious processing after polymerase chain reaction, which takes a long time.
  • the object of the present invention is achieved by the following principle: In order to detect the A1555G mutation in the maternal genetic mitochondrial gene sequence (SEQ ID NO: 5), a probe covering the 1555 bp site of the gene sequence is designed (the probe sequence span is 1543 bp to 1574 bp), and a fluorescent reporter group is labeled at the 5' end of the probe fragment, and a fluorescence quenching group is labeled at the 3' end, wherein: if the probe is replaced with G at the corresponding base A of 1555 bp, It is called a mutant real-time fluorescent quantitative probe (or mutant probe, mutant Taqman probe); if the probe is still A at the 1555 bp corresponding base, it is called wild-type real-time fluorescent quantitative probe (or wild Type probe, wild-type Taqman probe); due to the different 5'-end fluorescent reporter groups of the mutant and wild-type probes, that is, the fluorescence of the two different emission wavelengths (fluorescence of different wavelengths in real-time quantitative fluorescent
  • the mutant probe can be completed with the template with the A1555G mutation.
  • Matching does not match the wild-type template; wild-type probes do not match the template that produces the A1555G mutation, but only match the wild-type template; and the probe-binding template with different fluorescent reporter groups can emit different wavelengths. Fluorescence.
  • a forward primer and a reverse primer (which can be designed according to any biological primer software) for amplifying the maternal genetic mitochondrial deafness gene sequence SEQ ID NO 5 are added, and a mutant real-time fluorescence is added.
  • Quantitative probes if desired, can also be added to wild-type real-time fluorescent quantitative probes.
  • the 5'-end fluorescent reporter group can transfer energy to the adjacent 3'-end fluorescence quenching group, while the 3'-end fluorescence quenching group can Inhibition of the fluorescence emission of the 5'-end fluorescent reporter group. Therefore, when the probe is intact, that is, when the 5'-end fluorescent reporter group is not detached from the 3' fluorescence quenching group, the fluorescence emitted by the fluorophore at the 5' end of the probe is not detected.
  • the primer and the mutant probe and the wild type probe are simultaneously added to the real-time quantitative PCR reaction system.
  • both the primer and the probe are bound to the template.
  • the fluorescent reporter group attached to the 5' end of the probe is cleaved from the probe and freed from the reaction system, thereby being detached from the shielding of the 3'-end fluorescence quenching group and excited by the pupil.
  • the fluorescent signal that is, one fluorescent molecule is formed for each DNA strand amplified, and the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product, that is, the intensity of the fluorescent signal is linearly related to the amount of the corresponding PCR product.
  • the mutant probe will be fully matched with it.
  • the 5'-end fluorescent reporter group is continuously excised by Taq, and is detached from the 3'-end fluorescence quenching group. The fluorescence intensity increases as the amount of PCR product increases.
  • the wild type probe cannot be combined with one base difference, so only the fluorescent signal corresponding to the emission of the mutant probe is detected; when the template is A1555 A wild type, the wild type probe will fully match the template. In combination, the mutant probe cannot be bound by the difference of one base with it, so only the fluorescent signal corresponding to the emission of the wild type probe is detected. Since the 5' ends of the mutant and wild-type probes are respectively linked to different fluorescent reporter groups, the fluorescence can be distinguished by the fluorescence of different wavelengths recorded by different colors or labels recorded by the real-time PCR instrument analysis software. The corresponding template also detects whether the sample contains the maternal genetic mitochondrial deafness gene.
  • only the mutant probe may be added to the real-time quantitative PCR reaction system.
  • the fluorescent signal corresponding to the emission of the mutant probe is finally detected, thereby determining the sample content.
  • the maternal genetic mitochondrial deafness gene A1555G mutation determines that the patient has maternal mitochondrial genetic deafness disease; when the template is wild type, the fluorescent signal corresponding to the emission of the mutant probe is not detected, thus determining that the sample does not contain maternal genetic mitochondria
  • the deafness gene A1555G was mutated to confirm that the patient did not have maternal mitochondrial genetic deafness.
  • the first object of the present invention is to provide a real-time fluorescent quantitative probe for detecting a maternal genetic mitochondrial deafness gene A1555G mutation, wherein the 5' end of the probe is located in a maternal genetic mitochondrial deafness gene sequence.
  • the 3' end is located in the 1564-1574 bp region as shown in the mitochondrial gene sequence SEQ ID NO: 5 (preferably located 1567bp-1571bp, more preferably 1569bp), and a fluorescent reporter group is labeled at the 5' end of the probe fragment, a fluorescence quenching group is labeled at the 3' end, and a base A of 1555 bp is replaced with G, A mutant real-time fluorescent quantitative probe.
  • the nucleotide sequence of the mutant fluorescent probe is located in the 1548-1569 bp region as shown in the parental mitochondrial deafness gene sequence SEQ ID NO: 5 (such as the nucleoside shown in SEQ ID NO: l) The acid sequence), and the 5'-end fluorescent reporter group is a FAM fluorescent reporter group, and the 3'-end fluorescence quencher group is a TAMRA fluorescence quencher group.
  • a second object of the invention is to provide a wild type probe for use in conjunction with a mutant probe.
  • the wild-type real-time fluorescent probe is A
  • the 5'-end fluorescent reporter group is different from the 5'-end fluorescent reporter group of the mutant probe, except for the 1555 bp base.
  • the rest of the structure is the same.
  • the nucleotide sequence of the wild-type fluorescent probe is located in the 1548-1569 bp region as shown in the parental mitochondrial deafness gene sequence SEQ ID NO: 5 (such as the nucleoside shown in SEQ ID NO: 2) The acid sequence), and the 5'-end fluorescent reporter group is a HEX fluorescent reporter group, and the 3'-end fluorescence quencher group is a TAMRA fluorescence quencher group.
  • the 5'-end fluorescent reporter group and its corresponding 3'-end fluorescent quencher group can be any fluorophore available in the art, and those skilled in the art will select these fluorescent reporter groups and Their corresponding fluorescent quenching groups are also well known conventional techniques.
  • the fluorescent dyes FAM and HEX can be replaced by other fluorophores or dyes, such as VIC, JOE, and the like.
  • a third object of the invention is to provide PCR forward primers and reverse primers for use with real-time fluorescent quantitative probes.
  • the forward and reverse primers were used to amplify the maternal genetic mitochondrial deafness gene sequence SEQ ID NO: 5 containing the A1555G mutation.
  • the forward primer (about 15-24 bp long) 5' end is located in the l-1467 bp region of SEQ ID NO: 5 (upstream of A1555G), and the 3' end of the reverse primer (about 15-24 bp long) is located at SEQ ID NO: 5.
  • the 1668 bp to 16568 bp region (downstream of A1555G); preferably, the 5' end of the forward primer (about 15-24 bp long) is located in the 1000-1467 bp region of SEQ ID NO 5 (upstream of A1555G), and the reverse primer (about 15-length)
  • the 3' end of 24 bp) is located in the 1668 bp to 2000 bp region of SEQ ID NO: 5 (downstream of A1555G); more preferably, the 5' end of the forward primer (about 15-24 bp long) is located at SEQ ID NO: 5 (upstream of A1555G)
  • the 3' end of the reverse primer (about 15-24 bp long) is located in the 1668 bp to 1700 bp region of SEQ ID NO: 5 (downstream of A1555G); most preferably, the forward primer (about 15-24 bp long) The 5' end is located at the 1467 bp site of SEQ ID NO:
  • the forward primer is the 1467-1483 bp nucleotide sequence shown in SEQ ID NO: 5 of the maternal genetic mitochondrial deafness gene sequence, the sequence of which is SEQ ID NO: 3 in the sequence listing;
  • the reverse primer is Its nucleotide sequence of 1650-1668 bp, the sequence of which is SEQ ID NO: 4 in the Sequence Listing.
  • primers can be designed using a variety of biological software programs. Primers are preferably designed using the Genetool Lite program (software from Biotools, USA).
  • a fourth object of the present invention provides the use of the above probe for the preparation of a kit for diagnosing a maternal mitochondrial deafness genetic disease.
  • the inventors provide primers using the principles and design of the invention, as well as Taqman probes.
  • a kit for detecting a maternal genetic deafness mitochondrial gene A1555G mutation in vitro for diagnosis of maternal mitochondrial genetic deafness comprising:
  • PCR amplification reaction reagent mixture preferably Hotstar Mastermix mixture
  • the forward primer and the reverse primer of SEQ ID NO: 5 (preferably in equal proportions);
  • kits may further comprise the aforementioned wild type real-time fluorescent quantitative probe.
  • the forward primer and reverse primer structures in the kit described above are as described above.
  • it is selected from the nucleotide sequence shown in SEQ ID NO: 3
  • the reverse primer is preferably selected from the nucleotide sequence shown in SEQ ID NO: 4.
  • the mutant probe nucleotide sequence is preferably selected from the group consisting of the nucleotide sequence set forth in SEQ ID NO: 1, the fluorescent reporter group at the 5' end is a FAM fluorescent reporter group, 3 ' The fluorescent quenching group at the end is a TAMRA fluorescence quenching group.
  • the wild-type probe nucleotide sequence is preferably selected from the nucleotide sequence set forth in SEQ ID NO: 2, the fluorescent reporter group at the 5' end is a HEX fluorescent reporter group, 3 ' The fluorescent quenching group at the end is a TAMRA fluorescence quenching group.
  • the reagent for extracting blood sample DNA described in the above kit is a solution I for extracting DNA of the foot root, the main component of which is Chelex (product of ABI); or the reagent for extracting blood sample DNA is peripheral blood.
  • DNA extraction reagents are selected for use with commercially available products.
  • kit is also intended to include biological products having a different name than the kit, but having similar structures or components and biological functions as the kit.
  • the invention provides the use of the above probe or kit for detecting a maternal genetic mitochondrial deafness gene A1555G mutation in vitro.
  • the present invention also provides the above-described probe or kit for preparing a product (including drugs, reagents, kits, etc.) for diagnosing a maternal mitochondrial deafness genetic disease (ie, detecting a maternal genetic mitochondrial deafness gene A1555G mutation) In the product). Detection of the maternal genetic mitochondrial deafness gene A1555G mutation has the following advantages compared with the prior art (such as Hae III digestion identification method): 1.
  • the method for detecting mitochondrial deafness gene A1555G mutation and the reaction time of the invention have obvious advantages over the prior art (such as Hae III enzyme digestion identification method):
  • the invention can detect the presence of A1555G mutation only by one PCR process,
  • the prior art (such as Hae III digestion method) simplifies the agarose gel electrophoresis detection, the enzyme digestion reaction and the agarose gel electrophoresis detection after PCR, which not only reduces the post-PCR reaction band The risk of pollution has also shortened the reaction time from nearly 6 hours to 1.5 hours.
  • the two probes of the present invention are designed for the A1555G mutant and the A1555A wild type, respectively.
  • each probe binds only to the template whose sequence matches it perfectly, so the specificity is extremely strong.
  • the 5'-end fluorescence of a mutant probe such as FAM fluorescence
  • the 5'-end fluorescence (such as HEX fluorescence) of the wild-type probe is not detected as the A1555G mutant; On the contrary, it can be definitely A1555A wild type.
  • a mutation probe for the A1555G mutant template can be added to a system for diagnosis.
  • the double correction effect of the reverse correction can be performed, thereby making the result more accurate and reliable.
  • the high specificity of the probe technology and the sensitivity of the spectroscopy technique give the present invention the advantages of good repeatability, specificity, sensitivity, and ease of operation.
  • the A1555G mutation detection is carried out by using the kit or related product provided by the present invention, and the cost is lower than other conventional detection methods.
  • the probe and the kit thereof of the invention fill the gaps in the relevant domestic detection field, and the advantages of simple operation, intuitive interpretation, high specificity, high sensitivity, low price, etc. are to implement maternal inheritance nationwide.
  • Screening of the mitochondrial deafness gene A1555G mutation and pre-application prophylactic testing provide a more convenient condition to fundamentally reduce the chance of drug-induced deafness in individuals who are sensitive to aminoglycoside antibiotics.
  • Figure 1 shows partial results of real-time PCR detection of 132 different genotypes (Allelic data for Cycling A.FAM/Sybr, Cycling A JOE).
  • Figure 2 shows a legend of a partial result plot of real-time fluorescent quantitative PCR detection of 132 different genotypes. detailed description
  • Primer and Taqman probe sequences were designed using the Genetool Lite program according to the published mitochondrial gene sequence (Cambridge Sequence or NCBI human mitochondrial genome sequence NC_001807.4 or NT_006713.14, etc., or SEQ ID NO: 5), wherein:
  • the nucleotide sequence of the mutant fluorescent probe (T1) is located in a region of 1548-1569 bp as shown in the parental mitochondrial deafness gene sequence SEQ ID NO 5 (such as the nucleotide sequence shown in SEQ ID NO: 1), and
  • the fluorescent reporter group is a FAM fluorescent reporter group, and the fluorescence quenching group is a TAMRA fluorescence quenching group.
  • the probe structure is as follows:
  • the nucleotide sequence of the wild-type fluorescent probe (T2) is located in a region of 1548-1569 bp as shown in the parental mitochondrial deafness gene sequence SEQ ID NO 5 (such as the nucleotide sequence shown in SEQ ID NO: 2), and
  • the fluorescent reporter group is a HEX fluorescent reporter group, and the fluorescence quenching group is a TAMRA fluorescence quenching group.
  • the probe structure is as follows:
  • the forward primer (F1) is the 1467-1483 bp nucleotide sequence shown in SEQ ID NO: 5 of the maternal genetic mitochondrial deafness gene sequence, the sequence of which is SEQ ID NO: 3 in the sequence listing;
  • the reverse primer (R1) is The nucleotide sequence of 1650-1668 bp is shown, the sequence of which is SEQ ID NO: 4 in the Sequence Listing.
  • the probes T1, T2, forward primer were obtained by artificial synthesis.
  • F1 and the reverse primer R1 and performing real-time quantitative PCR amplification reaction using the peripheral blood DNA of the sample to be tested as a template:
  • a total of 32 A1555G mutations carrying mtDNA were detected by real-time fluorescence quantitative Taqman probe method in 132 subjects.
  • the 1555 bp sites of all tested individuals were verified by Alw26I digestion and direct sequencing. The results showed that 32 deaf patients carrying the A1555G mutation detected by real-time fluorescent quantitative Taqman probe method contained 1555 sites. None of the amplified products could be cleaved by Alw26I; 100 individuals who did not carry the A1555G mutation, which contained amplification products at 1555, were all cleaved by Alw26I. Direct sequencing confirmed the results of the Taqman probe method. The agreement rate of the three methods was 100%, indicating that the reliability and stability of the A1555G mutation were detected using the method of the present invention. See Table 2 for specific data.
  • the present invention comprises a probe for detecting a maternal genetic mitochondrial deafness gene A1555G mutation and related products, which can be used to more easily, quickly and accurately satisfy the need for extensive screening of the mtDNA A1555G mutation.

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Abstract

L'invention concerne une sonde fluorescente quantitative en temps réel destinée à détecter la mutation A1555G d'un gène de surdité mitochondrial hérité de la mère et son utilisation. Elle concerne également des sondes TaqMan mutantes et de type sauvage ainsi qu'une paire d'amorces. Le génotype de la mutation A1555G du gène de surdité mitochondrial hérité de la mère est analysé au moyen d'un procédé de sonde TaqMan, quantitative en temps réel. La surdité mitochondriale héritée de la mère peut ainsi être diagnostiquée.
PCT/CN2007/071003 2006-12-26 2007-11-01 Sonde destinée à détecter la mutation a1555g de surdité génétique mitochondriale héritée de la mère et son utilisation WO2008077329A1 (fr)

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CN200610169638.3A CN1987462B (zh) 2006-12-26 2006-12-26 检测母系遗传线粒体耳聋基因a1555g突变的试剂盒
CN200610169638.3 2006-12-26

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Cited By (2)

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CN102154481A (zh) * 2011-02-11 2011-08-17 智海生物工程（北京）有限公司 一种线粒体突变检测方法及试剂盒
CN112746105A (zh) * 2021-01-26 2021-05-04 上海博奥颐和医学检验所有限公司 一种遗传性耳聋基因检测方法

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CN1987462B (zh) * 2006-12-26 2015-03-25 金政策 检测母系遗传线粒体耳聋基因a1555g突变的试剂盒
CN101818193B (zh) * 2009-11-13 2012-09-05 中国人民解放军总医院 筛查检测线粒体dna片段缺失的序列及试剂盒
CN103571846B (zh) * 2012-07-18 2017-07-18 中国人民解放军总医院 Atp6v1b2基因突变体及其应用
CN102994643B (zh) * 2012-12-27 2014-05-07 北京市农林科学院 用于检测鸡fmo3基因a1034t突变的引物和探针
CN107287303A (zh) * 2017-06-28 2017-10-24 浙江大学 线粒体耳聋a1555g突变的荧光定量pcr检测试剂盒及其应用

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CN102154481B (zh) * 2011-02-11 2013-01-02 智海生物工程（北京）有限公司 一种线粒体突变检测方法及试剂盒
CN112746105A (zh) * 2021-01-26 2021-05-04 上海博奥颐和医学检验所有限公司 一种遗传性耳聋基因检测方法

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