CN114752667A - Primer group, probe and kit for quantitatively detecting heterogeneity of MT-ATP6m.9185 locus - Google Patents

Primer group, probe and kit for quantitatively detecting heterogeneity of MT-ATP6m.9185 locus Download PDF

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CN114752667A
CN114752667A CN202210438091.1A CN202210438091A CN114752667A CN 114752667 A CN114752667 A CN 114752667A CN 202210438091 A CN202210438091 A CN 202210438091A CN 114752667 A CN114752667 A CN 114752667A
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probe
heterogeneity
atp6m
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seq
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邹薇薇
纪冬梅
宗凯
曹云霞
刘雅静
章志国
梁春梅
梁丹
魏兆莲
周平
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First Affiliated Hospital of Anhui Medical University
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    • 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
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The invention discloses a primer group, a probe and a kit for quantitatively detecting heterogeneity of MT-ATP6m.9185 locus, and relates to the technical field of biology. The primer group comprises an upstream primer shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2; the probe comprises a wild type probe shown as SEQ ID NO. 3 and a mutant type probe shown as SEQ ID NO. 4. The kit comprises the primer group and the probe. The invention provides a primer group, a probe and a kit for quantitatively detecting the heterogeneity of an MT-ATP6m.9185 locus, which can realize high-accuracy, high-sensitivity and high-repeatability detection of the heterogeneity of the MT-ATP6m.9185 locus in a human single-cell sample.

Description

Primer group, probe and kit for quantitatively detecting heterogeneity of MT-ATP6m.9185 locus
Technical Field
The invention relates to the field of biotechnology, in particular to a primer group, a probe and a kit for quantitatively detecting heterogeneity of an MT-ATP6m.9185 locus.
Background
MT-ATP6 gene of mitochondrial DNA (mitochondrial DNA, mtDNA) encodes a critical enzyme F for mitochondrial energy metabolism1F0The ATP6 subunit of the ATP synthase complex. The MT-ATP6 mutation has been considered as one of the causative factors of mitochondrial diseases. m.9185T>The C mutation was first found in a 7 year old patient who developed sudden ptosis, ocular fundus paralysis and fatigue. Although m.9185T>At 85% heterogeneity, isolated axonal neuropathy occurs, but higher mutation loads result in more severe neural phenotypes including neurogenic muscle weakness, ataxia, retinitis pigmentosa, and early and late Leigh Syndrome (LS). And, m.9185T>C exhibits tissue specificity for a striking disease phenotype. LS is the most common mitochondrial disease, with clinical phenotypes including neurological symptoms with dysfunction of the basal ganglia and/or brainstem. LS usually occurs in infancy and patients exhibit T2 symmetric high signs of delayed growth, encephalopathy, dystonia, the basal ganglia and the brainstem. In addition, systemic problems outside the central nervous system, such as renal failure, cardiomyopathy, diabetes, etc., can occur, making LS a disease with a variety of clinical features.
At present, no special treatment method is available for mitochondrial diseases, only symptomatic treatment can be carried out, and the lethal disability rate of patients is high. Therefore, the prevention of the birth of mitochondrial disease children is the only way to block the transmission of mitochondrial disease. However, Prenatal Diagnosis (PD) and embryo Placement Genetics Diagnosis (PGD) face challenges in clinical applications that block transmission of mitochondrial diseases caused by mtDNA mutations, relative to genetic diseases caused by the nuclear genome. Since mtDNA follows maternal inheritance, the proportion of mutant mtDNA in offspring is correlated with the mother. However, due to genetic bottleneck effects of mtDNA during oocyte development, heterogeneous changes that are completely different from the mother may occur in the oocyte, and the bottleneck effects may cause offspring heterogeneity to be unpredictable to some extent. Moreover, since the heterogeneity of m.9185-related mutations is significantly related to the severity of the disease, accurate quantification of m.9185 mutation heterogeneity in cord blood, amniotic fluid, and single cell samples from embryo biopsies is crucial in prenatal diagnosis and pre-embryo implantation genetics.
Currently, there are a variety of methods that can detect mtDNA heterogeneity, including Next-generation sequencing (NGS), Sanger sequencing, high-performance liquid chromatography (HPLC), SNaPshot, pyrosequencing, fluorescent quantitative PCR, and the like. HPLC can detect mtDNA mutation site genotype but cannot quantify mutation load. Sanger sequencing and SNaPshot are less sensitive to samples with heterogeneity < 5%. The fluorescent quantitative PCR depends on a standard dissolution curve, so that relative quantification can only be realized, and the PCR has a plurality of influence factors and is easily influenced by PCR inhibitors such as various metal ions in a sample. Pyrosequencing and NGS, especially NGS, are widely used for quantification of mtDNA heterogeneity levels, especially for samples with very low heterogeneity, the results are accurate and sensitive, but the cost of the pyrosequencing and NGS is too high to be popularized in large-scale clinical samples.
Disclosure of Invention
The invention aims to provide a primer group, a probe and a kit for quantitatively detecting the heterogeneity of the MT-ATP6m.9185 locus, which are used for solving the problems in the prior art and realizing the high-accuracy, high-sensitivity and high-repeatability detection of the heterogeneity of the MT-ATP6m.9185 locus in a human single-cell sample.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a primer group and a probe for quantitatively detecting MT-ATP6m.9185 site heterogeneity of a human single cell sample, wherein the primer group comprises an upstream primer shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2; the probe comprises a wild type probe shown as SEQ ID NO. 3 and a mutant type probe shown as SEQ ID NO. 4.
The invention also provides a kit for quantitatively detecting the heterogeneity of the MT-ATP6m.9185 locus of the human single-cell sample, which comprises the primer group and the probe.
Further, the kit also comprises Taq enzyme, dNTPs and Mg2+。
The invention also provides application of the primer group and the probe in preparing a reagent or a kit for quantitatively detecting the heterogeneity of the MT-ATP6m.9185 site of the human single cell sample.
The invention discloses the following technical effects:
according to the invention, microdroplet treatment is carried out on the sample before PCR amplification, the accurate quantification can be reduced to 1-10 copies, and compared with the fluorescent quantitative PCR, the absolute quantification of mtDNA copy number can be realized without a standard dissolution curve. And the ddPCR is tolerant to PCR inhibitors, so that the detection kit is suitable for detecting various complex samples. Most importantly, ddPCR has three advantages of high sensitivity (the quantitative rate can be lower than 2% of mutation load), high accuracy (the quantitative result is highly consistent with NGS and has no statistical difference), and high repeatability (the three-time repeated result of each sample is highly consistent), and particularly, the ddPCR can accurately quantify the mutation load of the mtDNA with extremely low proportion. Compared with the commonly used method for quantifying mtDNA heterogeneity such as NGS, pyrosequencing and the like, the cost of ddPCR is obviously reduced, so that the method is very suitable for detecting a plurality of tissue samples and a plurality of embryos in the PD and PGD processes, and the detection cost is greatly reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a flow chart of ddPCR operation;
FIG. 2 is the number of wild type and mutant allele droplets: effective liquid drops are obtained when the fluorescence amplitude value is more than 1000; wherein A is a wild type positive droplet and negative droplet scatter diagram, positive droplets are at the amplitude of 5000, and negative droplets are at the amplitude of 1000; b is a mutant positive droplet and negative droplet scatter diagram, wherein the positive droplet is at the amplitude of 1500, and the negative droplet is at the amplitude of 1000; c is a peak diagram of the wild-type positive droplet and the wild-type negative droplet, the positive droplet is at the amplitude of 5000, and the negative droplet is at the amplitude of 1000; d is a mutant positive droplet and negative droplet scatter diagram, wherein the positive droplet is at the amplitude of 1500, and the negative droplet is at the amplitude of 1000;
FIG. 3 is a graph of wild type and mutant allele copy numbers calculated using QuantaSoft software;
fig. 4 is a comparison of ddPCR with NGS for quantification of mtDNA mutation heterogeneity, where a: comparing the ddPCR and NGS quantitative results of the biopsy single cell sample in a one-to-one correspondence manner; b: statistical analysis of ddPCR and NGS quantitation results for multiple samples;
FIG. 5 is a comparison of results of three duplicate detection of single-cell biopsy samples by ddPCR;
FIG. 6 is a comparison of the cost of detection of single samples by ddPCR and NGS.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated value or intervening value in a stated range, and any other stated or intervening value in a stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
Example 1
(1) Embryo biopsy:
the inverted microscope and micromanipulation system switches were first turned on and the embryo biopsy needle ((ID 30 μm)) and the egg holding needle were loaded on the needle holder of the micromanipulation system. Transferring the embryo/blastocyst to be biopsied at cleavage stage into a medium with 5% CO at 37 deg.C2、25％O2And 90% N2Well-balanced biopsy plates (prepared from cleavage stage medium/blastocyst medium, 5% PVP, and paraffin oil) in the incubator. The biopsy dish was then placed under an inverted microscope, the embryo to be examined was placed in the 3 o' clock position, and after the embryo was fixed using an egg-holding needle, the zona pellucida was perforated (laser pulse 400 μ s, intensity 100%) using a laser rupture apparatus (Hamilton Thorne, usa) at the zona pellucida on the right side of the embryo. The embryo biopsy needle is then passed through the zona pellucida cavity into the embryo, and the blastomere or blastocyst trophoblast cells are gently aspirated using the biopsy needle.The transparent belt hole can be pulled out by slightly sucking the blastomere; blastocyst trophoblast cells require continuous firing (laser pulses 700 mus, 100% intensity) of laser light directed at the intercellular junctions between the trophoblast cells being pulled apart while being pulled using the biopsy and egg holding needles, while continuing to pull the blastocyst and trophoblast cells gently to either side until the trophoblast cells separate from the blastocyst and are pulled out of the zona pellucida hole. The isolated blastomeres or trophoblasts obtained were placed in 4. mu.LPBS to obtain single cell samples.
(2) Single cell whole genome amplification:
3 mu L of lysis solution BufferD2 was added to the 4 mu L of single cell sample, and the mixture was incubated at 65 ℃ for 10 minutes, followed by addition of 3 mu of LStop solution, to obtain the lysate. mu.L of the lysate was added to 40. mu.L of the premix 40. mu.L of the lysate, and the lysate was incubated at 30 ℃ for 8 hours to complete amplification of the whole genome, followed by incubation at 65 ℃ for 3 minutes to inactivate Taq enzyme, thereby obtaining 50. mu.L of the whole genome amplification product.
Wherein:
buffer D2: 1 μ L of 50mM DTT and 11 μ L of 200mM KOH;
stop solution: 1M tris-HCL solution;
PCR premix 1 (40. mu.L) contained: mu.L of 5U/. mu.L Taq enzyme, 27.5. mu.L of 2.5mM dNTPs, 1.5. mu.L of 2mmol/LMg2+And 9 μ LH2O。
(3) Droplet digital PCR (operational flow diagram fig. 1):
and (3) taking the whole genome amplification product obtained in the step (2) as a DNA template, and preparing 20 mu L of PCR premix 2, wherein the PCR premix 2 comprises: 0.5. mu.L each of wild type and mutant type probes, 1. mu.L each of upstream and downstream primers, 2. mu.L of whole genome amplification product, 2. mu.L of 5U/. mu.L Taq enzyme, 5. mu.L of 2.5mM dNTPs, 1.5. mu.L of 2.0mmol/L Mg2+And 6.5. mu. L H2And O. Add 20. mu.L of PCR premix 2 to the microtiter plate. The droplet generation card is placed in a droplet generator which generates 20000 droplets in 2.5 minutes from 20. mu.L of reaction mixture. The droplets were then transferred to a 96-well PCR plate and amplified on a PCR instrument.
An upstream primer F: GCTGTCGCCTTAATCCAA (SEQ ID NO: 1);
a downstream primer R: TAGGCCGGAGGTCATTAG (SEQ ID NO: 2);
the LNA probe includes: wild type: FAM-tagtaagcctctacctgca-BHQ1(SEQ ID NO:3), mutant: VIC-tagtaagcccctacctgca-BHQ1(SEQ ID NO: 4).
After PCR amplification, the 96-well plate was placed in a microdroplet analyzer (BIO-RADQX 200). The droplet analyzer (BIO-RADQX200) starts to absorb droplets of each sample and passes them one by one through a two-color detector. Droplets with fluorescent signal were positive and droplets without fluorescent signal were negative and the software recorded the proportion of positive droplets in each sample (figure 2). Finally, the data was automatically analyzed by QuantaSoft software (fig. 3).
PCR amplification conditions: the first stage, 50 ℃/5min, 95 ℃/5 min; in the second stage, 45 cycles of 95 ℃/15sec, 60 ℃/1min are carried out; the third stage, storage at 98 deg.C/10 min and 4 deg.C.
ddPCR quantified m.9185 site heterogeneity in 8 actual single cell samples, and the results are shown in Table 1.
The ddPCR carries out microdroplet treatment on a sample before the traditional PCR amplification, can accurately quantify as low as 1-10 copies, and can realize the absolute quantification of mtDNA copy number without a standard dissolution curve compared with the fluorescence quantitative PCR. And the ddPCR is tolerant to PCR inhibitors, so that the detection kit is suitable for detecting various complex samples. Most importantly, ddPCR has three advantages of high sensitivity (the mutation load can be quantified by less than 2%, see table 1), high accuracy (the quantification result is highly consistent with NGS, no statistical difference exists, see figure 4), and high repeatability (the three-time repeated result of each sample is highly consistent, see figure 5), and particularly, the ddPCR can also be used for accurately quantifying the mutation load of mtDNA with extremely low proportion. Compared with the method commonly used for quantifying the heterogeneity of mtDNA, such as NGS, pyrosequencing and the like, the cost of ddPCR is obviously reduced (see figure 6), so that the method is very suitable for detecting a plurality of tissue samples and a plurality of embryos in the PD and PGD processes, and the detection cost is greatly reduced.
TABLE 1m.9185T > C heterogeneity quantitation
Figure BDA0003613675240000051
Figure BDA0003613675240000061
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> first subsidiary hospital of medical university of Anhui
<120> primer group, probe and kit for quantitatively detecting heterogeneity of MT-ATP6m.9185 locus
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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Claims (4)

1. A primer group and a probe for quantitatively detecting the heterogeneity of a human single cell sample MT-ATP6m.9185 site are characterized in that the primer group comprises an upstream primer shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2; the probe comprises a wild type probe shown as SEQ ID NO. 3 and a mutant type probe shown as SEQ ID NO. 4.
2. A kit for quantitatively detecting MT-ATP6m.9185 site heterogeneity of a human single cell sample, which is characterized by comprising the primer group and the probe of claim 1.
3. The kit of claim 2, further comprising Taq enzyme, dNTPs and Mg2+。
4. The use of the primer set and the probe as claimed in claim 1 in the preparation of a reagent or a kit for quantitatively detecting MT-ATP6m.9185 site heterogeneity of human single cell samples.
CN202210438091.1A 2022-04-25 2022-04-25 Primer group, probe and kit for quantitatively detecting heterogeneity of MT-ATP6m.9185 locus Pending CN114752667A (en)

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CN108949926A (en) * 2018-08-03 2018-12-07 张丽英 A kind of detection method based on digital pcr platform EGFR gene Exon19 deletion mutation
CN111705132A (en) * 2020-07-03 2020-09-25 南方医科大学南方医院 Primer probe set, kit and method for detecting liver cancer prognosis marker TP53R249S by ddPCR (double-stranded polymerase chain reaction)
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CN112888788A (en) * 2018-08-14 2021-06-01 艾梅尔生物治疗公司 Methods and compositions for treating mitochondrial diseases or disorders and heterogeneity

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US20170151287A1 (en) * 2015-11-30 2017-06-01 Flagship Ventures Management, Inc. Methods and compositions of chondrisomes
CN108949926A (en) * 2018-08-03 2018-12-07 张丽英 A kind of detection method based on digital pcr platform EGFR gene Exon19 deletion mutation
CN112888788A (en) * 2018-08-14 2021-06-01 艾梅尔生物治疗公司 Methods and compositions for treating mitochondrial diseases or disorders and heterogeneity
CN111705132A (en) * 2020-07-03 2020-09-25 南方医科大学南方医院 Primer probe set, kit and method for detecting liver cancer prognosis marker TP53R249S by ddPCR (double-stranded polymerase chain reaction)
CN112430647A (en) * 2020-12-10 2021-03-02 江苏吉诺思美精准医学科技有限公司 Primer, probe, kit and detection method for detecting ASXL1 gene c.1934dupG mutation

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金丹群等: "以急性肺出血为突发表现的Leigh综合征二例临床及基因分析", 中华儿科杂志, vol. 53, no. 4, pages 290 - 295 *

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