CN116837090B - Primer group, kit and method for detecting fetal bone dysplasia - Google Patents

Primer group, kit and method for detecting fetal bone dysplasia Download PDF

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CN116837090B
CN116837090B CN202310830480.3A CN202310830480A CN116837090B CN 116837090 B CN116837090 B CN 116837090B CN 202310830480 A CN202310830480 A CN 202310830480A CN 116837090 B CN116837090 B CN 116837090B
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detection
sequencing
sample
fetal
kit
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CN116837090A (en
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黄铨飞
朱晓琳
庾晓康
刘腾飞
唐睿
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CapitalBio Genomics Co Ltd
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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
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q1/6869—Methods for sequencing
    • 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
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Abstract

The invention discloses a primer group, a kit and a method for detecting fetal bone dysplasia. The product comprises the following components: primers shown as SEQ ID NO. 1-60; sequencing the adaptor; a tag linker; wherein, the primer, the sequencing joint and the tag joint are all connected with a section of public primer. The product can effectively detect the risks of three diseases of fetal ACH, TD and JWS, has high detection accuracy, can efficiently realize large-scale sample detection, and overcomes the defects of complex operation and low flux of the traditional detection method. And the detection can be realized only based on trace peripheral blood, so that the compliance and the detection experience of a subject are improved, and the method has extremely high practicability and clinical use value.

Description

Primer group, kit and method for detecting fetal bone dysplasia
Technical Field
The invention relates to the technical field of biology, in particular to a primer group, a kit and a method for detecting fetal bone dysplasia.
Background
Skeletal dysplasia, also known as dwarfism, is a general term for a group of diseases that result in short stature due to skeletal or cartilage abnormalities. Skeletal dysplasia, which is currently found, is more than 200, including osteogenesis imperfecta, achondroplasia (ACH), lethal achondroplasias (TD), jackson-Weiss syndrome, and the like. Research shows that most skeletal development abnormality is caused by gene mutation, and prenatal and postnatal care can be realized by detecting whether the fetus has the mutation, so that the birth of the child can be effectively avoided.
Achondroplasia (ACH), lethal achondroplasias (Thanatophoric Dysplasia, TD) and Jackson-Weiss syndrome (JWS) are all rare autosomal dominant genetic diseases. ACH is mainly characterized by short stature, thick limbs, finger in the shape of 'three-fork' and the like, and the incidence rate is 25-66/100 ten thousand. TD is mainly characterized by extremely short limbs, large trunk, short ribs, narrow chest and the like, and the incidence rate is about 1/60000.JWS is mainly characterized by foot abnormalities and premature skull fusion, associated with mutations in the FGFR2 gene. FGFR2 gene encodes a receptor tyrosine kinase protein, which is mainly involved in cell proliferation and differentiation, and mutations in it affect normal development of bone and skull.
At present, an effective treatment means for the skeletal dysplasia of the fetus caused by genetic mutation is not available, and the birth of the infant brings heavy burden to society and families of patients. Gene detection is used as a gold standard for skeletal dysplasia caused by genetic mutation, and methods such as PCR (polymerase chain reaction) combined with first-generation sequencing, multiple ligation probe amplification technology (Multiplex Ligation-dependent Probe Amplification, MLPA), gene chips and the like are commonly adopted. Usually, high-risk pregnant women need to be subjected to villus or amniotic fluid puncture, and PCR (polymerase chain reaction) is combined with first-generation sequencing to detect whether the fetus carries related gene mutation. However, the method has the defects of complicated operation, long time consumption, low flux and the like, and the abortion risk exists. Therefore, the development of a safer, effective and noninvasive detection method for the genetic skeletal dysplasia of the fetus has important clinical significance.
Disclosure of Invention
The present invention aims to solve at least one of the above technical problems in the prior art. Therefore, the invention aims to provide a product for detecting genetic skeletal dysplasia of a fetus, and a detection method and application thereof. The invention adopts a multiplex PCR amplification technology to capture 30 special sites in the fetal free DNA of the peripheral blood of the pregnant woman, combines a sequencing technology to realize the detection of the existing risk of the target diseases, has good stability, high sensitivity and extremely high accuracy, and can realize the noninvasive prenatal screening of three diseases of the undergrowth of cartilage, the lethal undergrowth of cartilage and the Jackson-Weiss syndrome.
In a first aspect of the present invention, there is provided a detection kit comprising:
primers shown as SEQ ID NO. 1-60;
a sequencing adapter for sequencing of the library;
a tag linker for labelling libraries constructed from different samples;
wherein, the primer, the sequencing linker and the tag linker are all connected with a section of public sequence.
In the invention, the primer pair shown in SEQ ID NO. 1-60 is used for amplifying high-frequency heterozygous SNP loci and associated genetic mutation loci of achondroplasia, lethal achondroplasia and Jackson-Weiss syndrome, and the corresponding information is shown in table 1 in the specification.
In some embodiments of the invention, the public sequence is as set forth in SEQ ID NO: shown at 61.
In some embodiments of the invention, the public sequences are ligated to the 5' end of the primers shown in SEQ ID NOS.1-60.
In some embodiments of the invention, the nucleotide sequence of the sequencing linker is: 5'-CCTCTCTATGGGCAGTCGGTGAT-3' (SEQ ID NO: 62).
In some embodiments of the invention, the common primer is attached to the 3' end of the sequencing linker.
In some embodiments of the invention, the nucleotide sequence of the tag linker is: 5'-CCATCTCATCCCTGCGTGTCTCCG ACTCAGNNNNNNNNGAT-3' (SEQ ID NO: 63), wherein N represents a degenerate base (A/G/C/T).
In some embodiments of the invention, nnnnnnnnnn in the tag linker is used to identify libraries of different sample constructs.
In some embodiments of the invention, the common primer is attached to the 3' end of the tag linker.
In some embodiments of the invention, the detection kit further comprises: purification reagents and PCR amplification reagents.
In some embodiments of the invention, the purification reagents are purification magnetic beads commonly used in the art, such as: ampure XP magnetic beads.
In some embodiments of the invention, the PCR amplification reagents are multiplex PCR buffers commonly used in the art, such as: multiplex PCR Kit, 2G Fast Multiplex PCR Kit.
In a second aspect of the invention, there is provided a detection system comprising a detection kit according to the first aspect of the invention and a sequencing module.
In some embodiments of the invention, the sequencing module sequences the amplified product obtained by the detection kit according to the first aspect of the invention to obtain an Allele Frequency (AF) value of the mutant Allele.
In some embodiments of the invention, the ratio of fetal DNA in the test sample is verified prior to obtaining the gene mutation AF value.
In some embodiments of the invention, the test results are reliable and valid only when the fetal DNA fraction in the test sample is not less than 4%.
In some embodiments of the invention, the sequencing module comprises a sequencing platform conventionally used in the art, including, but not limited to, proton.
In a third aspect, the invention provides the use of the detection kit according to the first aspect of the invention for preparing a product for detecting genetic skeletal dysplasia in infants.
In some embodiments of the invention, the fetal genetic skeletal dysplasia comprises achondroplasia, lethal achondroplasia and Jackson-Weiss syndrome
In some embodiments of the invention, the method of using the fetal genetic skeletal dysplasia detection product is:
(1) Extracting DNA from a sample, and performing polymerase chain reaction (Polymerase Chain Reaction, PCR) by using the detection kit according to the first aspect of the invention to obtain an amplification product;
(2) Sequencing the amplified product to obtain a gene mutation AF value, comparing the AF value with a threshold value, and judging the risk of fetal genetic skeletal dysplasia.
In some embodiments of the invention, in step (1), the sample is taken from 12 +0 Pregnant women around and above.
In some embodiments of the invention, the sample comprises blood.
In some embodiments of the invention, the sample is peripheral blood plasma.
In some embodiments of the invention, the fetal DNA fraction in the sample is 4% or greater.
In some embodiments of the invention, in step (2), the amplified product is purified prior to sequencing.
In some embodiments of the invention, the purification is performed using a purification reagent.
In some embodiments of the invention, the purification reagents are purification magnetic beads commonly used in the art, such as: ampure XP magnetic beads.
In some embodiments of the present invention, in step (2), if the AF value is 1% or more, then determining that the sample is positive, and there is a risk of fetal genetic skeletal dysplasia (i.e., there is a risk of disease of at least one of ACH, TD, and JWS); if the AF value is less than 1%, the sample is judged to be a negative sample, and the risk of genetic bone development abnormality of the fetus is avoided.
In the present invention, the inventors found that when a mutation frequency threshold value of 1.00% is set, the threshold value can accurately and effectively distinguish positive samples from negative samples and has extremely high reliability when the fetal DNA ratio is ensured to be 4% or more.
The beneficial effects of the invention are as follows:
1. the invention is developed for the first time to obtain a product capable of effectively detecting the risks of three diseases of fetal ACH, TD and JWS, has high detection accuracy, can efficiently realize large-scale sample detection, and overcomes the defects of complicated operation and low flux of the traditional detection method.
2. The detection method can realize detection by only trace amount of free DNA of peripheral blood plasma, and does not need invasive detection sampling operations with risk such as villus or amniotic fluid puncture sampling, so that the compliance and detection experience of a subject are improved, and the detection method has extremely high practicability and clinical use value.
Drawings
Fig. 1 shows the correlation of AF values at different thresholds.
FIG. 2 is a correlation of fetal DNA duty cycle to real duty cycle.
FIG. 3 shows the result of uniformity verification.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The starting materials, reagents or apparatus used in the examples and comparative examples were either commercially available from conventional sources or may be obtained by prior art methods unless specifically indicated. Unless otherwise indicated, assays or testing methods are routine in the art.
Selection of detection sites and design of specific primers therefor
In the examples of the present invention, the inventors obtained 4 target SNPs and 26 high frequency heterozygous site regions that could be used to detect fetal genetic skeletal dysplasia (achondroplasia, lethal achondroplasia and Jackson-Weiss syndrome) through data studies, and related corresponding specific primers based on the screened regions. And the free DNA is taken as a target detection sample, and 30 pairs of primers with high amplification efficiency and good specificity are finally obtained after screening, optimizing and verifying (shown in table 1).
TABLE 1 regions for detection and corresponding primer information
Fetal genetic skeletal dysplasia detection product based on maternal peripheral blood fetal free DNA
In this example, a method based on multiplex PCR capture technology and the specific primer set in the above example is exemplarily provided according to the principle of semiconductor (chip) sequencing by detection 12 +0 A detection kit for judging the risk of hereditary skeletal dysplasia of fetus by peripheral blood fetal free DNA of pregnant women and pregnant women above.
The detection kit comprises the following components:
(1) Primer group:
the 5' -ends of the primers shown in SEQ ID NOS.1 to 60 in the above examples were each ligated with a common sequence (5'-AAATGGGCGGTAGGCTTG-3' (SEQ ID NO: 61)) to obtain a primer set.
(2) Sequencing adaptors:
the nucleotide sequence of the sequencing linker is: 5'-CCTCTCTATGGGCAGTCGGTGAT-3' (SEQ ID NO: 62), wherein the same public sequence as in the primer set is ligated at the 3' -end of the sequencing adapter.
(3) And (3) tag joint:
the nucleotide sequence of the tag linker is: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNGAT-3' (SE Q ID NO: 63), wherein N represents a degenerate base (A/G/C/T). Nnnnnnnnnnnnn was used to identify libraries constructed from different samples. The 3' -end of the tag linker is ligated to the same common sequence as in the primer set.
(4) PCR amplification reaction reagent:
commercially available multiplex PCR buffers can be used, for example: multiplex PCR Kit, 2G Fast Multiplex PCR Kit.
(5) Purifying reagent:
commercially available purified magnetic beads can be used, for example: ampure XP magnetic beads.
The specific detection method based on the kit comprises the following steps:
(1) Extraction of maternal peripheral blood fetal free DNA:
fetal free DNA in a peripheral blood sample of a pregnant woman was extracted using a commercially available cfDNA (circulating free DNA) kit (e.g., QIAamp Circulating Nucleic Acid Kit, magnetic Serum/Plasma DNA Maxi Kit, etc.).
(2) Multiplex PCR amplification:
the maternal peripheral blood fetal free DNA extracted in the above steps was used as a template for detection using the detection kit of this example, wherein the multiplex PCR amplification system is shown in table 2.
TABLE 2 multiplex PCR amplification System
Component (A) Content of
cfDNA template ≥10ng
Primer set 0.8pM
Sequencing adapter 10pM
Label joint 10pM
PCR amplification reaction reagent 25μL
Total volume of 50μL
Wherein, different label linkers (for adjusting the base selection of NNNNNNNNNN) are used for different samples, and the content ratio of each primer in the primer group is 0.125-8.
The reaction procedure for multiplex PCR amplification was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 60℃for 90s, extension at 72℃for 30s,35 cycles; extension was carried out at 72℃for 1min and maintained at 4 ℃. Obtaining an amplification product.
According to the number of PCR amplified samples, taking equal volumes of PCR amplified products of different samples to obtain 100 mu L of mixed library with different labels, and purifying the PCR products by using a purifying reagent to obtain a purified mixed library.
(3) Sequencing:
using Qubit TM dsDNA Hs Assay Kit the purified mixed library was quantified and sequenced on a machine according to the quantitative concentration. In this example, sequencing was performed using Proton platform, and sequencing template preparation and enrichment can be seen in Ion PI TM Hi Q TM OT2 200Kit (A26434) instructions, microbeads (hybrid library) carrying template molecules were loaded onto Ion Proton TM Sequencing was performed on the semiconductor chip of the sequencer, see Ion PITM Hi QTMSequence, 200Kit instructions for detailed procedures.
(4) Data analysis:
in this example, the threshold mutation frequency was set to 1.00%, and the inventors found that below this threshold, when the fetal DNA ratio was 4% and above, the threshold was effective to distinguish between positive and negative samples (as shown in fig. 1).
When a sample positive for pathogenic mutation is detected, the present embodiment uses mutation frequency information of high-frequency mutation SNP sites of the sample, estimates the fetal DNA duty ratio using the maximum likelihood method, observes whether the quantitative relationship between the mutation frequency of pathogenic sites and the fetal DNA duty ratio meets theoretical expectations, and measures the credibility of the positive result in combination with whether the fetal DNA duty ratio is lower than 4% (as shown in fig. 2, there is a moderate correlation between the fetal DNA duty ratio and the real duty ratio).
Verification of detection effect of fetal genetic skeletal dysplasia detection product
In order to be able to fully demonstrate that the fetal genetic skeletal dysplasia detection product in the above-described examples can be used for fetal genetic skeletal dysplasia risk detection, the inventors performed the following verification test.
For blind testing, 12 positive samples (verified to have relevant SNP mutations, 6 2% af SNP mutations and 6 5% af SNP mutations, respectively) and 75 negative samples (verified to have no mutation) were selected, respectively. The samples were tested separately according to the method described in the examples above, with sequencing depths greater than 1000×, and after sequencing was completed, the AF values were counted, and the positive threshold was set at 1.00%, with AF values less than 1.00% being negative.
The results are shown in Table 3.
TABLE 3 actual detection Effect of fetal genetic skeletal dysplasia detection products
From the results, when the mutation frequency of the detected sample is more than or equal to 2%, 12 positive standard substances are detected; the results of the mutation-free samples are all negative, which shows that the method has excellent detection accuracy.
Further, the uniformity of amplification was verified, and the detection was performed according to the above method, and after multiplex PCR library construction using the primer set, uniformity evaluation was performed by counting the amplification depth obtained for a sample in one run.
The results are shown in FIG. 3.
The uniformity of amplicon depth of 26 high-frequency heterozygous sites and 4 SNP mutant sites is good, which indicates that the primer set in the embodiment has high specificity and small mutual interference during amplification.
The method of the above example was repeated by re-selecting 17 non-mutated samples and 16 mutated samples (3 replicates per sample) and the test method was the same as the above example. The test results are shown in Table 4.
Table 4 results of repeatability evaluation
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The results show that the detection products based on the above examples have high detection stability and good repeatability.
The inventor also evaluates the success rate of database establishment and the data utilization rate. The procedure was followed in the above examples with 68 plasma samples, with a stock build cfDNA input of > 10ng. The results are shown in Table 5.
Table 5 success rate of database creation and evaluation result of data utilization
It was found that the average depth of amplicons of all test samples was > 1000×, and the effective utilization of the average data of the found samples was calculated to be 44.88%, and the success rate of library construction was 100%.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (9)

1. The detection kit is characterized by comprising the following components:
primers shown as SEQ ID NO. 1-60;
a sequencing adapter for sequencing of the library;
a tag linker for labelling libraries constructed from different samples;
wherein, the primer, the sequencing linker and the tag linker are all connected with a section of public sequence.
2. The test kit of claim 1, further comprising: purification reagents and PCR amplification reagents.
3. A detection system, characterized in that it comprises the detection kit of claim 1 or 2 and a sequencing module;
the sequencing module is used for sequencing the amplification product obtained by the detection kit according to claim 1 or 2 to obtain the gene mutation AF value.
4. Use of the detection kit of claim 1 or 2 in the preparation of a fetal genetic skeletal dysplasia detection product; the fetal genetic skeletal dysplasia is at least one of achondroplasia, lethal achondroplasia and Jackson-Weiss syndrome.
5. The use according to claim 4, wherein the method of using the fetal genetic skeletal dysplasia detection product is:
(1) Extracting DNA in a sample, and performing polymerase chain reaction by using the detection kit of claim 1 or 2 to obtain an amplification product;
(2) Sequencing the amplified product to obtain a gene mutation AF value, comparing the AF value with a threshold value, and judging the risk of fetal genetic skeletal dysplasia.
6. The use according to claim 5, wherein in step (1) the sample is taken from pregnant women with a pregnancy of 12 weeks, day 0 and more.
7. The use of claim 6, wherein the fetal DNA fraction in the sample is 4% or more.
8. The use according to claim 5, wherein in step (2) the amplified product is purified before it is sequenced.
9. The use according to claim 5, wherein in step (2), if the AF value is 1% or more, a positive sample is determined to be at risk of fetal genetic skeletal dysplasia; if the AF value is less than 1%, the sample is judged to be a negative sample, and the risk of genetic bone development abnormality of the fetus is avoided.
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