CN106319079B - Method for detecting 22q11.2 copy number loss - Google Patents
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Abstract
The invention relates to a method for detecting 22q11.2 copy number deletion by using limited dNTP competitive PCR combined HRM technology, which comprises the step of detecting CLTCL1, SNAP29, KLHL22, PI4KA and CFTR genes. The invention also provides a primer combination and a kit related to the method. Its advantages are: the operation is simple, convenient and quick, only the PCR reaction and the subsequent HRM analysis process are included, and the detection period is shortened; the closed-tube operation is realized, and because the fluorescent dye is added in the PCR reaction, the HRM curve analysis can be carried out without other treatment after the amplification of the detection fragment is finished, thereby effectively avoiding pollution; only conventional PCR reagents and a small amount of fluorescent dye are needed in the reaction, and special detection and analysis instruments are not needed; the test on the control samples of 99 patients and normal people shows that the sensitivity and specificity reach 100 percent, and the stability and accuracy of the system are verified.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method for detecting 22q11.2 copy number deletion by combining limited dNTP competitive PCR with HRM technology.
Background
The 22q11.2 microdeletion syndrome is the most common genetic syndrome in clinic, and the syndrome is caused by the deletion of the 22 # chromosome long arm centromere end microchip 22q11.21-q 11.23. The core region of the deletion is located between the low copy repeat sequences LCR22-A to LCR22-D in the 22q11.2 region and is 3Mb in size, and about 90% of patients with the syndrome show an overall deletion of one copy number of the fragment, and some patients show a deletion combination between different low copy repeat sequences. According to clinical manifestations and characteristics, patients with the syndrome can be preliminarily diagnosed, and the detection of 22q11.2 deletion fragments is an important basis for the accurate diagnosis of the disease.
at present, Copy Number Variation (CNV) detection methods mainly comprise a chip-based whole genome scanning technology and a PCR-based candidate CNV detection technology, and indexes such as reliability, operation difficulty and economy of different methods are different. The chip-based CNV detection technology mainly comprises comparative genome hybridization, single nucleotide polymorphism chips and new generation sequencing technology, and the technologies are suitable for high-throughput detection of CNV, are favorable for discovering new copy number variation related to diseases, but have high cost to limit the wide application of the CNV. In the detection of diseases with clear deletion fragments such as 22q11.2 microdeletion syndrome, the candidate CNV detection technology is more suitable.
Limited dNTP competitive PCR is an improvement over conventional PCR in that the target gene and the reference gene are amplified simultaneously in the same reaction tube, wherein the amount of dNTP is limited, and due to competition, when the amount of one template is gradually increased, the amplification products of the other template are relatively gradually reduced, and the ratio of the amplification products is consistent with the relative quantification of the templates in their initial state.
High Resolution Melting curve technology (HRM) is a new technology for gene mutation detection first proposed in 2003 by Wittwer et al, university of utah, usa. The technology directly runs high-resolution melting after PCR is finished, monitors the melting process of nucleic acid through a saturated dye to obtain a characteristic melting curve, and judges the difference of the properties of the nucleic acid according to the difference of the melting curves.
chinese patent document CN101555528A discloses a method for determining microdeletion and microduplication of chromosome 22q11.2 region: extracting genome DNA; performing multiplex fluorescence quantitative PCR multiplex amplification, wherein a multiplex amplification system comprises 5 STR markers: D22S873, 22D _5_1, 22D _4_5, 2D _4_4, 22D _4_3 and an internal reference G6 PDH; taking PCR amplification products for denaturation to convert double-chain products into single chains which can be analyzed by capillary electrophoresis, and analyzing the length and the quantity of the products of the denaturation products by capillary electrophoresis; and judging whether the sample is normal, micro-missing or micro-repeated by adopting a method of quantitative ratio analysis and/or a method of analyzing the position and the number of peaks. Chinese patent document CN102533974A discloses a gene chip for detecting 22q11 microdeletion syndrome, which comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier. However, the method for detecting 22q11.2 copy number deletion by using limited dNTP competitive PCR combined with HRM technology, which has high sensitivity, specificity, stability and accuracy, is not reported at present.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a kit for detecting 22q11.2 copy number loss.
it is a further object of the present invention to provide a method for detecting a 22q11.2 copy number deletion.
It is another object of the present invention to provide a primer combination for detecting the deletion of 22q11.2 copy number.
the fourth purpose of the invention is to provide the application of the primer combination.
in order to achieve the purpose, the invention adopts the technical scheme that: a kit for detecting 22q11.2 copy number deletion, which comprises a reagent for detecting the copy numbers of CLTCL1, SNAP29, KLHL22 and PI4KA genes by using a CFTR gene as a control gene.
further, the kit comprises an instruction book, and the instruction book is recorded with the following contents: the detection reaction comprises two double PCR reactions and one triple PCR reaction, the CLTCL1 and CFTR genomes are detected to be combined into a double PCR reaction system, the SNAP29 and CFTR genomes are detected to be combined into a double PCR reaction system, and the KLHL22, PI4KA and CFTR genomes are detected to be combined into a triple PCR reaction system.
Further, the primers in the kit are respectively as follows: the sequence of the CLTCL1 upstream primer is shown as SEQ ID NO. 1, and the sequence of the CLTCL1 downstream primer is shown as SEQ ID NO. 2; the sequence of the SNAP29 upstream primer is shown as SEQ ID NO. 3, and the sequence of the SNAP29 downstream primer is shown as SEQ ID NO. 4; the sequence of the upstream primer of KLHL22 is shown as SEQ ID NO. 5, and the sequence of the downstream primer of KLHL22 is shown as SEQ ID NO. 6; the sequence of the PI4KA upstream primer is shown as SEQ ID NO. 7, and the sequence of the PI4KA downstream primer is shown as SEQ ID NO. 8; the sequence of the CFTR upstream primer is shown as SEQ ID NO. 9, and the sequence of the CFTR downstream primer is shown as SEQ ID NO. 10.
Further, the kit comprises Mg2+Taq enzyme, dNTP, fluorescent dye and primer.
Further, the kit contains limited amount of dNTP.
Further, the operation instruction records the following contents: the PCR reaction system is：2mM MgCl20.4U KlenaTaq enzyme, 6.25. mu.M dNTP, 1Xplus dye, 50ng of genome DNA, and the primer concentrations are respectively: CFTR 0.125. mu.M; CLTCL 10.5. mu.M; SNAP 291. mu.M; KLHL 221. mu.M; PI4KA 0.5. mu.M.
in order to achieve the second object, the invention adopts the technical scheme that: a method of detecting a 22q11.2 copy number deletion, said method comprising the steps of: limited dNTP competitive PCR reaction is carried out first, and then HRM analysis is carried out.
Further, PCR reaction and HRM analysis are both carried out on a real-time fluorescence quantitative PCR analyzer, and the PCR reaction conditions are pre-denaturation at 95 ℃ for 1 minute, denaturation at 95 ℃ for 10s for 35 cycles and annealing at 64 ℃ for 30 s; after the PCR amplification is finished, the temperature of the reaction system is continuously programmed to 65 ℃ to 95 ℃ in a fluorescent quantitative PCR analyzer, the fluorescent signal is recorded once when the temperature rises by 0.2 ℃, and the fluorescent signal forms a high-resolution melting curve along with the change of the temperature.
In order to achieve the third object, the invention adopts the technical scheme that: a primer combination for detecting 22q11.2 copy number deletion, the primer combination comprises primers for detecting CLTCL1, SNAP29, KLHL22, PI4KA and CFTR genes, and the primer sequences are respectively as follows: the sequence of the CLTCL1 upstream primer is shown as SEQ ID NO. 1, and the sequence of the CLTCL1 downstream primer is shown as SEQ ID NO. 2; the sequence of the SNAP29 upstream primer is shown as SEQ ID NO. 3, and the sequence of the SNAP29 downstream primer is shown as SEQ ID NO. 4; the sequence of the upstream primer of KLHL22 is shown as SEQ ID NO. 5, and the sequence of the downstream primer of KLHL22 is shown as SEQ ID NO. 6; the sequence of the PI4KA upstream primer is shown as SEQ ID NO. 7, and the sequence of the PI4KA downstream primer is shown as SEQ ID NO. 8; the sequence of the CFTR upstream primer is shown as SEQ ID NO. 9, and the sequence of the CFTR downstream primer is shown as SEQ ID NO. 10.
In order to achieve the fourth object, the invention adopts the technical scheme that:
Use of a primer combination as described above in the preparation of a reagent or kit for detecting a deletion in the copy number of 22q 11.2.
The invention has the advantages that:
1. The detection method is simple, convenient and quick to operate, the whole detection process only comprises a PCR reaction and a subsequent HRM analysis process, and the detection period is shortened.
2. the detection method realizes the closed-tube operation, and because the fluorescent dye is added in the PCR reaction, the HRM curve analysis can be carried out without other treatment after the amplification of the detection fragment is finished, thereby effectively avoiding pollution.
3. the detection method of the invention has low cost, only needs conventional PCR reaction reagent and a small amount of fluorescent dye in the reaction, does not need special detection and analysis instruments, and greatly reduces the cost investment.
4. the invention selects proper genes, designs proper primer sequences, has reasonable PCR reaction system and program setting, analyzes the known 22q11.2 copy number loss patients and normal control samples by adopting the detection method of the invention, compares the result with the MLPA detection result, further proves the high accuracy and feasibility of the technology, and has great advantages in 22q11.2 microdeletion detection and large-scale population screening.
Drawings
FIG. 1: 22q11.2 region low copy repeat sequence distribution and target gene localization.
FIG. 2 is a drawing: the results of the CLTCL1 copy number detection are shown in the figure, and the curves are CFTR reference sequence melting peak and CLTCL1 detection sequence melting peak respectively. In this case, curve 1 is a sample with a missing copy number (copy number 1), and curve 2 is a normal control sample (copy number 2). -dF/dT: the first negative derivative of fluorescence with respect to temperature.
FIG. 3: the SNAP29 copy number detection result shows that the curves in the graph are a CFTR reference sequence melting peak and a SNAP29 detection sequence melting peak respectively. In this case, curve 1 is a sample with a missing copy number (copy number 1), and curve 2 is a normal control sample (copy number 2). -dF/dT: the first negative derivative of fluorescence with respect to temperature.
FIG. 4 is a drawing: the curves in the graph are respectively a CFTR reference sequence melting peak and PI4KA and KLHL22 detection sequence melting peaks according to the detection results of PI4KA and KLHL22 copy numbers. The curve 1 is a copy number loss sample (copy number 1), the curve 2 is a normal control sample (copy number 2), and the curve 3 is a PI4KA gene copy number normal or KLHL22 gene copy number loss sample. -dF/dT: the first negative derivative of fluorescence with respect to temperature.
Detailed Description
The following detailed description of the present invention will be made with reference to the accompanying drawings.
The invention utilizes limited dNTP competitive PCR to combine HRM technology, establishes a 22q11.2 copy number deletion rapid detection method, and carries out copy number variation detection on specific genes on a core region. In order to determine the length range of the deleted fragment while detecting deletion, four genes located between different low-copy repeat sequences were selected during primer design, with different combinations of copies corresponding to different deletion ranges.
example 1 detection method
1. Designing a primer:
Aiming at the disease characteristics of 22q11.2 copy number deletion, genes (LCR22A-B: CLTCL 1; LCR22B-C: KLHL 22; LCR22C-D: PI4KA/SNAP29) positioned between different Low Copy Repeats (LCR) in the 22q11.2 region of a chromosome are selected as detection objects (figure 1), four pairs of PCR amplification primers are designed, and are respectively used for amplifying fragments in the genes, CFTR genes positioned on different chromosomes are simultaneously selected, and the primers are designed to be used as reference sequences. In the primer design, the length of the PCR product is controlled to 50 to 120bps while predicting the melting curve and Tms value of the product, and the Tm difference between the target sequence and the reference sequence is set to between 2 ℃ and 10 ℃. The specificity of the target and reference primers was confirmed by searching the human genome database while the amplified fragments avoided the SNP sites as much as possible. According to the above primer design principle, PCR primers meeting the conditions are selected, and the PCR primer sequences and the product length information are shown in Table 1:
TABLE 1 PCR primer sequences and product Length information
2. PCR amplification System:
the PCR was performed in a total volume of 10. mu.l, with upstream and downstream primers 0.125. mu.M-1. mu.M, and 2mM MgCl20.4U KlenaTaq enzyme, 6.25. mu.M dNTP, 1Xplus dye and 50ng of genomic DNA. The total detection system comprises two double PCR reactions and one triple PCR reaction, and the concentrations of the primers are respectively as follows: CFTR 0.125. mu.M; CLTCL 10.5. mu.M; SNAP 291. mu.M; KLHL 221. mu.M; PI4KA 0.5. mu.M.
3. PCR amplification and HRM conditions:
PCR and HRM reactions were performed on a Rotor-Gene Q real-time fluorescent quantitative PCR analyzer under conditions of pre-denaturation at 95 ℃ for 1 minute, 35 cycles of denaturation at 95 ℃ for 10s, and annealing at 64 ℃ for 30 s. After the amplification is finished, the temperature of the reaction system is continuously programmed to 65 ℃ to 95 ℃ in a fluorescent quantitative PCR analyzer, the fluorescent signal is recorded once when the temperature rises by 0.2 ℃, and the high-resolution melting curve is formed by the change of the fluorescent signal along with the temperature.
Example 2 verification of the detection method
Material
Rotor-Gene Q real-time fluorescent quantitative PCR analyzer (Qiagen), KlenaQ enzyme (Ab Peptides),Plus dye (BioFire Defense), primers (Huada Gene Biotech Co., Ltd.), human genomic DNA samples (extracted from 22q11.2 microdeletion patients and normal controls).
Method of producing a composite material
To validate the assay of example 1, 99 cases of 22q11.2 region copy number-deficient patients and normal control samples were tested using the established system, each sample was subjected to three experimental replicates, and the results were validated using Multiplex Ligation-dependent probe amplification (MLPA).
results
The research utilizes limited dNTP competitive PCR to combine with HRM technology to establish a 22q11.2 copy number deletion rapid detection method, and the system utilizes three competitive PCR reactions to detect the copy number conditions of four genes positioned between 22q11.2 different low copy repeat sequences, so as to judge the deletion condition of the chromosome region and determine the deletion range. The CLTCL1 gene and SNAP29 gene are located in the LCR22A-LCR22B and LCR22C-LCR22D regions, and are combined with the CFTR reference sequence to form a double PCR reaction, and the KLHL22 and PI4KA are located in the LCR22B-LCR22C and LCR22C-LCR22D regions, and are combined with the CFTR reference sequence to form a triple PCR reaction system for copy number detection. In the multiplex PCR reaction system, due to different Tm values of different amplicons, fluorescence rapid decrease regions are distributed in different temperature intervals and are mutually distinguished. Meanwhile, the research effectively and reliably controls PCR amplification by reducing the concentration of dNTP, and relative quantitative information of different amplification products can be reflected by the strength of a fluorescence signal, so that after double or triple PCR, relative quantification of copy number can be carried out by marking reference PCR products positioned on different chromosomes. Referring to fig. 2, 3 and 4, which are graphs showing the results of the above three detection reactions, each detection process includes a normal control sample with 2 copies of the target gene and a deletion patient with 1 copy of the target gene, and we can clearly identify the copy number of CLTCL1, KLHL22, PI4KA and SNAP29 by labeling CFTR gene. The results of the method for detecting 99 cases of patients with the loss of the copy number of the 22q11.2 region and normal human control samples (38 cases of LCR22A-LCR22D, 3 cases of LCR22A-LCR22B, 2 cases of LCR22A-LCR22C and 56 cases of normal control samples) are analyzed, so that the results of three repeated experiments of the system on each sample are consistent, the results are the same as those of MLPA, the sensitivity and the specificity reach 100%, and the stability and the accuracy of the system are proved.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
SEQUENCE LISTING
<110> Shanghai university of traffic medical college affiliated Shanghai Children's medical center
<120> a method for detecting 22q11.2 copy number loss
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Claims (6)
1. A kit for detecting 22q11.2 copy number deletion, which is characterized by comprising a reagent for specifically detecting the copy numbers of CLTCL1, SNAP29, KLHL22 and PI4KA genes by taking a CFTR gene as a reference gene and an operation instruction, wherein the operation instruction is recorded with the following contents: the detection reaction comprises two double PCR reactions and a triple PCR reaction, the detection of CLTCL1 and CFTR genomes is a double PCR reaction system, the detection of SNAP29 and CFTR genomes is a double PCR reaction system, the detection of KLHL22, PI4KA and CFTR genomes is a triple PCR reaction system, and the primers in the kit are respectively:
The sequence of the CLTCL1 upstream primer is shown in SEQ ID NO. 1,
The sequence of the downstream primer of CLTCL1 is shown in SEQ ID NO. 2;
The sequence of the SNAP29 upstream primer is shown in SEQ ID NO. 3,
The sequence of the SNAP29 downstream primer is shown as SEQ ID NO. 4;
The sequence of the upstream primer of KLHL22 is shown as SEQ ID NO. 5,
The sequence of the downstream primer of KLHL22 is shown as SEQ ID NO. 6;
The sequence of the PI4KA upstream primer is shown as SEQ ID NO. 7,
The sequence of the PI4KA downstream primer is shown as SEQ ID NO. 8;
The sequence of the CFTR upstream primer is shown as SEQ ID NO. 9,
the sequence of the CFTR downstream primer is shown as SEQ ID NO. 10.
2. the kit of claim 1, wherein the kit comprises Mg2+Taq enzyme, dNTP, fluorescent dye and primer.
3. the kit of claim 2, wherein the kit contains a limited amount of dntps.
4. The kit of claim 3, wherein the kit comprises instructions for performing the following: the PCR reaction system is as follows: 2mM MgCl20.4U KlenaTaq enzyme, 6.25. mu.M dNTP, 1 Xplus dye, 50ng of genomic DNA, the primer concentrations are respectively: CFTR 0.125. mu.M; CLTCL 10.5. mu.M; SNAP 291. mu.M; KLHL 221. mu.M; PI4KA 0.5. mu.M.
5. A primer combination for detecting 22q11.2 copy number deletion is characterized in that the primer combination is used for detecting CLTCL1, SNAP29, KLHL22, PI4KA and CFTR genes, and the primer sequences are respectively as follows:
The sequence of the CLTCL1 upstream primer is shown in SEQ ID NO. 1,
The sequence of the downstream primer of CLTCL1 is shown in SEQ ID NO. 2;
The sequence of the SNAP29 upstream primer is shown in SEQ ID NO. 3,
The sequence of the SNAP29 downstream primer is shown as SEQ ID NO. 4;
the sequence of the upstream primer of KLHL22 is shown as SEQ ID NO. 5,
The sequence of the downstream primer of KLHL22 is shown as SEQ ID NO. 6;
the sequence of the PI4KA upstream primer is shown as SEQ ID NO. 7,
the sequence of the PI4KA downstream primer is shown as SEQ ID NO. 8;
The sequence of the CFTR upstream primer is shown as SEQ ID NO. 9,
The sequence of the CFTR downstream primer is shown as SEQ ID NO. 10.
6. Use of a primer combination according to claim 5 for the preparation of a reagent or kit for detecting a 22q11.2 copy number deletion.
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| CN114592056A (en) * | 2022-04-15 | 2022-06-07 | 常州市妇幼保健院 | 22q11 micro-deletion and/or micro-repetition detection primer group, primer probe composition, kit and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101555528A (en) * | 2008-09-28 | 2009-10-14 | 南京市妇幼保健院 | Method for testing chromosome 22q11.2 microdeletion and microduplication |
| CN102533974A (en) * | 2011-12-09 | 2012-07-04 | 上海交通大学 | Gene chip for detecting 22q11 microdeletion syndrome and application method and kit thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101555528A (en) * | 2008-09-28 | 2009-10-14 | 南京市妇幼保健院 | Method for testing chromosome 22q11.2 microdeletion and microduplication |
| CN102533974A (en) * | 2011-12-09 | 2012-07-04 | 上海交通大学 | Gene chip for detecting 22q11 microdeletion syndrome and application method and kit thereof |
Non-Patent Citations (1)
| Title |
|---|
| Comparative mapping of the 22q11.2 deletion region and the potential of simple model organisms;Alina Guna等;《Journal of Neurodevelopmental Disorders》;20150701;第7卷;第18号,第1-16页 * |
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