A primer composition for detecting a PNH gene and application thereof

Abstract

The application relates to the field of biomedical detection technology, and provides a primer composition for detecting a PNH gene and application thereof, which comprises a primer composition for detecting a PNH gene, the PNH gene comprising one or both of PIGA or PIGT, the primer set is designed according to the characteristics of the mutant gene, has the advantages of wide coverage and high detection efficiency, and can detect 57 gene mutation sites of two mutant genes at one time; in combination with a high-throughput sequencing technology, auxiliary detection of PNH is realized in addition to bone marrow morphology and blood routine examination, especially has special advantages for making a prediction on a subsequent treatment direction and effect of the disease, and can make up for the deficiency of clinical PNH in the aspects of molecular diagnosis, disease evolution, treatment, prognosis and medication guidance; has the advantages of simple operation, high precision, high accuracy, high sensitivity and the like, and solves the technical defects of low monitoring accuracy and limited detection method.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a primer composition for detecting the PNH gene and its application. Background Technology

[0002] Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disease caused by acquired somatic PIGA or PIGT gene mutations in hematopoietic stem cells. Clinically, it mainly manifests as chronic intravascular hemolysis, hematopoietic failure, and recurrent thrombosis; some patients may progress to acute myeloid leukemia. Flow cytometry detection of PNH clonal content is the gold standard for diagnosing PNH. Approximately 15% of PNH cases are dominant hereditary diseases caused by the PIGT gene. Traditional methods are difficult to use to identify these types of hereditary PNH. Gene mutation analysis is an important standard for the diagnosis of hereditary PNH, and it can currently detect approximately 95% of PNH patients.

[0003] Currently, methods for detecting PNH mainly involve complete blood counts, bone marrow cell morphology, flow cytometry, and molecular biology. Among these, molecular biology methods primarily include nested PCR and first-generation sequencing. PCR cannot directly obtain specific mutation sequences, while first-generation sequencing is limited by sequencing throughput and cannot simultaneously detect all exon regions of multiple genes. Second-generation sequencing, as a high-throughput detection method, can accurately measure the exon sequences of multiple genes at once, enabling precision medicine. Currently, research on the application of second-generation sequencing for gene detection and identification of PNH is scarce, but the detection of PNH at the gene diagnostic level is particularly important.

[0004] Therefore, there is an urgent need for a more accurate and precise PNH detection kit and method to compensate for the shortcomings of clinical PNH in guiding molecular diagnosis, disease progression, treatment, prognosis, and medication. Summary of the Invention

[0005] To address the aforementioned shortcomings, this invention provides a primer composition for detecting PNH genes and its application. Using the primer composition described in this invention, the detection of genes related to paroxysmal nocturnal hemoglobinuria (PNH) offers advantages such as ease of operation, high precision, accuracy, and sensitivity, thus overcoming the limitations of clinical PNH in guiding molecular diagnosis, disease progression, treatment, prognosis, and medication.

[0006] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0007] On the one hand, a primer composition for detecting the PNH gene is provided, wherein the PNH gene includes one or both of PIGA or PIGT.

[0008] Specifically, the mutation sites of the PNH gene are shown in Table 1 below:

[0009] Table 1 PNH gene mutation sites

[0010] PIGA p.H65P PIGA p.C183Y PIGA p.L60P PIGA p.R233G PIGA p.P207R PIGA p.Q327X PIGA p.Y98X PIGA p.L280Cfs*11 PIGA p.G64Afs*2 PIGA c.1188+1G>C PIGA p.P447Nfs*12 PIGA p.S164L PIGA p.G47E PIGA p.L243P PIGA p.L347Yfs*6 PIGA p.T123Nfs*7 PIGA p.R415Lfs*8 PIGA p.V403Wfs*21 PIGA p.C36Y PIGA p.G263R PIGA p.H154Ifs*18 PIGA p.C183F PIGA c.715+2T>C PIGA p.Q252X PIGA p.R81P PIGA c.715+1G>A PIGA p.G86Vfs*14 PIGA p.S155F PIGA p.S351X PIGA p.C348Wfs*5 PIGA p.S368X PIGA p.V67Lfs*2 PIGA p.V352X PIGA p.K382X PIGA p.L113R PIGT p.T52A PIGA p.E120A PIGA p.I116V PIGT p.Q417E PIGA p.F384Lfs*11 PIGA p.E120K PIGT p.P194L PIGA c.848+1G>A PIGA c.981+1G>A PIGT p.P382L PIGA p.S127X PIGA p.L359W PIGT p.Y281C PIGA p.L437_I450dup PIGA p.S309F PIGT p.R573X PIGA p.R77X PIGA p.N379fs PIGT p.P92L PIGA p.E338X PIGA c.1189-2A>G PIGT p.P518L .

[0011] Specifically, the primer composition consists of the primers shown in Table 2 below:

[0012] Table 2 Primer Compositions

[0013]

[0014]

[0015]

[0016] On the other hand, the present invention provides the application of a primer composition for detecting the PNH gene in the preparation of products for detecting the PNH gene.

[0017] In another aspect, the present invention provides a product for detecting the PNH gene, the product comprising the primer composition according to any one of claims 1-3.

[0018] Specifically, the product also includes DNA polymerase, buffer, ddH2O, and dNTPs.

[0019] Specifically, the product is a standalone reagent or a kit.

[0020] In another aspect, the present invention provides the application of the above primer composition or the above product in the detection of the PNH gene.

[0021] In another aspect, the present invention provides a method for detecting the PNH gene, which is a non-disease diagnosis and treatment method, and the method includes detecting the PNH gene in the sample to be tested using the above primer composition or the above product.

[0022] Specifically, the method includes the following steps:

[0023] S1. Extract whole-genome DNA from the sample;

[0024] S2. Amplification: Amplify the whole genome DNA in step S1 using the primer composition according to any one of claims 1-3;

[0025] S3. Constructing a DNA library;

[0026] S4, library sequencing and data processing.

[0027] The advantages of this invention are:

[0028] (1) This invention provides a primer composition for detecting PNH genes. Utilizing the gene cluster of PNH-related gene mutations provided by this invention, primer combinations are designed targeting the characteristics of the mutated genes, which have the advantages of wide coverage and high detection efficiency. The primer composition can detect 57 gene mutation sites of the two genes at once, with wide coverage and high detection efficiency. Combined with high-throughput sequencing technology, it enables auxiliary detection of PNH in addition to bone marrow morphology and routine blood tests. In particular, it has special advantages in guiding the subsequent treatment direction and effect of the disease, and can make up for the shortcomings of clinical PNH in molecular diagnosis, disease evolution, treatment, prognosis, and medication guidance.

[0029] (2) The present invention provides a kit and method for detecting PNH gene, which has the advantages of simple operation, high precision, high accuracy and high sensitivity, and solves the technical defects of low monitoring accuracy and limited detection methods. Attached Figure Description

[0030] Figure 1 This is a schematic diagram showing the detection results of the c.1052C>A nucleotide mutation in the PIGA gene. Detailed Implementation

[0031] The present invention will be described in further detail below. The embodiments given are merely illustrative and not intended to limit the scope of the invention. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available. Unless otherwise specified, in the following examples, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA / RNA.

[0033] Example 1: PNH gene, mutation site, and primer composition

[0034] (1) The dbSNP database (http: / / www.ncbi.nlm.nih.gov / snp / ), the 1000 Genomes database (http: / / www.1000genomes.org / ), the Exome Integrated Database ExAC (http: / / exac.broadinstitute.org / ), the Human Genetic Mutation Database HGMD (http: / / www.hgm d.cf.ac.uk / ac / index.php), and the functional prediction tool PolyPhen (http: / / genetics.bwh.harvard.e The following principles were used to screen pathogenic mutation sites: Principle 1: Based on the genomic location and type of the mutation, mutations that do not affect the protein product sequence were filtered out; Principle 2: Using 1000 Genomes data and ExAC data, the proportion of each mutation in the population was annotated. If the proportion was less than or equal to 1%, it was not considered a polymorphic site; Principle 3: The human gene mutation database was searched to see if a mutation was recorded in HGMD and if it appeared in PNH or similar areas. Regarding the disease; Principle 4: Use protein function prediction software PolyPhen-2 and SIFT to predict whether the mutation affects protein function; Principle 5: If, after following Principles 2, 3, and 4, a mutation meets at least two of the following three criteria: "non-polymorphism," "documented in PNH," and "affects protein function," it will be judged as potentially related to the disease; Principle 6: If a mutation does not meet Principle 5 but is documented in HGMD, it will be interpreted as a mutation of unknown significance; If a mutation is clearly documented in the literature as highly related to the disease, it will be directly judged as a hotspot mutation.

[0035] The following genes were finally screened: (1) PIGA gene: its mutation is related to paroxysmal nocturnal hemoglobinuria (PNH) type 1, which is a somatic mutation; (2) PIGT gene: its mutation is related to paroxysmal nocturnal hemoglobinuria (PNH) type 2, which is a somatic mutation or dominant inheritance.

[0036] The mutation sites corresponding to each mutated gene are shown in Table 1.

[0037] (2) Primer compositions were designed based on the above-mentioned genes and mutation sites. The specific primer sequences of the primer compositions are shown in Table 2. The forward primer sequence is SEQ IN NO.1-34, and the reverse primer sequence is SEQ IN NO.35-68. The working concentration of each primer in the primer composition is 0.1 μM.

[0038] (3) PCR amplification of the complex system: Mix the above primers into a reaction system and select the most suitable primer concentration under the condition of ensuring a certain template concentration, so that each pair of primers in the complex system can achieve optimal amplification.

[0039] Example 2: A kit for detecting PNH and its usage method

[0040] Step 1. Amplification primers in the multiplex amplification system

[0041] In the multiplex amplification system, the forward and reverse amplification primers for each gene and mutation site are shown in Table 2. In order to make the amplification efficiency of each gene and mutation site as consistent as possible, the concentration of each primer pair in the multiplex system was adjusted, and the final adjusted concentration of each primer was 0.1 μM.

[0042] Step 2. DNA extraction: Extract whole-genome DNA from peripheral blood or bone marrow samples using a DNA extraction kit.

[0043] Step 3. Perform multiplex PCR amplification on the extracted DNA using the primer composition provided in Example 1.

[0044] The multiplex amplification system is shown in Table 3 below.

[0045] Table 3. Multiplex amplification system

[0046] <![CDATA[AgriSeq TM Amplification Mix]]> 2μL <![CDATA[Ion AmpliSeq TM Primer Pool]]> 5μL Diluted DNA sample (10ng / μL) 3μL

[0047] The DNA was derived from the sample being tested. The primer pool was a mixture of the primers mentioned above, with each primer having a concentration of 0.1 μM.

[0048] The multiplex amplification reaction program is as follows: 99℃, 2 min; 99℃, 15 s, 62℃, 4 min, 2 cycles; 99℃, 15 s, 60℃, 8 min, 14 cycles; 10℃, store.

[0049] Step 4. Use a kit to prepare the amplification products into a DNA library that can be sequenced by a sequencing platform, thus completing library construction.

[0050] Step 4.1 Digest the amplification product with enzymes. The enzyme digestion system is shown in Table 4 below.

[0051] Table 4 Enzyme digestion system

[0052] Pre-ligation 2μL

[0053] The enzyme digestion reaction procedure was as follows: 50℃, 20 min; 55℃, 20 min; 60℃, 20 min; 10℃, storage. After the reaction, the product was purified by magnetic bead method.

[0054] The pre-ligation was purchased from ThermoFisherScientific, item number A34141.

[0055] Step 4.2 End-of-phase repair & addition of A. The reaction system is shown in Table 5 below.

[0056] Table 5 End-stage repair & Adding A

[0057] End Repair&A-Tailing Enzyme 4μL End Repair&A-Tailing Buffer 6μL

[0058] The reaction procedure is as follows: 20℃, 30 min; 65℃, 30 min; 4℃, storage.

[0059] Among them, End Repair & A-Tailing Enzyme and End Repair & A-Tailing Buffer were purchased from Naonda, item number 1002103.

[0060] Step 4.3 Connector connection. The reaction system is shown in Table 6 below.

[0061] Table 6 Connector Connections

[0062] IDT UDI adapter (15μM) 2μL Ligation Buffer 26μL DNA Ligase 2μL

[0063] The reaction procedure was as follows: 20℃, 20 min. After the reaction was completed, the product was purified by magnetic bead purification.

[0064] Among them, the IDT UDI adapter (15μM), Ligation Buffer, and DNA Ligase were purchased from Naonda (catalog number 1003227), and the Ligase (catalog number 1002103).

[0065] Step 4.4 Library enrichment. The reaction system is shown in Table 7 below.

[0066] Table 7 Library Enrichment

[0067] Amplification Primer Mix (10μM) 5μL Adapter-ligated library 20μL

[0068] The amplification reaction program was as follows: 98℃, 2 min; 98℃, 15 s, 60℃, 30 s, 72℃, 30 s, 7 cycles; 72℃, 2 min; 4℃, storage. After the reaction, the product was purified by magnetic bead method.

[0069] Among them, the 2X HiFi PCR Master Mix was purchased from Naonda, item number 1002103.

[0070] Step 5. Prepare templates for the DNA library for sequencing, obtain sequencing data, and perform biological analysis on the sequencing data.

[0071] Example 3 Repeatability Test

[0072] One patient diagnosed with PNH based on clinical symptoms and relevant laboratory tests was selected. Among them, the PIGA gene c.1052C>A nucleotide mutation was detected by first-generation sequencing, and its amino acid mutation was p.S351X.

[0073] The sample was tested three times using the primer composition and kit described in this application. The specific steps and detection methods are as follows:

[0074] 1. DNA extraction: Whole genome DNA was extracted from bone marrow samples using the Tiangen DNA Extraction Kit (catalog number: DP318-03).

[0075] 2. The extracted DNA was amplified by multiplex PCR according to the primer compositions and kits provided in Examples 1 and 2.

[0076] 3. Prepare the amplification products into a DNA library suitable for sequencing on the Ion Torrent sequencing platform. For detailed operating procedures, please refer to the instruction manual of Life Sciences kit (name: Ion AmpliSeq™ Library Kit, catalog number: 4480441).

[0077] 4. Prepare templates for the obtained DNA library and then sequence it to obtain the data: After the constructed library is treated with water in oil, it is subjected to high-throughput sequencing on the Ion Torrent platform. The water in oil treatment method and high-throughput sequencing method are performed in accordance with the instruction manual of the Ion Torrent high-throughput sequencer and its supporting equipment One Touch.

[0078] 5. Bioinformatics analysis of the sequencing data: First, low-quality sequencing fragments were filtered using Ion Report software (v4.6, ThermoFisher, Carlsbad, CA, USA), and qualified sequencing fragments were aligned to the human reference genome hg19 (http: / / hgdownload.cse.ucsc.edu / downloads.html). Mutation sites were detected using the Torrent VariantCaller (v4.6.0.7) subroutine, with the software parameters set to default. This software has been optimized to handle error types specific to the Ion Torrent sequencing platform. Finally, the identified mutations, including SNPs and Indels, were annotated using ANNOVAR (http: / / annovar.openbioinformatics.org / en / latest / ). The annotations included the mutation's location in the genome, associated genes, gene exon numbers, nucleotide level variations, corresponding protein level variations, and annotations of the mutation in the dbSNP database (http: / / www.ncbi.nlm.nih.gov / snp / ), the 1000 Genomes database (http: / / www.1000genomes.org / ), the Exome Integration Database ExAC (http: / / exac.broadinstitute.org / ), and the Human Genetic Mutation Database HGMD (http: / / www.hgmd.cf.ac.uk / ac / index.php), as well as functional prediction results from PolyPhen (http: / / genetics.bwh.harvard.edu / pph2 / ) and SIFT (http: / / sift.jcvi.org / ).

[0079] As shown in Table 8 below, all three tests showed a positive result for the PIGA gene c.1052C>A nucleotide mutation. Figure 1 As shown, through Figure 1 Detection and verification showed that the primer composition and kit described in this application have good reproducibility.

[0080] Table 8 Repeatability Test Results

[0081] Mutant gene PIGA PIGA PIGA Nucleotide change c.1052C>A c.1052C>A c.1052C>A Amino acid change p.S351X p.S351X p.S351X Mutation position NM_002641:Exon5 NM_002641:Exon5 NM_002641:Exon5 Number of sequencing reads 834622039 835632678 832453258 Target area coverage 98.52％ 98.54％ 98.51％ Average sequencing depth (×) 967 982 959 Uniformity 96.58％ 96.61％ 96.56％

[0082] Example 4 Specificity Detection

[0083] 4-1. Using the primers and concentrations given in Example 1, the DNA sample extracted in Example 3 was amplified using the amplification system and amplification program given in Example 2, sequenced using Ion Torrent, and further analyzed.

[0084] 4-2. The primer concentrations given in Example 1 were all modified to 0.05 μM. The DNA samples extracted in Example 3 were amplified using the same amplification system and amplification program. Sequencing was performed using Ion Torrent, and further analysis was conducted.

[0085] 4-3. The primer concentrations given in Example 1 were all modified to 0.2 μM. The DNA samples extracted in Example 3 were amplified using the same amplification system and amplification program. Sequencing was performed using Ion Torrent, and further analysis was conducted.

[0086] The test results for 4-1, 4-2, and 4-3 above are shown in Table 9 below.

[0087] Table 9 Specificity Detection Results

[0088] Mutant genes PIGA PIGA PIGA Nucleotide alteration c.1052C>A c.1052C>A c.1052C>A Amino acid changes p.S351X p.S351X p.S351X mutation location NM_002641:Exon5 NM_002641:Exon5 NM_002641:Exon5 Number of sequencing reads 834622039 636158758 657547333 Target area coverage 98.52％ 74.85％ 76.31％ Average sequencing depth (×) 967 703 736 Uniformity 96.58％ 79.92％ 82.65％

[0089] As shown in Table 7, when the primer concentration is too low or too high, the results of sequencing read count, sequencing depth and uniformity are all poor.

[0090] Example 5 Sensitivity Detection

[0091] According to the kit provided in Example 2, DNA samples of different concentrations extracted in Example 3 were amplified, sequenced using Ion Torrent, and further analyzed. The DNA template concentrations were: 5 ng / μL, 2.5 ng / μL, 1.25 ng / μL, 0.625 ng / μL, 0.3125 ng / μL, and 0.15625 ng / μL.

[0092] The test results are shown in Table 10 below:

[0093] Table 10 Sensitivity Detection Results

[0094]

[0095]

[0096] The results show that the multiplex amplification system provided by this invention can accurately detect samples with a concentration of 0.15625 ng / μL, with a sensitivity of 0.64%, which is far superior to the sensitivity of first-generation sequencing (10%), and has strong recognition ability.

[0097] Example 6 Accuracy Detection

[0098] A total of 29 patients diagnosed with PNH at Jiankang Huamei Medical Diagnostic Center based on clinical symptoms and relevant laboratory tests were selected. The 29 patients were numbered sequentially from No. 1 to No. 29. 3 mL of peripheral blood was collected from each patient to extract whole-genome DNA for testing. This test was approved by the center's ethics committee and with the consent of the patients.

[0099] Samples were tested according to the kit and its usage method provided in Example 2, and the test results are shown in Table 11 below.

[0100] Table 11 Accuracy Test Results

[0101]

[0102]

[0103]

[0104] As shown in the table above, PNH was detected in all 29 patients. Patients numbered 1, 2, 3, 6, 8, 9, 12, 21, 22, and 24 had at least two mutation sites on the same gene. Among them, 24 patients had PIGA gene mutations and 5 patients had PIGT gene mutations, and the conclusions were consistent with the final clinical diagnosis. Therefore, this test kit can effectively diagnose patients with paroxysmal nocturnal hemoglobinuria, offering rapid, accurate, and informative results.

[0105] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A primer composition for detecting the PNH gene, characterized in that, The primer composition consists of primers for detecting PIGA gene mutation sites and PIGT gene mutation sites; The mutation sites of the PIGA gene include: p.H65P, p.C183Y, p.L60P, p.R233G, p.P207R, p.Q327X, p.Y98X, p.L280Cfs11, p.G64Afs2, c.1188+1G>C, p.P447Nfs12, p.S164L, p.G47E, p.L243P, p.L347Yfs6, p.T123Nfs7, p.R415Lfs8, p.V403Wfs21, p.C36Y, p.G263R, p.H154Ifs18, p.C183F, c.715+2T>C, p.Q252X, p.R81P. c.715+1G>A,p.G86Vfs14, p.S155F, p.S351X, p.C348Wfs5, p.S368X, p.V67Lfs2, p.V352X,p.K382X, p.L113R, p.E120A, p.I116V, p.F384Lfs11, p.E120K, c.848+1G>A, c.981+1G>A, p.S127X, p.L359W, p.L437_I450dup, p.S309F, p.R77X, p.N379fs, p.E338X,c.1189-2A>G; The mutation sites of the PIGT gene include: p.T52A, p.Q417E, p.P194L, p.Y281C, p.P382L, p.R573X, p.P92L, p.P518L; Furthermore, the primer composition consists of the primers shown in SEQ ID NO.1-68.

2. The use of the primer composition for detecting the PNH gene as described in claim 1 in the preparation of products for detecting the PNH gene.

3. A product for detecting the PNH gene, characterized in that, The product comprises the primer composition of claim 1.

4. The product for detecting the PNH gene according to claim 3, characterized in that, The product also includes DNA polymerase, buffer, ddH2O, and dNTPs.

5. A product for detecting the PNH gene according to claim 4, characterized in that, The product is either a standalone reagent or a kit.

6. The use of the primer composition of claim 1 or the product of claim 3 in the detection of the PNH gene, wherein the detection is not for disease diagnosis and treatment.

7. A method for detecting the PNH gene, said method being a non-disease diagnosis and treatment method, characterized in that, The method includes detecting the PNH gene in the sample to be tested using the primer composition of claim 1 or the product of claim 3.

8. The method for detecting the PNH gene according to claim 7, characterized in that, The method includes the following steps: S1. Extract whole-genome DNA from the sample; S2. Amplification: The whole genome DNA in step S1 is amplified using the primer composition described in claim 1. S3. Constructing a DNA library; S4, library sequencing and data processing.

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