Primer group, probe group, and kit and use thereof

Abstract

The present invention relates to the technical field of biology, and in particular to a primer group, a probe group, and a kit and a use thereof. The primer group comprises: a first primer pair comprising a first forward primer and a first reverse primer; and a second primer pair comprising a second forward primer and a second reverse primer, wherein the first forward primer has a nucleic acid sequence as shown in SEQ ID NO: 1 or a nucleic acid sequence having at least 80% homology to SEQ ID NO: 1, the first reverse primer has a nucleic acid sequence as shown in SEQ ID NO: 2 or a nucleic acid sequence having at least 80% homology to SEQ ID NO: 2, the second forward primer has a nucleic acid sequence as shown in SEQ ID NO: 3 or a nucleic acid sequence having at least 80% homology to SEQ ID NO: 3, and the second reverse primer has a nucleic acid sequence as shown in SEQ ID NO: 4 or a nucleic acid sequence having at least 80% homology to SEQ ID NO: 4. On the basis of the specific primer sequences, accurate and stable detection of gene mutation sites in sickle cell anemia can be achieved.

Description

Primer set, probe set, kit and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular, the present application relates to primer set, probe set, kit and application thereof. BACKGROUND

[0002] Sickle cell anemia, also known as sickle cell disease (SCD), is an autosomal recessive hemoglobinopathy, which is clinically manifested as chronic hemolytic anemia, susceptibility to infection and recurrent painful crisis, causing chronic ischemia and leading to organ and tissue damage. According to literature reports, about 300,000 children are born worldwide each year, and about 5 million carriers, mainly in Africa, India, and African American populations. In many areas of Africa, the prevalence of sickle cell trait (HbS / C / D / O) is as high as 25%-35%, and it is estimated that 15 million Africans are affected by SCD, and SCD accounts for 16% of deaths in children under five in West Africa. Sickle cell disease is the most common genetic blood disease in the United States. About one in every 300-500 African Americans born in the United States has SCD; 1 in 1000 to 1400 Hispanic Americans has SCD.

[0003] The molecular definition of SCD includes a group of diseases characterized by the presence of at least one hemoglobin S (HbS) allele and a second pathogenic variant of the HBB gene, resulting in abnormal hemoglobin polymerization. Common SCD-related HBB genotype mutation information is shown in Table 1:

[0004] Table 1

[0005] Screening and detection of pathogenic variants of sickle cell anemia is the best way to prevent birth defects and effectively treat the disease as soon as possible. The main pathogenic mutations of sickle cell anemia include HbS (c.20A>T), HbC (c.19G>A), HbD (c.364G>C), HbO (c.364G>A), and HbE (c.79G>A). Detection of these five variant sites can achieve the purpose of effective screening and molecular detection.

[0006] Currently, the detection and screening of sickle cell anemia are mainly based on isoelectric focusing (IEF), high performance liquid chromatography (HPLC), immunochromatographic assay and other technologies, which detect and analyze the characteristics of proteins. These detection methods have the disadvantages of poor specificity, expensive equipment, complex operation, inability to achieve precise molecular typing, and susceptibility to subjective judgment of the detection personnel, thus are not suitable for clinical application and popularization.

[0007] The nucleic acid molecular detection technology for single gene genetic diseases mainly includes Sanger sequencing, high-throughput sequencing, chip hybridization and other technologies. These detection methods have the disadvantages of few detection sites, low throughput, high cost, complicated operation steps, long cycle and are not suitable for clinical application and popularization.

[0008] Therefore, the screening and detection method of pathogenic mutations of SCD still needs to be improved.

[0009] SUMMARY

[0010] The present application is proposed by the inventors based on the following problems and facts:

[0011] The mutation detection technology based on the melting curve method is a relatively mature gene detection technology. Based on this technology, nucleic acid gene mutation detection can be realized, but there is no mature product or service applied to SCD detection.

[0012] Therefore, the present application provides a product and method for high-throughput and accurate detection of sickle cell anemia gene mutation sites.

[0013] Specifically, the present application provides the following technical solutions:

[0014] In one aspect of the present application, a primer set is provided. According to the embodiments of the present application, the primer set comprises: a first primer pair comprising a first upstream primer and a first downstream primer; and a second primer pair comprising a second upstream primer and a second downstream primer; wherein the first upstream primer has a nucleic acid sequence as shown in SEQ ID NO: 1 or has at least 80% homology with SEQ ID NO: 1; the first downstream primer has a nucleic acid sequence as shown in SEQ ID NO: 2 or has at least 80% homology with SEQ ID NO: 2; the second upstream primer has a nucleic acid sequence as shown in SEQ ID NO: 3 or has at least 80% homology with SEQ ID NO: 3; and the second downstream primer has a nucleic acid sequence as shown in SEQ ID NO: 4 or has at least 80% homology with SEQ ID NO: 4. In some examples of the present application, the specific primer sequences described above can realize accurate and stable detection of sickle cell anemia gene mutation sites.

[0015] In some examples of the present application, the first primer pair described above is used to specifically amplify HbS mutation sites, HbC mutation sites and HbE mutation sites, which helps to improve the specificity and sensitivity of detection, exclude interference, and also helps to save cost and time.

[0016] In some examples of the present application, the foregoing second primer pair is used for specifically amplifying the HbD mutation site and the HbO mutation site, which helps to improve the specificity and sensitivity of the detection, eliminate interference, and also helps to save cost and time.

[0017] In some examples of the present application, the foregoing primer set can further comprise at least one of the following technical features:

[0018] In some examples of the present application, the primer set further comprises: a third primer pair comprising a third upstream primer and a third downstream primer; the third upstream primer has a nucleic acid sequence as shown in SEQ ID NO: 5 or has at least 80% homology with SEQ ID NO: 5; the third downstream primer has a nucleic acid sequence as shown in SEQ ID NO: 6 or has at least 80% homology with SEQ ID NO: 6. In some examples of the present application, the foregoing third primer pair is used for amplifying a reference gene. In a specific example of the present application, the foregoing third primer pair is used for amplifying the ATCB gene, which is used for quality control of the experiment and helps to explain the experimental results.

[0019] In some examples of the present application, sequences with a similarity higher than 80% to the foregoing primer sequences can also be used for accurate and stable detection of the sickle cell anemia gene mutation site.

[0020] In a second aspect of the present application, a reagent set is provided, according to an embodiment of the present application, the reagent set comprises: a first reagent comprising the primer set according to the first aspect of the present application. In some examples of the present application, the foregoing reagent set can be used for accurate and stable detection of the sickle cell anemia gene mutation site.

[0021] In some examples of the present application, the foregoing reagent set can further comprise at least one of the following technical features:

[0022] In some examples of the present application, the reagent set further comprises: a second reagent comprising a first probe, a second probe and / or a third probe. In some examples of the present application, the probe carries a fluorescent group, which is used for high specificity, sensitivity and multiplex detection of the sickle cell anemia gene mutation site.

[0023] In some examples of the present application, the first probe has a nucleic acid sequence as shown in SEQ ID NO: 7 or has at least 80% homology with SEQ ID NO: 7. In some examples of the present application, the foregoing first probe is used for specifically targeting the HbS and / or HbC mutation site, ensuring the specificity, accuracy and sensitivity of the detection.

[0024] In some examples of the present application, the second probe has a nucleic acid sequence as shown in SEQ ID NO: 8 or has at least 80% homology with SEQ ID NO: 8. In some examples of the present application, the aforementioned second probe is used to specifically target the HbD and / or HbO mutation site, ensuring the specificity, accuracy and sensitivity of the detection.

[0025] In some examples of the present application, the third probe has a nucleic acid sequence as shown in SEQ ID NO: 9 or has at least 80% homology with SEQ ID NO: 9. In some examples of the present application, the aforementioned third probe is used to specifically target the HbE mutation site, ensuring the specificity, accuracy and sensitivity of the detection.

[0026] In some examples of the present application, sequences with a similarity higher than 80% to the above-mentioned probe sequences can also be used for accurate and stable detection of the thalassemia gene mutation site.

[0027] In some examples of the present application, the second reagent further comprises: a fourth probe.

[0028] In some examples of the present application, the fourth probe has a nucleic acid sequence as shown in SEQ ID NO: 10 or has at least 80% homology with SEQ ID NO: 10. In some examples of the present application, the aforementioned fourth probe is used to specifically target the target region of the internal reference gene, which can be used for quality control of the experiment.

[0029] In some examples of the present application, the reagent set further comprises: a reaction solution, the reaction solution comprising: at least one of a DNA polymerase, a buffer and dNTPs.

[0030] In some examples of the present application, the aforementioned DNA polymerase is selected from Ex Taq HS DNA polymerase.

[0031] In some examples of the present application, the aforementioned buffer is selected from 10x Buffer (plus Mg 2+ ).

[0032] In a third aspect of the present application, the present application provides use of the primer set of the first aspect or the reagent set of the second aspect in the preparation of a kit for detecting a thalassemia gene mutation site. In some examples of the present application, the kit containing the above-mentioned primer set or reagent set can be used for accurate and specific detection of the mutant type of the thalassemia gene mutation site in the sample to be tested.

[0033] In the fourth aspect of the present application, the present application provides a use of the primer set of the first aspect or the reagent set of the second aspect in the preparation of a kit for detecting sickle cell anemia. In some examples of the present application, the kit containing the primer set or the reagent set described above can be used to accurately and specifically detect whether the sample to be tested has sickle cell anemia.

[0034] In the fifth aspect of the present application, the present application provides a use of the primer set of the first aspect or the reagent set of the second aspect in the preparation of a kit for detecting the genotype of sickle cell anemia. In some examples of the present application, the kit containing the primer set or the reagent set described above can be used to accurately and specifically detect the genotype of the sample to be tested.

[0035] In the sixth aspect of the present application, the present application provides a kit. According to the embodiments of the present application, the kit comprises the primer set of the first aspect or the reagent set of the second aspect. In some examples of the present application, the foregoing kit can be used to accurately and specifically detect whether the sample to be tested has sickle cell anemia and the specific genotype of sickle cell anemia at a low cost and high throughput. Further, the mutation site of the specific genotype of sickle cell anemia can also be detected. The kit is simple to operate, fast in detection, suitable for clinical application, and has important significance for birth defect prevention and control and early and effective treatment of diseases.

[0036] In the seventh aspect of the present application, the present application provides a method for detecting a mutation type of a sickle cell anemia related gene. According to the embodiments of the present application, the method comprises: performing a qPCR reaction on a sample to be tested by using the reagent set of the second aspect or the kit of the sixth aspect, wherein the probes in the reagent set or the kit independently carry different fluorescent groups respectively; the first probe carries a first fluorescent group, the second probe carries a second fluorescent group, and the third probe carries a third fluorescent group; and the mutation type of the sickle cell anemia related gene is determined based on the Tm value difference of the detection channels of the first fluorescent group, the second fluorescent group and the third fluorescent group. In some examples of the present application, the foregoing method realizes synchronous multiplex analysis and detection of multiple target sites of SCD in one reaction well, greatly improving the detection throughput. The process is more simple and the result is more stable by directly reading the Tm value for variation result analysis. In addition, it can also be used for non-diagnostic purposes, such as scientific research, to confirm whether the sample to be tested contains SCD related mutations. If the sample to be tested contains SCD related mutations, the sample to be tested can be used to further study the signal transduction mechanism of SCD related mutations.

[0037] In some examples of the present application, the foregoing detection method can further comprise at least one of the following technical features:

[0038] In some examples of the present application, the sample to be tested can be blood, tissue, cells, etc., but is not limited thereto.

[0039] In some examples of the present application, the concentration of the upstream primer in the qPCR reaction system is 0.1-2 μΜ.

[0040] In some examples of the present application, the concentration of the downstream primer in the qPCR reaction system is 1-10 μΜ.

[0041] In some examples of the present application, based on the concentrations of the upstream primer and the downstream primer, the target strand bound by the probe can be amplified more effectively under the premise of effective amplification, so as to achieve the purposes of amplifying the target signal and suppressing the interference of non-specific signals.

[0042] In some examples of the present application, the concentration of the probe in the qPCR reaction system is 0.1-2 μΜ.

[0043] In some examples of the present application, the concentration of the DNA polymerase in the qPCR reaction system is 2-3 U.

[0044] In some examples of the present application, the concentration of the dNTPs in the qPCR reaction system is 0.1-1 mM.

[0045] In some examples of the present application, the first fluorescent group, the second fluorescent group and the third fluorescent group are different in the qPCR reaction system.

[0046] In some examples of the present application, the first fluorescent group, the second fluorescent group and the third fluorescent group are each independently selected from at least one of FAM, HEX, ROX, CY5, Atto 425, TET, JOE, VIC, R6G, Yakima Yellow, Quasar570, Quasar670, Cy3, NED, Cy5.5, Cy7, Texas Red, Atto 590, IR Dye 650 and IR Dye 750. In some examples of the present application, the above fluorescent group-labeled probes can be freely selected.

[0047] In some examples of the present application, when the Tm value of the first fluorescent group detection channel is 59-63, it is an indication that the sample to be tested does not contain a mutation of a sickle cell anemia-related gene. The Tm value can be optionally a rational number, which includes positive integers and positive fractions. Exemplarily, the Tm value can be optionally 59, 60, 61, 62, 63, 59.5, 60.5, 61.5, 62.5, 59.55 or 60.55, etc.

[0048] In some examples of the application, the Tm value of the first fluorescent group detection channel is 52-56, which is an indication that the sample to be tested contains HbS mutation. Wherein, the Tm value is optionally a rational number, the rational number includes positive integers and positive fractions, exemplarily the Tm value is optionally 52, 53, 54, 55, 56, 52.5, 53.5, 54.5, 55.5, 52.55 or 55.55, etc.

[0049] In some examples of the application, the Tm value of the first fluorescent group detection channel is 45-49, which is an indication that the sample to be tested contains HbC mutation. Wherein, the Tm value is optionally a rational number, the rational number includes positive integers and positive fractions, exemplarily the Tm value is optionally 52, 53, 54, 55, 56, 52.5, 53.5, 54.5, 55.5, 52.55 or 55.55, etc.

[0050] In some examples of the application, the Tm value of the second fluorescent detection channel is 55-59, which is an indication that the sample to be tested does not contain Sickle Cell Anemia related gene mutation. Wherein, the Tm value is optionally a rational number, the rational number includes positive integers and positive fractions, exemplarily the Tm value is optionally 55, 56, 57, 58, 59, 55.5, 56.5, 57.5, 58.5, 55.55 or 56.55, etc.

[0051] In some examples of the application, the Tm value of the second fluorescent detection channel is 40-44, which is an indication that the sample to be tested contains HbD mutation. Wherein, the Tm value is optionally a rational number, the rational number includes positive integers and positive fractions, exemplarily the Tm value is optionally 40, 41, 42, 43, 44, 40.5, 41.5, 42.5, 43.5, 40.55 or 41.55, etc.

[0052] In some examples of the application, the Tm value of the second fluorescent detection channel is 46-50, which is an indication that the sample to be tested contains HbO mutation. Wherein, the Tm value is optionally a rational number, the rational number includes positive integers and positive fractions, exemplarily the Tm value is optionally 46, 47, 48, 49, 50, 46.5, 47.5, 48.5, 49.5, 46.55 or 47.55, etc.

[0053] In some examples of the present application, the Tm value of the third fluorescence detection channel is 68-72, which is an indication that the sample to be tested does not contain a sickle cell anemia related gene mutation. Wherein, the Tm value is optionally a rational number, the rational number includes positive integers and positive fractions, and the Tm value is optionally 68, 69, 70, 71, 72, 68.5, 69.5, 70.5, 71.5, 68.55 or 69.55, etc.

[0054] In some examples of the present application, the Tm value of the third fluorescence detection channel is 61-65, which is an indication that the sample to be tested contains HbE mutation. Wherein, the Tm value is optionally a rational number, the rational number includes positive integers and positive fractions, and the Tm value is optionally 61, 62, 63, 64, 65, 61.5, 62.5, 63.5, 64.5, 61.55 or 62.55, etc.

[0055] It should be noted that the above Tm value is a judgment standard obtained based on the above-mentioned primer and probe of the present application, and the change of the primer and probe sequence will also cause the change of the above-mentioned Tm value. The protection scope of the present application is not limited to specific primer sequences and probe sequences, and other detection methods for multiple sickle cell anemia related gene mutation types through one reaction system of the present application also belong to the protection scope of the present application.

[0056] In addition, in some other examples of the present application, one or more reaction wells can be added, and based on the detection method of the present application, simultaneous detection of multiple samples and / or multiple mutation sites can be realized.

[0057] Based on the above judgment method, it is possible to identify five mutation types (c.20A>T, c.19G>A, c.364G>C, c.364G>A and c.79G>A) of SCD in one reaction system at one time.

[0058] For the convenience of understanding, the detection method of the sickle cell anemia related gene mutation type is exemplarily described as follows:

[0059] 1. Extract gDNA of the sample to be tested;

[0060] In the present application, the method for extracting gDNA of the sample to be tested is not specifically limited, and the experimenter can extract gDNA based on the experimental requirements or the existing extraction scheme in the laboratory.

[0061] 2. Prepare qPCR reaction solution

[0062] DNA template: 2-5 ng; DNA polymerase: 2-3 U; 10x Buffer (containing Mg 2+) : 1-3 mM; dNTP mixture: 0.1-1 mM; primer (S / C / D / O / E / ACTB): 9-11 μM; probe (S / C / D / O / E / ACTB): 1-3 μM; nuclease-free water: supplemented to 10-50 μL.

[0063] Primer: HBS / C / E-F: SEQ ID NO: 1; HBS / C / E-R: SEQ ID NO: 2; HBD / O-F: SEQ ID NO: 3; HBD / O-R: SEQ ID NO: 4; ACTB-F: SEQ ID NO: 5; ACTB-R: SEQ ID NO: 6;

[0064] Probe: HBS / C-P: SEQ ID NO: 7; HBD / O-P: SEQ ID NO: 8; HBE-P: SEQ ID NO: 9; ACTB-P: SEQ ID NO: 10;

[0065] Fluorescent group: FAM, ROX and CY5;

[0066] In some examples of the present application, the above-mentioned reaction system range can achieve accurate and specific detection of sickle cell anemia related gene mutation type. It is known to those skilled in the art that the optimal concentration may be slightly different in different samples. The present application aims to protect the detection method, not the optimal concentration of each component in the above qPCR reaction solution, therefore, the optimal concentration of the experiment is not specifically limited, and the experimental personnel can select the most suitable concentration within the above concentration range based on the experimental requirements.

[0067] 3. qPCR reaction program (Table 2)

[0068] Table 2

[0069] 4. Result analysis

[0070] After the reaction is completed, the Tm value of each fluorescence channel of the sample to be tested is obtained, and the detection result is interpreted according to the Tm value. It should be noted that different Tm values may be produced by changes in primer and probe sequences, and the experimental personnel can confirm the final interpretation standard after studying the positive threshold based on the known type samples.

[0071] In some examples of the present application, the above-mentioned reagent group, primer, probe and reaction system are taken as examples, and the following reference interpretation results (Table 3) are obtained.

[0072] Table 3

[0073] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features described in detail below (such as the embodiments) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0074] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the references to the figures, in which:

[0075] Fig. 1 is a schematic diagram of the agarose gel electrophoresis result of the PCR amplification product provided by the embodiment of the present application;

[0076] Fig. 2 is a schematic diagram of the melting curve of sample S1 of primer probe combination 1 provided by the embodiment of the present application;

[0077] Fig. 3 is a schematic diagram of the melting curve of sample S2 of primer probe combination 1 provided by the embodiment of the present application;

[0078] Fig. 4 is a schematic diagram of the melting curve of sample S3 of primer probe combination 1 provided by the embodiment of the present application;

[0079] Fig. 5 is a schematic diagram of the melting curve of sample S4 of primer probe combination 1 provided by the embodiment of the present application;

[0080] Fig. 6 is a schematic diagram of the melting curve of sample S5 of primer probe combination 1 provided by the embodiment of the present application;

[0081] Fig. 7 is a schematic diagram of the melting curve of sample S1 of primer probe combination 2 provided by the embodiment of the present application;

[0082] Fig. 8 is a schematic diagram of the melting curve of sample S2 of primer probe combination 2 provided by the embodiment of the present application;

[0083] Fig. 9 is a schematic diagram of the melting curve of sample S3 of primer probe combination 2 provided by the embodiment of the present application;

[0084] Fig. 10 is a schematic diagram of the melting curve of sample S4 of primer probe combination 2 provided by the embodiment of the present application;

[0085] Fig. 11 is a schematic diagram of the melting curve of sample S5 of primer probe combination 2 provided by the embodiment of the present application;

[0086] Fig. 12 is a schematic diagram of the melting curve of the known negative sample detected by the experimental group 1 provided by the embodiment of the present application, wherein the CY5 channel has a melting peak at the Tm of the internal reference and the wild type Tm; the FAM channel has a melting peak at the wild type Tm; and the ROX channel has a melting peak at the wild type Tm;

[0087] Fig. 13 is a melting curve diagram of detecting a known HbS heterozygous sample according to an embodiment of the present application. The CY5 channel has a melting peak at the internal reference Tm, the wild type Tm and the mutant-HbS Tm; the FAM channel has a melting peak at the wild type Tm; and the ROX channel has a melting peak at the wild type Tm.

[0088] Fig. 14 is a melting curve diagram of detecting a known HbC heterozygous sample according to an embodiment of the present application. The CY5 channel has a melting peak at the internal reference Tm, the wild type Tm and the mutant-HbC Tm; the FAM channel has a melting peak at the wild type Tm; and the ROX channel has a melting peak at the wild type Tm.

[0089] Fig. 15 is a melting curve diagram of detecting a known HbD heterozygous sample according to an embodiment of the present application. The CY5 channel has a melting peak at the internal reference Tm and the wild type Tm; the FAM channel has a melting peak at the wild type Tm and the mutant-HbD Tm; and the ROX channel has a melting peak at the wild type Tm.

[0090] Fig. 16 is a melting curve diagram of detecting a known HbO heterozygous sample according to an embodiment of the present application. The CY5 channel has a melting peak at the internal reference Tm and the wild type Tm; the FAM channel has a melting peak at the wild type Tm and the mutant-HbO Tm; and the ROX channel has a melting peak at the wild type Tm.

[0091] Fig. 17 is a melting curve diagram of detecting a known HbE heterozygous sample according to an embodiment of the present application. The CY5 channel has a melting peak at the internal reference Tm and the wild type Tm; the FAM channel has a melting peak at the wild type Tm; and the ROX channel has a melting peak at the wild type Tm and the mutant-HbE Tm. DETAILED DESCRIPTION

[0092] Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein the embodiments of the application are shown in example. The embodiments described below are examples of the present application, and are not intended to limit the present application.

[0093] In addition, the terms "first", "second", "third", etc. are used herein only to describe different instances, and are not used to indicate or imply relative importance or a number of indicated technical features. Thus, the features defined with "first", "second", etc. can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, such as two, three, etc., unless otherwise specifically limited.

[0094] In the present context, the terms "homology", "identity" or "similarity" are used to describe or compare the degree of nucleotide similarity of two or more nucleotide sequences, unless otherwise indicated. The percentage of "sequence homology" between a first sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical to the nucleotides at the corresponding positions] by [the number of nucleotides in the second sequence]. [The number of nucleotides in the second sequence] minus [the total number of nucleotides in the first sequence] is then multiplied by [100%], wherein each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - relative to the first nucleotide sequence - is considered as a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, using standard settings. Some other techniques, computer algorithms and settings for determining the degree of sequence identity are for example described in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768-A.

[0095] In the present context, one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) nucleotides can be added to, and / or deleted from, and / or replaced in, a primer or probe sequence of the application, by the skilled person, without materially affecting the accuracy of the quantitative PCR detection, provided that the primer or probe sequence is a variant of the primer or probe sequence. They are all considered to be included within the scope of protection of the application. Nucleotides having similar properties are replaced in the variable region. The variant sequences described in the present application can have at least 80% homology to the reference sequences, meaning at least 80%, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology to each reference sequence. The sequence identity described in the present application can be measured using sequence analysis software, for example the computer program BLAST, in particular BLASTP or TBLASTN, using default parameters. The nucleic acid sequences described in the present application are all shown in the N-terminal to C-terminal direction.

[0096] In the present application, the Tm values are all in °C.

[0097] The present application will be described below with reference to specific examples, which are intended to be illustrative only and not limiting of the present application in any way. Unless otherwise indicated, technical or conditions not specified in the examples were performed according to techniques or conditions described in the literature or according to the product instructions. Unless otherwise indicated, reagents or instruments not specified by the manufacturer were all commercially available conventional products.

[0098] Example 1: Design, screening and optimization of primer and probe sequences

[0099] According to the technical solution of the present application, specific primers are used to amplify the target region, and probes modified with different fluorescent groups are used to specifically recognize and bind to the target mutation site. By analyzing the melting curve, different Tm values of different fluorescent signal channel probes are obtained, so as to judge the variation information of the sample to be detected. At the same time, specific primers and probes are used for the detection of internal standard genes, which are used for quality control and evaluation of the reaction system. The present example aims to screen the most suitable primer and probe sequences to realize accurate detection of sickle cell anemia gene mutations.

[0100] Experiment 1: Design and screening of primer sequences

[0101] According to the characteristics of the genomic location of the target to be detected, the present example uses a first primer pair to specifically amplify the regions of HbS mutation site, HbC mutation site and HbE mutation site, a second primer pair to specifically amplify the regions of HbD mutation site and HbO mutation site, and a third primer pair to specifically amplify the target region of the internal reference ACTB gene.

[0102] Among them, the specific primer sequences are shown in Table 4.

[0103] Table 4 Specific amplification primer sequences

[0104] The genomic DNA extracted from 2 human whole blood samples was subjected to PCR amplification reaction using the above primer pairs, and the PCR amplification products were detected by 1.0% agarose gel electrophoresis method, and the results are shown in Figure 1.

[0105] The test results show that the aforementioned primer pair combination can work efficiently in the same reaction system and achieve specific amplification of the 5 target site regions and the internal reference gene.

[0106] Experiment 2: Design, screening and optimization of probe sequences

[0107] According to the genomic position characteristics of the target to be detected, one fluorescent group labeled hydrolysis probe is used to target mark the HbS mutation site and the HbC mutation site, a second fluorescent group labeled hydrolysis probe is used to target mark the HbD mutation site and the HbO mutation site, a third fluorescent group labeled hydrolysis probe is used to target mark the HbE mutation site, and a fourth fluorescent group labeled hydrolysis probe is used to target mark the target region of the internal reference ACTB. At the same time, by optimizing and adjusting the sequences of each probe and the overall reaction program, different melting temperature values (Tm values) are obtained in the temperature rising and melting process of the hydrolysis probe after binding with the target, and different combinations of fluorescent groups are used to realize the detection of five mutant types at one time in one reaction system.

[0108] In order to design, screen and optimize the fluorescent probe, according to the principle of fluorescent probe PCR melting curve analysis and the basic principles of probe design, two sets of fluorescent probe combinations (Table 5) are designed for testing and selection in combination with the specific amplification primers determined in Experiment 1.

[0109] Table 5 Specific probe sequences of target genes and internal reference genes

[0110] Based on the above designed primers (Table 4) and fluorescent probes (Table 5), the genomic DNA extracted from human whole blood samples is subjected to fluorescent PCR melting curve analysis.

[0111] Based on the technical solutions of the present application and the principle of fluorescent PCR melting curve analysis, the Tm values of the samples to be tested in different fluorescent signal channels are used for variation detection and typing.

[0112] Among them, different types of samples in different fluorescent signal channels should obtain melting peaks and Tm values that can be used for typing as shown in Table 6.

[0113] Table 6 Test sample information and corresponding fluorescent signal channel theoretical melting peaks Note: WideType represents wild type; taking S2 as an example, HbAS represents sickle cell anemia type; HBB represents sickle cell anemia gene; c.20A>T represents the 20th nucleotide position of cDNA sequence, A is mutated to T; Het represents heterozygous mutation.

[0114] Two groups of primer probe combinations are used to perform fluorescent PCR melting curve analysis on 5 samples, and the melting peak diagram and Tm value of each fluorescent signal channel are obtained.

[0115] 1) Test results of probe combination 1

[0116] The primer probe combination 1 is used to perform fluorescent PCR melting curve analysis on 5 samples (S1-S5), and the melting peak diagram of each sample (3 times) is shown in Figures 2-6.

[0117] The fluorescence PCR melting curve analysis was performed on 5 samples using probe combination 1 (Table 5, Group-1), and the Tmvalues of each fluorescence signal channel are shown in Table 6, and the test results are shown in Table 7.

[0118] Table 7 Test results of probe combination 1

[0119] 2) Test results of probe combination 2

[0120] The fluorescence PCR melting curve analysis was performed on 5 samples using primer probe combination 2 (Table 5, Group-2), and the melting peak graphs of each sample (3 replicates) are shown in Figures 7-11.

[0121] The fluorescence PCR melting curve analysis was performed on 5 samples using primer probe combination 2, and the Tmvalues of each fluorescence signal channel are shown in Table 8.

[0122] Table 8 Test results of probe combination 2

[0123] By comparing the test results of primer probe combination 1 and primer probe combination 2, it can be seen that probe combination 1 can obtain melting peak and Tmvalue results consistent with the detection principle and design scheme, and can be used for variant detection. Probe combination 2 cannot obtain melting peak and Tmvalue results consistent with the expected results.

[0124] In summary, the present application optimizes the design and screening of primers and probes according to the sequence characteristics of the target to be detected, and determines the primer and probe sequences that meet the expected requirements of detection, as shown in Table 9. Among them, the fluorescence group and the quencher group carried by the probe sequence can be adjusted and optimized according to the actual situation. It should be noted that the primers and probes are not limited to this sequence, such as sequences with more than 80% homology to the primers and probes.

[0125] Table 9 Determined primer and probe sequences after screening and optimization

[0126] Example 2: Optimization of reaction system and reaction procedure

[0127] In this embodiment, in view of the detection purpose and the sequence characteristics of the primers / probes, an asymmetric amplification method is used in the reaction system. By adjusting the concentration and ratio of the upstream and downstream primers, the target strand combined with the probe can be more advantageously amplified under the premise of effective amplification, so as to achieve the purpose of signal method and inhibition of non-specific signal interference.

[0128] Experiment 1: Reaction system optimization

[0129] After testing and optimization, the final concentration of primers in the reaction system is preferably determined as follows: the final concentration of the upstream primer is 0.1-2 μM; the final concentration of the downstream primer is 1-10 μM; and the final concentration of the fluorescent probe is 0.1-2 μM. The reaction enzyme is TaKaRa Ex Taq Hot Start Version from Beijing Zixiaosheng Biotechnology Co., Ltd., which can be adjusted according to the actual situation. Hot Start Version, which can be adjusted according to the actual situation.

[0130] Experiment Two: Reaction Procedure Optimization

[0131] Based on the technical solution of the present application and the principle of fluorescent PCR melting curve analysis, the reaction procedure mainly includes three main links: target region amplification ((95℃-60℃-72℃)*50 cycle), melting and cooling (95℃-35℃), and warming and melting curve analysis (35℃-90℃). According to the technical principle of the detection method of the present application, the reaction procedure parameters of the target region amplification and the melting and cooling two links are mainly determined by the sequence characteristics of the target region and the working temperature of the reaction enzyme, and have less effect on the detection performance of the method of the present application. Therefore, the program parameter setting of the melting segment warming rate is mainly tested and optimized.

[0132] The melting segment warming rate in the fluorescent PCR melting curve analysis reaction procedure is set as Continuous 0.02℃ / s, Continuous 0.04℃ / s, and Continuous 0.06℃ / s (Table 10), respectively. Six human genomic DNA samples are selected, and the reaction procedure with three different melting segment warming rates is used for fluorescent PCR melting curve analysis, and the melting peak graphs and Tm values of different fluorescent signal channels are statistically analyzed.

[0133] Table 10: Test scheme for optimizing the melting segment warming rate of the reaction procedure Note: Y means yes, and N means no.

[0134] According to the detection scheme of the present application and the principle of fluorescent PCR melting curve analysis, the Tm values of the samples to be tested in different fluorescent signal channels are used for variation detection and typing.

[0135] Among them, different type samples in different fluorescent signal channels should obtain melting peaks and Tm values that can be used for typing (shown in Table 11).

[0136] Table 11: Test sample information and corresponding fluorescent signal channel theoretical melting peaks

[0137] The six samples were analyzed by using the melting segment heating rate scheme in the three groups of reaction procedures, respectively, and the Tm values of each fluorescence signal channel were obtained.

[0138] 1) Group 1 (Continuous 0.02℃ / s) test results

[0139] The six samples were analyzed by using the melting segment heating rate Continuous 0.02℃ / s (Group 1) reaction procedure, and the Tm values of each fluorescence signal channel were shown in Table 12.

[0140] Table 12 Group 1 (Continuous 0.02℃ / s) test results

[0141] 2) Group 2 (Continuous 0.04℃ / s) test results

[0142] The six samples were analyzed by using the melting segment heating rate Continuous 0.04℃ / s (Group 2) reaction procedure, and the Tm values of each fluorescence signal channel were shown in Table 13.

[0143] Table 13 Group 2 (Continuous 0.04℃ / s) test results

[0144] 3) Group 3 (Continuous 0.06℃ / s) test results

[0145] The six samples were analyzed by using the melting segment heating rate Continuous 0.06℃ / s (Group 3) reaction procedure, and the Tm values of each fluorescence signal channel were shown in Table 14.

[0146] Table 14 Group 3 (Continuous 0.06℃ / s) test results

[0147] By comparing the test results of the three groups of different reaction procedures, the melting segment heating rate Continuous 0.04℃ / s and the melting segment heating rate Continuous 0.06℃ / s can obtain the melting peak and Tm value results consistent with the detection principle and design scheme, which can be used for variation detection.

[0148] After the test optimization of the reaction procedure, the finally determined reaction system was shown in Table 15

[0149] Table 15 Reaction procedure

[0150] Example 3: Sickle cell trait detection

[0151] This example aims to verify the effectiveness of the above-mentioned primers, probes, reaction system and reaction procedure.

[0152] Experimental samples: negative samples and positive samples with known HbS, HbC, HbD, HbO, HbE site mutations were used for detection, and the known variation information of the samples is shown in Table 16.

[0153] Table 16

[0154] Kit components: probe primers (Table 17), DNA polymerase, 10X Buffer (buffer), dNTP mixture and ddH2O.

[0155] Table 17 Probe primers

[0156] Experimental steps:

[0157] 1. Configure the reaction system according to Table 18;

[0158] Table 18 Reaction system

[0159] 2. Fluorescent PCR detection;

[0160] Add the sample to be detected into the reaction system, and perform amplification and melting curve analysis detection according to the reaction procedure in Table 19;

[0161] Table 19

[0162] 3. Result interpretation

[0163] Based on Figures 12-17, the detection result data of the six experimental groups of samples were exported (Table 20), and the detection results were judged according to the Tm value. The results showed that the kit of the present application can accurately detect the mutation of HbS, HbC, HbD, HbO, HbE sites on the HBB gene.

[0164] Table 20

[0165] In summary, the application adopts the technical scheme of multiple fluorescence probes combined with melting curve, realizes the purpose of one-time closed tube simultaneous detection of five pathogenic mutations of sickle cell anemia, including HbS (c.20A>T), HbC (c.19G>A), HbD (c.364G>C), HbO (c.364G>A), and HbE (c.79G>A). At the same time, an internal reference gene (ACTB gene) is set in the one-tube reaction for quality control. The method of the application improves the basic melting curve method, and combines multiple PCR and multiple fluorescence channels according to the sequence characteristics of the pathogenic mutation sites of sickle cell anemia genes, greatly improves the detection throughput; in addition, through testing and optimization adjustment, the optimal reaction system and reaction program are determined, and a standardized executable quality control system and result interpretation standard are established, which has the advantages of accurate result, good specificity, high sensitivity, good repeatability, simple operation, low cost, short cycle, etc.

[0166] In the description of the present specification, the description referring to the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0167] Although the embodiments of the present application have been shown and described above, it should be understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A primer set, characterized by, Comprising: a first primer pair comprising a first upstream primer and a first downstream primer; and a second primer pair comprising a second upstream primer and a second downstream primer; wherein, the first upstream primer has a nucleic acid sequence as set forth in SEQ ID NO: 1 or at least 80% homologous to SEQ ID NO: 1; the first downstream primer has a nucleic acid sequence as set forth in SEQ ID NO: 2 or at least 80% homologous to SEQ ID NO: 2; the second upstream primer has a nucleic acid sequence as set forth in SEQ ID NO: 3 or at least 80% homologous to SEQ ID NO: 3; the second downstream primer has a nucleic acid sequence as set forth in SEQ ID NO: 4 or at least 80% homologous to SEQ ID NO:

4.

2. The primer set of claim 1, wherein Further comprising: a third primer pair comprising a third upstream primer and a third downstream primer; the third upstream primer has a nucleic acid sequence as set forth in SEQ ID NO: 5 or at least 80% homologous to SEQ ID NO: 5; the third downstream primer has a nucleic acid sequence as set forth in SEQ ID NO: 6 or at least 80% homologous to SEQ ID NO:

6.

3. A reagent set, characterized by Comprising: a first reagent comprising the primer set of claim 1 or 2.

4. The reagent set according to claim 3, characterized by Further comprising: a second reagent comprising a first probe, a second probe, and / or a third probe; optionally, the first probe has a nucleic acid sequence as set forth in SEQ ID NO: 7 or at least 80% homologous to SEQ ID NO: 7; optionally, the second probe has a nucleic acid sequence as set forth in SEQ ID NO: 8 or at least 80% homologous to SEQ ID NO: 8; optionally, the third probe has a nucleic acid sequence as set forth in SEQ ID NO: 9 or at least 80% homologous to SEQ ID NO:

9.

5. The reagent set according to claim 4, characterized by the second reagent further comprises: a fourth probe; optionally, the fourth probe has a nucleic acid sequence as set forth in SEQ ID NO: 10 or at least 80% homologous to SEQ ID NO:

10.

6. The reagent set according to claim 3, characterized by the reagent set further comprises: a reaction solution comprising at least one of a DNA polymerase, a buffer, and dNTPs.

7. Use of the primer set of claim 1 or 2 or the reagent set of any one of claims 3-6 in the manufacture of a kit for detecting a sickle cell anemia gene mutation site.

8. Use of the primer set of claim 1 or 2 or the reagent set of any one of claims 3-6 in the manufacture of a kit for detecting sickle cell anemia.

9. Use of the primer set of claim 1 or 2 or the reagent set of any one of claims 3-6 in the manufacture of a kit for determining a sickle cell anemia genotype.

10. A kit characterized in that, Comprising: the primer set of claim 1 or 2 or the reagent set of any one of claims 3-6.

11. A method for detecting a mutation of a sickle cell anemia-related gene, characterized by, Comprising: The reagent set of any one of claims 3-6 or the kit of claim 10 is used for qPCR reaction of a sample to be tested, wherein the probes in the reagent set or the kit independently carry different fluorescent groups, respectively; the first probe carries a first fluorescent group, the second probe carries a second fluorescent group, and the third probe carries a third fluorescent group. The mutation type of the sickle cell anemia related gene is determined based on the Tm value difference of the first fluorescent group, the second fluorescent group, and the third fluorescent group detection channel.

12. The method of claim 11, wherein, The qPCR reaction is performed in a qPCR reaction system, wherein the concentration of the upstream primer is 0.1-2 μM. Optionally, in the qPCR reaction system, the concentration of the downstream primer is 1-10 μM. Optionally, in the qPCR reaction system, the concentration of the probe is 0.1-2 μM. Optionally, in the qPCR reaction system, the concentration of the DNA polymerase is 2-3 U. Optionally, in the qPCR reaction system, the concentration of the dNTPs is 0.1-1 mM.

13. The method of claim 12, wherein, In the qPCR reaction system, the first fluorescent group, the second fluorescent group, and the third fluorescent group are different. Optionally, the first fluorescent group, the second fluorescent group, and the third fluorescent group are independently selected from at least one of FAM, HEX, ROX, CY5, Atto 425, TET, JOE, VIC, R6G, Yakima Yellow, Quasar570, Quasar670, Cy3, NED, Cy5.5, Cy7, Texas Red, Atto 590, IR Dye 650, and IR Dye 750.

14. The method of claim 11, wherein, When the Tm value of the first fluorescent group detection channel is 59-63, it is an indication that the sample to be tested does not contain a mutation of a sickle cell anemia related gene; Optionally, when the Tm value of the first fluorescent group detection channel is 52-56, it is an indication that the sample to be tested contains HbS mutation; Optionally, when the Tm value of the first fluorescent group detection channel is 45-49, it is an indication that the sample to be tested contains HbC mutation; Optionally, when the Tm value of the second fluorescent group detection channel is 55-59, it is an indication that the sample to be tested does not contain a mutation of a sickle cell anemia related gene; Optionally, when the Tm value of the second fluorescent group detection channel is 40-44, it is an indication that the sample to be tested contains HbD mutation; Optionally, when the Tm value of the second fluorescent group detection channel is 46-50, it is an indication that the sample to be tested contains HbO mutation; Optionally, when the Tm value of the third fluorescent group detection channel is 68-72, it is an indication that the sample to be tested does not contain a mutation of a sickle cell anemia related gene; Optionally, when the Tm value of the third fluorescent group detection channel is 61-65, it is an indication that the sample to be tested contains HbE mutation.

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