Primer probe composition, kit and method for noninvasive prenatal detection of Southeast Asia deletion (--SEA) type alpha-thalassemia and application of primer probe composition, kit and method

Abstract

The invention relates to a primer probe composition, a kit and a method for noninvasive prenatal detection of Southeast Asia deletion (--SEA) type alpha-thalassemia and application of the primer probe composition, the kit and the method. According to the application, a strategy for non-invasive prenatal diagnosis of SEA type alpha-thalassemia based on micro-droplet digital gap-PCR (dd gap-PCR) is created for the first time, the operation steps are simple, the sensitivity is high, the specificity is strong, and the stability is good; on the basis of low cost, noninvasive prenatal diagnosis can be carried out on all families possibly having the SEA type alpha-thalassemia pregnancy risk only by 1-2 mL of plasma, safety and effectiveness are achieved, the defect of invasive injury caused by invasive puncture is overcome, and physiological pain and economic burden of pregnant women are relieved. Repeated tests show that the detection strategy is good in stability, high in sensitivity and high in specificity, and is expected to be used for noninvasive prenatal diagnosis of alpha-thalassemia and other single-gene hereditary diseases.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a primer-probe composition, a kit, a method and applications thereof for non-invasive prenatal detection of Southeast Asian deletion (--SEA) type α-thalassemia. Background Art

[0002] α-thalassemia is caused by the deletion or point mutation of the human α-globin gene (HBA1 and HBA2) located at the end of chromosome 16, 16p13.3, which causes the synthesis of the corresponding globin chain to be inhibited to varying degrees or the function to be lost. Clinically, most common α-thalassemia is caused by large-segment deletion of the globin gene, and a few are point mutations. Among them, the large-segment deletion is mainly caused by the Southeast Asian deletion (-- SEA ) type has the highest incidence rate. When both spouses are -- SEA If the person is a carrier of the gene, there is a 1 / 4 chance that their offspring will have Hb Bart's fetal hydrops syndrome (-- SEA / -- SEA ), the affected fetus has all four α-globin genes missing and cannot synthesize α-globin peptide chains. Fetuses with Hb Bart's hydrops fetalis syndrome may have edema, hepatosplenomegaly, pleural effusion, etc. Most of them die in utero during the second and third trimesters or within a few hours after delivery. A very small number of them may survive to birth, but their life span is extremely short. The mother may develop complications such as hypertension, preeclampsia, polyhydramnios, and bleeding, which brings great psychological burden and economic pressure to the patient and the family.

[0003] At present, there is no effective treatment for severe α-thalassemia (Hb Bart's fetal edema syndrome) at home and abroad. Prenatal diagnosis through the commonly used method for diagnosis of deletional α-thalassemia - gap-PCR technology and termination of pregnancy are the preferred management measures at home and abroad. Existing prenatal diagnosis methods are mainly performed through invasive operations such as amniocentesis, chorionic villus sampling, and umbilical vein puncture. SEA Prenatal diagnosis of α-thalassemia is difficult to achieve, but these procedures can easily lead to intrauterine infection, miscarriage, or even fetal death. Therefore, it is crucial to find a safe and effective non-invasive prenatal diagnosis (NIPD) method.

[0004] Digital Droplet PCR (ddPCR) technology is the third generation of PCR detection technology that has developed rapidly after the first generation of PCR and quantitative real-time PCR (Q-PCR). The main principle of ddPCR is: before the PCR amplification of nucleic acid molecules, droplet processing is performed to produce tens of thousands of "oil-in-water" droplets of similar volume and uniform size. Each droplet contains no or one or more target molecules. Then, the fluorescence signal of a specific probe is used to detect each droplet after PCR amplification. The droplets with fluorescent signals are positive droplets containing target DNA molecules, and the negative droplets are negative droplets. The number of target molecules in the sample can be calculated by the number of positive and negative fluorescent signal droplets detected using the Poisson distribution principle. Compared with traditional PCR technology, dd PCR technology can achieve absolute quantification of target DNA molecules with higher accuracy and sensitivity, providing more accurate detection of low-concentration cff DNA in maternal plasma, and has broad application prospects in NIPD. Researchers only need to absolutely quantify the trace fetal DNA contained in the cfDNA of pregnant women's plasma to rule out whether the fetus carries a paternal deletion mutation. dd PCR technology has been proven to be applicable to NIPD of various single genetic diseases, but most of them are achieved by detecting paternal mutations in maternal plasma, and are not suitable for NIPD where parents carry homologous mutations or maternal mutations.

[0005] So far, clinical use for -- SEA The technologies for prenatal diagnosis of type α-thalassemia are all based on invasive puncture. There is no relevant patent for the use of microdroplet digital gap-PCR (dd gap-PCR) to detect Southeast Asian deletion type α-thalassemia, and there is no non-invasive prenatal diagnosis technology or product with high diagnostic efficiency.

[0006] In summary, there is an urgent need for a non-invasive prenatal diagnostic technology or product with high diagnostic efficiency and applicable to all Southeast Asian deletional α-thalassemia NIPDs that conform to Mendelian genetic variations, so as to provide technical support for the early, safe, and rapid non-invasive prenatal diagnosis of thalassemia, as well as for the non-invasive prenatal diagnosis of single gene diseases. Summary of the invention

[0007] In view of the shortcomings of the prior art mentioned above, the present invention provides a primer probe composition, a kit, a method and its application for non-invasive prenatal detection of Southeast Asian deletion (--SEA) type α-thalassemia, so as to achieve prenatal diagnosis of Southeast Asian deletion type α-thalassemia applicable to paternal variation, maternal variation and parent-carried homologous variation, thereby providing technical support for early, safe and rapid non-invasive prenatal diagnosis of thalassemia, and also providing technical support for non-invasive prenatal diagnosis of monogenic diseases.

[0008] To achieve the above objectives, the scheme of this application is as follows:

[0009] In the first aspect, the present invention provides a primer-probe composition for detecting Southeast Asian deletion type α-thalassemia, characterized in that it comprises a first primer set, a first probe, a second primer set and a second probe, the first primer set comprises a first upstream primer and a first downstream primer, the second primer set comprises a second upstream primer and a second downstream primer, the nucleotide sequence of the first upstream primer is shown in SEQ ID NO.1, the nucleotide sequence of the first downstream primer is shown in SEQ ID NO.2, the nucleotide sequence of the first probe is shown in SEQ ID NO.3, the nucleotide sequence of the second upstream primer is shown in SEQ ID NO.4, the nucleotide sequence of the second downstream primer is shown in SEQ ID NO.5, the nucleotide sequence of the second probe is shown in SEQ ID NO.6, the first probe is modified with a first fluorescent reporter group and a first fluorescent quencher group, and the second probe is modified with a second fluorescent reporter group and a second fluorescent quencher group.

[0010] Optionally, the first primer set can specifically bind to a normal α-globin gene, and the first probe can specifically recognize the normal α-globin gene.

[0011] Optionally, the second primer set can specifically bind to the Southeast Asian deletion break site sequence, and the second probe can specifically recognize the Southeast Asian deletion break site sequence.

[0012] Optionally, the first fluorescent reporter group is selected from at least one of 6-carboxyfluorescein (FAM), phosphoramidite (VIC), (hexachlorofluorescein) active ester (HEX), sulfocyanine 5 (CY5) and maleimide (ROX), preferably HEX; the first fluorescent quenching group is selected from at least one of minor groove binder (MGB), black hole quencher (BHQ), phosphoamide ester (Eclipse) and 5-tetramethylrhodamine-succinimidyl ester (TAMRA), preferably BHQ; the second fluorescent reporter group is selected from at least one of 6-carboxyfluorescein (FAM), phosphoramidite (VIC), (hexachlorofluorescein) active ester (HEX), sulfocyanine 5 (CY5) and maleimide (ROX), preferably FAM; the second fluorescent quenching group is selected from at least one of minor groove binder (MGB), black hole quencher (BHQ), phosphoamide ester (Eclipse) and 5-tetramethylrhodamine-succinimidyl ester (TAMRA), preferably BHQ.

[0013] In this application, BHQ includes but is not limited to: BHQ1 and other substances.

[0014] In a second aspect, the present application also provides use of the primer-probe combination described above in the preparation of a reagent for detecting Southeast Asian deletion type α-thalassemia.

[0015] In a third aspect, the present application also provides a kit for detecting Southeast Asian deletion α-thalassemia, comprising the primer-probe combination as described above.

[0016] Optionally, in the kit, the molar ratio of the first upstream primer to the first probe is 450-1350:200-300, the molar ratio of the first downstream primer to the first probe is 450-1350:200-300, the molar ratio of the second upstream primer to the second probe is 450-1350:200-300, and the molar ratio of the second downstream primer to the second probe is 450-1350:200-300.

[0017] Optionally, the kit for detecting Southeast Asian deletion α-thalassemia further includes the following components: DNA polymerase, dNTP.

[0018] In a fourth aspect, the present application also provides a method for detecting Southeast Asian deletion α-thalassemia for the purpose of non-disease diagnosis and / or treatment, comprising the following steps:

[0019] The primer-probe combination as described above, or the kit as described above, is used to perform a polymerase chain reaction amplification test across the break site on the template DNA of the sample to be tested to determine whether Southeast Asian deletion type α-thalassemia exists.

[0020] Optionally, the amplification conditions are: first activation treatment at 92-100°C for 2-4 min, preferably first activation treatment at 95°C for 3 min; then denaturation treatment at 95-100°C for 20-40 s and then annealing and extension at 58-64°C for 30-120 s for 40 cycles, preferably denaturation treatment at 98°C for 30 s and then annealing and extension at 62°C for 1 min for 40 cycles; then treatment at 95-100°C for 8-12 min, preferably further treatment at 98°C for 10 min.

[0021] Beneficial effects of the present invention:

[0022] This application designs wild-type and Southeast Asian deletion-specific primers and fluorescent probes based on the α-globin gene sequence, and develops a rapid and highly sensitive -- SEA Type α-thalassemia microdroplet digital gap-PCR prenatal diagnosis kit, the detection kit can be used for - SEA Compared with other commercial kits, the prenatal diagnosis of α-thalassemia type 2 is simpler to operate and can be analyzed rapidly. It can detect Southeast Asian deletion type and wild-type DNA at concentrations as low as 0.415 copies / μL and 1.05 copies / μL, respectively, with high sensitivity.

[0023] The detection kit of this application detects in paternal deletion NIPD-- SEA The copy number is strongly correlated with the fetal DNA concentration (R 2 >0.99) and feasibility, detecting as few as 2.6% of fetuses -- SEA Compared with Q-PCR, it has better sensitivity and reproducibility.

[0024] The detection kit of this application combines the relative mutation dosage (RMD) strategy in maternal deletion and both parents carry-- SEA The feasibility of NIPD was good, and the fetal concentration had a good correlation with the RMD value (R 2 >0.98), which has better sensitivity and reproducibility than Q-PCR.

[0025] The detection kit of this application can be used in all possible-- SEA It has certain feasibility, high sensitivity and specificity, and better stability than Q-PCR in NIPD for the risk of fetal pregnancy with type α-thalassemia.

[0026] The detection kit of this application is rapid, safe, sensitive and specific, and can be used for-- SEA Noninvasive prenatal clinical diagnosis of α-thalassemia type 2.

[0027] This application pioneered a method based on dd gap-PCR for the first time. SEA The strategy for non-invasive prenatal diagnosis of type α-thalassemia has simple operation steps, high sensitivity, strong specificity and good stability. On the basis of low cost, only 1-2mL of plasma is needed to detect all possible SEA Non-invasive prenatal diagnosis for families at risk of type α-thalassemia is safe and effective. It not only overcomes the disadvantages of invasive puncture and reduces the physiological pain and economic burden of pregnant women.

[0028] Repeated tests have shown that the detection strategy of the present application has good stability, high sensitivity and strong specificity, and is expected to be used for non-invasive prenatal diagnosis of α-thalassemia and other monogenic genetic diseases. Currently, most of these monogenic genetic diseases rely on paternal mutations, and prenatal diagnosis of the same deletion or mutation carried by the fetus and the mother has not yet been possible. It is expected to be an important technical means to prevent the birth of children with monogenic diseases in addition to puncture.

[0029] The detection strategy of the present application is a non-invasive prenatal diagnostic method that can quickly determine the fetal genotype. It is harmless to pregnant women and fetuses, has high safety, has a high diagnostic accuracy rate and high sensitivity in determining whether the fetus carries a mutation or deletion, and is of great significance for the clinical development of non-invasive prenatal diagnosis of thalassemia and other monogenic diseases. The development and application of this technology will greatly improve the efficiency of screening for monogenic diseases, and its promotion in areas with a high carrier rate of α-thalassemia and nationwide has far-reaching clinical medical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a schematic diagram of the present invention, Pregnant women represents pregnant women, Plasma represents plasma, isolation represents isolation, Primers represents primers, Probes represents probes, oil represents oil, Signal represents signal, Total copy number of represents total copy number, preparation of dd gap-PCR reaction mixture represents preparation of dd gap-PCR reaction premix, Droplet generation represents droplet generation, Amplification represents amplification, Fluorescence Detection represents fluorescence detection, and represents and, Data Analysis represents data analysis, Relative mutation dosage determination represents relative mutation dosage determination, and exclusion diagnostic represents exclusion diagnosis;

[0031] Figure 2 This is a graph of the test results during the primer and probe concentration screening process. Amplitude represents the fluorescence intensity value of each droplet, Event number represents the number of droplets in the sample, and Events represents the number of positive droplets;

[0032] Figure 3 This is a graph showing the test results during the annealing temperature screening process;

[0033] Figure 4 This is a graph of the detection results during the annealing time screening process;

[0034] Figure 5 This is a graph of specificity test results;

[0035] Figure 6 This is a graph showing the results of linearity and sensitivity tests.

[0036] Figure 7 For the purpose of accurate verification of test results, Bart's represents Hb Bart's fetal hydrops syndrome (-- SEA / -- SEA ), Heterozygous means heterozygous -- SEA α-thalassemia (-- SEA / αα), Wild type means wild type (αα / αα); Specificity means specificity, Sensitivity means sensitivity, the same below;

[0037] Figure 8 To simulate that the mother is α wild type and the father is -- SEA Carriers, dd gap-PCR for paternal lineage -- SEA The test results of the feasibility of exclusionary non-invasive prenatal diagnosis are shown in Figure 1. Figure 8 A is the dd gap-PCR droplet distribution and linear analysis results. Figure 8 B is the Q gap-PCR detection ct value result and linear analysis result diagram;

[0038] Fig. 9 To simulate motherhood-- SEA For carriers of α-thalassemia type 2 and fetuses with severe α-thalassemia, dd gap-PCR combined with relative mutation strategy was used to perform SEA The feasibility test results of non-invasive prenatal differential diagnosis of α-thalassemia type 2 Fig. 9 A is the dd gap-PCR droplet distribution and linear analysis results. Fig. 9 B is the Q gap-PCR detection ct value result and linear analysis result diagram;

[0039] Fig.10 To simulate motherhood-- SEA The fetus is wild type. The fetal non-invasive prenatal differential diagnosis of SEA type α-thalassemia was performed using dd gap-PCR combined with relative mutation strategy. Fig.10 A is the dd gap-PCR droplet distribution and linear analysis results. Fig.10 B is the Q gap-PCR detection ct value result and linear analysis result diagram;

[0040] Fig.11 For 22 paternal lines -- SEA The results of non-invasive prenatal exclusionary diagnostic test of clinical pregnant women's plasma samples are shown in Figure 2. Fig.11 A is a binary classification matrix comparing the results of dd gap-PCR non-invasive prenatal puncture and clinical invasive puncture. Fig.11 B is the ROC curve of ddgap-PCR test results; Predicted means measured, Actual means actual, the same below;

[0041] Fig.12 For 40 maternal lines -- SEA The results of non-invasive prenatal diagnosis of clinical maternal plasma samples. Fig.12 A is the distribution of RMD values ​​obtained by ddgap-PCR detection, Fig.12 B is the heterozygous type detected by dd gap-PCR -- SEA Mean RMD values ​​of α-thalassemia and wild-type fetuses, Fig.12 C is the binary classification matrix comparing the results of dd gap-PCR non-invasive prenatal puncture and clinical invasive puncture. Fig.12 D is the ROC curve of dd gap-PCR detection results;

[0042] Fig.13 For 45 cases, both parents are -- SEA Non-invasive prenatal diagnosis test results of clinical pregnant women's plasma samples for α-thalassemia carriers. Fig.13 A is the RMD value distribution obtained by dd gap-PCR detection, Fig.13 B is severe α-thalassemia, heterozygous type detected by dd gap-PCR SEA Mean RMD values ​​of α-thalassemia and wild-type fetuses, Fig.13 C is the ROC curve of the differential diagnosis of severe α-thalassemia by ddgap-PCR. Fig.13 D is dd gap-PCR differential diagnosis of whether the fetus is carrying -- SEA ROC curve plot of gene results. DETAILED DESCRIPTION

[0043] The following will describe the embodiments of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, not for limiting the scope of protection of the present invention.

[0044] It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and thus the drawings only show components related to the present invention rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed arbitrarily, and the component layout may also be more complicated.

[0045] In the following description, numerous details are discussed to provide a more thorough explanation of embodiments of the present invention; however, it is apparent to one skilled in the art that embodiments of the present invention may be practiced without these specific details.

[0046] (I) Establishment and optimization of dd gap-PCR detection method

[0047] 1 Materials and methods

[0048] 1.1 Sample source and extraction of plasma free DNA

[0049] 1.1.1 Sample Source

[0050] All human-related peripheral blood samples and puncture specimens used in this application were obtained from the First Affiliated Hospital of Guangxi Medical University;

[0051] 1.1.2 Collection of plasma samples and DNA extraction

[0052] Collect 2-3 mL of EDTA-K2 anticoagulated whole blood sample and separate it within 24 hours after sampling. Centrifuge it in a low-temperature centrifuge at 4°C and 1600×g for 10 min, and carefully transfer the upper plasma to a new clean 1.5 mL centrifuge tube; then, centrifuge it in a low-temperature centrifuge at 4°C and 16000×g for 10 min, carefully transfer the upper plasma to a clean 1.5 mL centrifuge tube, and store it at -80°C for later use;

[0053] This application adopts The plasma free DNA extraction kit is used to extract plasma free DNA. The specific extraction process is as follows

[0054] 1) Prepare buffer ACB, ACW1 and ACW2 according to the instructions;

[0055] 2) Transfer the separated plasma to a clean 15 mL centrifuge tube, add 100 μL proteinase K, 1.8 mL buffer ACL containing 1.0 μg carrier RNA (buffer and RNA need to be prepared before each experiment), cover tightly, and vortex to mix for 15 seconds;

[0056] 3) Briefly centrifuge and incubate in a 60°C constant temperature water bath for 35 min, mixing by inverting for 10 s every 10 min to ensure complete lysis;

[0057] 4) After incubation, centrifuge briefly in a centrifuge, unscrew the lid, and add 1.8 mL of buffer ACB to obtain a mixture of lysate-buffer ACB;

[0058] 5) Incubate the mixture of lysate and buffer ACB on ice for 5 minutes to obtain an incubated mixture of lysate and buffer ACB;

[0059] 6) Insert the QIAamp Mini column into the connector on the vacuum pump and insert a 20 mL extender into the opened QIAamp Mini column, making sure the extender is firmly inserted into the QIAamp Mini column to avoid sample leakage;

[0060] 7) Carefully add the incubated lysate-buffer ACB mixture to the extension tube of the QIAamp Mini column, turn on the vacuum pump, and when the liquid has completely passed through the column, turn off the vacuum pump and release the pressure to 0 mbar, then slowly remove and discard the extension tube;

[0061] 8) Add 600 μL of buffer ACW1 to the QIAamp Mini column, keep the cover of the adsorption column open, and turn on the vacuum pump. When all the buffer ACW1 is completely pumped out through the QIAamp Mini column, turn off the vacuum pump and release the pressure to 0 mbar;

[0062] 9) Add 750 μL of buffer ACW2 to the QIAamp Mini column; keep the cover of the adsorption column open and turn on the vacuum pump. When all the buffer ACW2 has been pumped out through the QIAamp Mini column, turn off the vacuum pump and release the pressure to 0 mbar;

[0063] 10) Add 750 μL of anhydrous ethanol to the QIAamp Mini column, keep the cover of the adsorption column open, and turn on the vacuum pump. When all the anhydrous ethanol has been pumped out through the column, turn off the vacuum pump and release the pressure to 0 mbar;

[0064] 11) Close the cap of the QIAamp Mini column and slowly and carefully remove it from the vacuum pump and discard the vacuum connector. Place the QIAamp Mini column in a new clean 2 mL collection tube and centrifuge at 20,000 × g for 3 min.

[0065] 12) Remove the QIAamp mini column and place it in a new clean 2 mL collection tube. Open the lid and place the collection tube in a 56°C metal bath for 10 min to allow the membrane to dry completely.

[0066] 13) Discard the 2 mL collection tube in step 12, place the QIAamp mini column in a clean 1.5 mL centrifuge tube, add 25 μL of buffer AVE to the center of the column membrane, cover the lid, and incubate at room temperature for 3 min;

[0067] 14) centrifuging the product obtained in step (13) at 20,000×g for 1 min in a high-speed centrifuge to elute the nucleic acid, and storing at -20°C for later use;

[0068] 15) Add the eluted nucleic acid back to the center of the QIAamp mini column membrane, cover the column, and incubate at room temperature for 3 minutes;

[0069] 16) Repeat step 14) to elute the nucleic acid and store it at -20°C for future use.

[0070] 1.2 Primers and probes

[0071] This application designs specific wild-type α primers and probes based on the α-globin gene sequence and designs specific primers for both ends of the Southeast Asian deletion breakpoint. SEA Type primers and probes, all primers and probes were synthesized by biological companies, the sequences of primers and probes are shown in Table 1:

[0072] Table 1 Primer and probe sequences

[0073]

[0074] like Figure 1 The principles of this application are shown as follows:

[0075] The plasma free DNA in the peripheral blood of pregnant women contains free fetal DNA. The present invention uses the couple or one of them to carry - SEA of-- SEAThe α-thalassemia family was selected as the research object, and the free DNA in the plasma of pregnant women was separated and extracted; gap-PCR is often used to detect large fragment deletions. The present invention designs two pairs of specific primers and probes, the first upstream primer and the first downstream primer can specifically bind to the α-globin gene, and the first probe can specifically identify the α-globin gene; the second upstream primer and the second downstream primer can specifically bind to the Southeast Asian deletion of the large fragment deletion of the α-globin gene (-- SEA ) break site sequence, the second probe can specifically identify the Southeast Asian deletion break site sequence; after dd gap-PCR amplification, the droplet containing α wild-type DNA can produce a green fluorescent signal, containing-- SEA The positive droplets of type DNA can produce blue fluorescent signals, and the negative droplets without amplification do not produce fluorescent signals. The copy numbers (copies / uL) of α wild-type and --SEA type DNA molecules in the plasma free DNA of pregnant women can be calculated by bringing the percentage of positive droplets into the algorithm based on Poisson distribution.

[0076] According to the inheritance law of single gene diseases, when the mother is α wild type and the father is -- SEA When a carrier of α-thalassemia is diagnosed, the mother's plasma free DNA can be tested to see if it contains -- SEA Type α-thalassemia DNA, fetal- SEA Non-invasive prenatal diagnosis of α-thalassemia type 2; when the mother is - SEA Type α-thalassemia carriers, when the father is wild-type or both spouses are - SEA When the carriers of α-thalassemia type 2 are combined with the relative mutation dosage analysis strategy, SEA Analysis of the relative allele ratios of α and α can be used to analyze the fetus- SEA Non-invasive prenatal diagnosis of α-thalassemia, that is, when the fetus and the mother have the same genotype and are both heterozygous, the free DNA in the pregnant woman's plasma SEA The type gene is balanced with the α wild allele, and the ratio is about 1; if the fetal genotype is pure and missing type (severe α-thalassemia), its -- SEA The ratio of the wild-type gene to the α wild-type allele will increase; if the fetal genotype is wild-type, its -- SEA The ratio of the type gene to the α wild-type allele will decrease.

[0077] 1.3 Establishment of droplet number digital gap-PCR detection method

[0078] 1.3.1 Droplet digital gap-PCR amplification reaction system

[0079] The concentration and ratio of primers and probes in the dd gap-PCR reaction system will have a certain impact on the experimental results. If the amount is too small, it will lead to incomplete amplification, and if the amount is too large, it may lead to primer dimers.

[0080] The dd gap-PCR reaction system of the present application includes a premix, a first upstream primer, a first downstream primer, a first probe, a second upstream primer, a second downstream primer, a second probe and DNA, and is prepared according to the volume of the dd gap-PCR reaction system being 20 μL, and supplemented to 20 μL with dd H2O, and the primer concentration (herein, the final concentration in the reaction system) and the probe concentration (herein, the final concentration in the reaction system) are screened, and the specific steps are as follows:

[0081] 10 μL of the prepared commercially available premix corresponding to the droplet digital PCR instrument, the first upstream primer, the first downstream primer, the first probe, the second upstream primer, the second downstream primer and the second probe were added to the premix, and the concentrations of the first upstream primer, the first downstream primer, the first probe, the second upstream primer, the second downstream primer and the second probe were all 10 μM (all using dd H2O as solvent). First, add the first probe and the second probe to the reaction system, and add 0.9 μL of the first upstream primer, 0.9 μL of the first downstream primer, 0.9 μL of the second upstream primer and 0.9 μL of the second downstream primer respectively, so that the final concentration of each probe in the reaction system is 250 nM, and the final concentration of each primer in the reaction system is 450 nM. Correspondingly, add 1.8 μL or 2.7 μL of the first upstream primer, the first downstream primer, the second upstream primer and the second downstream primer, so that the final concentration of each primer in the reaction system is 900 nM or 1350 nM, prepare several reaction systems, and perform amplification (the specific conditions of the amplification reaction are as follows), and perform screening according to the screening conditions (the specific screening conditions are as follows) to obtain the optimal primer concentration (here refers to the final concentration in the reaction system). Then, based on the results of the optimal primer concentration obtained in the early stage, primers with the optimal final concentration were added to the reaction system, and 0.4 μL of the first probe and 0.4 μL of the second probe were added respectively, so that the final concentration of each probe in the reaction system was 200 nM. Correspondingly, 0.5 μL or 0.6 μL of the first probe and the second probe were added, so that the final concentration of each probe in the reaction system was 250 nM or 300 nM. Several reaction systems were prepared, and amplification was carried out (the specific conditions of the amplification reaction are as follows), and screening was carried out according to the screening conditions (the specific screening conditions are as follows) to obtain the optimal probe concentration.

[0082] The specific process of amplification is as follows: 1 μL gDNA is added to each reaction system, and dd H2O is used to make up to 20 μL, mixed, and then transferred to the sample wells of the droplet generation card, and then 70 μL of droplet generation oil is added to the droplet wells, and the raw card is placed in the droplet generator to generate droplets, and then the generated droplets are transferred to the 96 PCR reaction plate and sealed with aluminum foil, and the enzyme is activated (i.e., pretreated) at 95°C for 3 min on a common PCR instrument, and denatured at 98°C for 30 s and then annealed at 60°C for 1 min as one cycle, and the cycle is repeated 40 times, and then the enzyme is inactivated at 98°C for 10 min, and then stored at 10°C for standby use, and the PCR amplification results are analyzed on the droplet analyzer;

[0083] The fluorescence signals of FAM and HEX were detected, and the software automatically counted the proportion of positive droplets in the PCR reaction, and the starting template in the reaction system was quantified, and then the primer concentration (here refers to the final concentration in the reaction system) and the probe concentration (here refers to the final concentration in the reaction system) were screened. The specific screening conditions were: ① the positive droplets and the negative droplets were clearly separated; ② based on the obvious separation of positive droplets and droplets, it had higher amplification efficiency and lower experimental costs. The results are shown in the figure. Figure 2 shown.

[0084] like Figure 2 As shown, when the basic amplification requirements are met, the amount of primers has no significant effect on the experimental results; appropriately increasing the concentration of the probe in the reaction system can increase the fluorescence intensity of the droplets, so that the positive droplets and negative droplets can achieve a better separation effect, but too high a proportion of probes will lead to an increase in raindrops, affecting the clear separation of positive and negative droplet clusters. In order to achieve a better separation effect of positive and negative droplet clusters at a low experimental cost, the preferred concentrations of the first upstream primer, the first downstream primer, the second upstream primer, and the second downstream primer (here refers to the final concentration in the reaction system) finally determined by this application are all 900nM, and the preferred concentrations of the first probe and the second probe (here refers to the final concentration in the reaction system) are both 250nM.

[0085] The preferred reaction system of this application is shown in Table 2.

[0086] Table 2 dd gap-PCR optimal amplification reaction system

[0087] Components volume <![CDATA[2×dd PCR TM Supermix for Probes (i.e. premix)]]> 10μL Forward Primer (i.e. first upstream primer or second upstream primer) (10 μM) 1.8μL Reverse Primer (i.e. first downstream primer or second downstream primer) (10 μM) 1.8μL Probe (i.e., first probe or second probe) (10 μM) 1μL <![CDATA[dd H2O]]> Make up to 20 μL

[0088] According to the preferred concentrations of the first upstream primer, the first downstream primer, the second upstream primer and the second downstream primer (herein, the final concentration in the reaction system) being 900 nM, and the preferred concentrations of the first probe and the second probe (herein, the final concentration in the reaction system) being 250 nM, 10 μL of the premix, the first upstream primer, the first downstream primer, the first probe, the second upstream primer, the second downstream primer, the second probe and dd H2O are prepared into several reaction systems, and then the annealing temperature is screened, specifically: except that each reaction system is annealed at 58°C, 59°C, 60°C, 61°C, 62°C and 63°C, respectively, amplification is performed according to the above conditions, and the PCR amplification results are analyzed on a droplet analyzer;

[0089] Detect the fluorescence signals of FAM and HEX, and the software automatically counts the proportion of positive droplets in the PCR reaction, and quantifies the starting template in the reaction system, and then screens the annealing temperature. The screening conditions are the same as above. The results are as follows Figure 3 shown.

[0090] like Figure 3 As shown, as the annealing temperature decreases, the amplification efficiency of the two pairs of primers gradually increases, but non-specific amplification is prone to occur when the temperature is too low. In order to achieve the best amplification effect, the preferred annealing temperature selected in this application is 62°C.

[0091] The length of annealing time will affect the result of PCR. If the annealing time is too long, the two deformed DNA chains will be reconnected. If the annealing time is too short, the primer may not be completely matched with the DNA chain, resulting in extension failure or increased mismatch rate. The present application then screens the annealing time. The specific screening conditions are the same as above. According to the preferred concentrations of the first upstream primer, the first downstream primer, the second upstream primer and the second downstream primer (herein, the final concentration in the reaction system) are all 900nM, and the preferred concentrations of the first probe and the second probe (herein, the final concentration in the reaction system) are both 250nM. 10μL of the premix, the first upstream primer, the first downstream primer, the first probe, the second upstream primer, the second downstream primer, the second probe and ddH2O are prepared into several reaction systems, and then the annealing time is screened. Specifically, except that each reaction system is annealed at 62°C for 30s, 1min and 2min respectively, amplification is performed according to the above conditions, and the PCR amplification results are analyzed on a droplet analyzer;

[0092] Detect the fluorescence signals of FAM and HEX, and the software automatically counts the proportion of positive droplets in the PCR reaction, and quantifies the starting template in the reaction system, and then screens the annealing time. The screening conditions are the same as above. The results are as follows Figure 4 shown.

[0093] like Figure 4As shown, as the annealing time increases, the amplification efficiency of dd gap-PCR increases, and the separation of positive droplets and negative droplets becomes clearer and more obvious, but when the time is too long, the amplification decreases instead of increases. After comprehensive consideration, the present application selects 1 min as the preferred annealing time.

[0094] The preferred amplification reaction conditions of the present application are shown in Table 3.

[0095] Table 3 Optimal amplification reaction conditions for dd gap-PCR

[0096]

[0097] (II) Droplet digital gap-PCR detection specificity test

[0098] This application is respectively in the homozygous deletion type (-- SEA / -- SEA ), heterozygous (-- SEA The specificity of dd gap-PCR detection technology was verified in DNA of wild type (αα / αα), wild type (αα / αα) and negative control (sterile enzyme-free water). The specific steps are as follows:

[0099] Several reaction systems were prepared according to the optimal reaction system, and 1 μL of three different genotypes (i.e.: SEA / -- SEA 、-- SEA / αα, αα / αα), add 1μL of dd H2O to the negative control well as a control, then use dd H2O to make up each reaction system to 20μL, mix well, and transfer to the sample wells of the droplet generation card respectively, then add 70μL of droplet generation oil to the droplet well, put the raw card into the droplet generator to generate droplets, and then transfer the generated droplets to a 96PCR reaction plate and seal it with aluminum foil. On a common PCR instrument, activate the enzyme (i.e., pretreatment) at 95°C for 3min, denature at 98°C for 30s and then anneal at 62°C for 1min as one cycle, cycle 40 times, and then inactivate the enzyme at 98°C for 10min for amplification, and then store at 10°C for standby use. The PCR amplification results are analyzed on a droplet analyzer. The results are as follows Figure 5 shown.

[0100] like Figure 5 As shown, -- SEA / -- SEA No wild-type α-specific positive droplets were found in αα / αα. SEA Type-specific positive droplets, SEA / αα can detect at the same time-- SEAThe positive droplets of the wild type and the α wild type were detected, and the number of positive droplets of the two genotypes was basically equal, and no positive droplets appeared in the negative control. This result shows that the detection strategy of the present application (ie, the ddgap-PCR method) has good specificity.

[0101] (III) Linearity and sensitivity test of droplet digital gap-PCR detection

[0102] The genomic DNA (i.e., plasma free DNA) was extracted using method (a);

[0103] The genomic DNA was broken into fragments of about 300 bp in length using a non-contact ultrasonic disruptor, and the droplet digital gap-PCR experiment was performed to simulate maternal plasma free DNA. The specific steps are as follows:

[0104] A non-contact ultrasonic disruptor was used to fragment 50 μL of high-purity, high-quality gDNA with a concentration of 20 ng / μL. The fragments were run at a frequency of 90w for 20 min to obtain fragmented genomic DNA with a length of about 300 bp. Several reaction systems were prepared according to the optimal reaction system.

[0105] The genomic DNA sample was quantitatively diluted to about 10 ng / μL and then directly diluted in multiples, with the concentrations being 10 ng / μL, 5 ng / μL, 2.5 ng / μL, 1.25 ng / μL, 0.725 ng / μL, 0.3125 ng / μL and 0 (blank control). Then, 1 μL of genomic DNA sample was added to each reaction system, and amplified according to the optimal amplification reaction conditions in (I). The PCR amplification results were analyzed on a droplet analyzer. The fluorescence signals of FAM and HEX read by the droplet analyzer were detected, and the proportion of PCR-positive droplets was automatically counted by the software, and the target gene in the reaction system (i.e.: - SEA The starting template concentrations of α and αα were quantitatively calculated, and SPSS software was used to perform linear analysis based on the actual measured DNA concentration (copies / μL) of the added sample and the measured number of positive droplets and the theoretical measured DNA concentration of the added sample. The results are shown in Figure 6 shown.

[0106] like Figure 6 As shown, -- SEA The copy number detection of both α-type and α-wild type alleles showed a good linear relationship, and its R 2 0.9947 and 0.9938 respectively, which can detect as low as 0.415 copies / μL - SEA The results show that the detection strategy of the present application (ie, dd gap-PCR method) has excellent sensitivity.

[0107] (IV) Accuracy test of droplet digital gap-PCR detection

[0108] dd gap-PCR was used to analyze 52 couples in which both partners were SEA The DNA samples of high-risk families with α-thalassemia / α-α who have undergone biopsy diagnosis and whose fetal genotypes are known are tested and compared with the detection method of the clinical commercial kit (fluorescence PCR melting curve method). The specific steps are as follows:

[0109] Several reaction systems were prepared according to the optimal reaction system. 1 μL of clinical puncture sample DNA was added to each reaction system. Then, each reaction system was supplemented to 20 μL with dd H2O, mixed, and transferred to the sample wells of the droplet generation card. Then, 70 μL of droplet generation oil was added to the droplet wells. The raw card was placed in the droplet generator to generate droplets. The generated droplets were then transferred to a 96 PCR reaction plate and sealed with aluminum foil. Amplification was performed according to the optimal amplification reaction conditions in (I), and then stored at 10°C for future use. The PCR amplification results were analyzed on a droplet analyzer. The fluorescence signals of FAM and HEX read by the droplet analyzer were detected, and the proportion of positive droplets in the PCR reaction was automatically counted by the software, and the target gene in the reaction system (i.e.: - SEA and αα) are quantitatively calculated by calculating -- SEA and the value of αα copy number to determine the fetus's -- SEA The results of the α-thalassemia genotype test were compared with those of the clinical commercial kit (fluorescence PCR melting curve method). Figure 7 shown.

[0110] like Figure 7 As shown in the figure, the test results of 52 puncture prenatal diagnosis samples from families with high reproductive risk of severe α-thalassemia are completely consistent with the test results of the clinical commercial kit (fluorescence PCR melting curve method). The results show that in clinical puncture prenatal diagnosis samples with confirmed fetal genotypes, the detection strategy of this application (ie, dd gap-PCR method) can reach 100% accuracy, 100% sensitivity, and 100% specificity. Compared with the fluorescence PCR melting curve method, the detection strategy of this application (ie, dd gap-PCR method) takes less time and has simpler data analysis, which helps to improve -- SEA Efficiency of prenatal diagnosis of α-thalassemia type 2.

[0111] (V) Patrilineal SEA Noninvasive prenatal diagnosis of exclusion

[0112] The dd gap-PCR test was performed using clinical plasma sample DNA as a template to simulate maternal plasma free DNA to verify the primer-probe combination of the present application in the paternal lineage. SEA The feasibility of application in non-invasive prenatal diagnosis, the specific steps are:

[0113] To verify that this application is in the paternal line-- SEA The feasibility of application in non-invasive prenatal exclusion diagnosis was investigated by using plasma DNA of clinical samples with wild-type α (αα / αα) to simulate free plasma DNA of wild-type maternal plasma and heterozygous (-- SEA / αα) clinical plasma samples simulated carrying paternal lineage-- SEA fetal free DNA; qubit4.0 was used to detect the concentration of α wild-type and heterozygous plasma free DNA, and diluted to the same concentration. The heterozygous plasma free DNA was added to the α wild-type plasma free DNA to prepare maternal plasma free DNA containing 15%, 12%, 9%, 6%, 3%, and 0% fetal proportion concentrations, respectively. According to the fetal concentration DNA ratio and the measured - SEA The ratio was linearly analyzed, and each concentration was repeated three times. According to the theoretical fetal concentration DNA ratio and the actual measured SEA The ratio was linearly analyzed and compared with the Q gap-PCR test results. Figure 8 shown.

[0114] Depend on Figure 8 It can be seen that the measured-- SEA The ratio showed a good linear relationship with the fetal concentration ratio (R 2 =0.997), dd gap-PCR can detect as low as 2.6% paternal SEA The plasma free DNA had good stability, and the proportions detected in three repeated tests were basically consistent. However, Q gap-PCR failed to detect the proportion of low-concentration fetal DNA, and had poor stability.

[0115] In summary, the detection strategy of this application is in the paternal line-- SEA It has certain feasibility in non-invasive prenatal exclusion diagnosis, and has good sensitivity and stability.

[0116] (VI) NIPD test using dd gap-PCR combined with RMD strategy

[0117] Since the fetus of severe α-thalassemia dies in the womb or a few hours after birth, the present application uses a non-contact ultrasonic crusher (the detailed operation steps are as shown in (III)) to respectively... SEA / -- SEA 、-- SEA / αα, αα / αα genomic DNA was broken into fragments of about 300 bp in length and diluted to the same concentration. SEA / αα-type fragmented genomic DNA was used as a template to simulate heterozygous maternal plasma free DNA, using -- SEA / -- SEA and αα / αα type fragmented genomic DNA respectively simulated severe α-thalassemia and α wild-type fetal free DNA, and performed dd gap-PCR experiments to verify the present invention in maternal- SEA Deletion and both parents carry -- SEA The feasibility of non-invasive prenatal diagnosis, including two possible genetic types ①- SEA / ααmatrix and-- SEA / -- SEA Fetus; ②-- SEA / αα mother and αα / αα fetus, the specific steps are:

[0118] ①Will-- SEA / -- SEA Type DNA added to -- SEA / αα type DNA, respectively prepared to contain 15%, 12%, 9%, 6%, 3%, 0% fetal ratio of maternal plasma free DNA, according to the fetal concentration DNA ratio and the measured - SEA The linear analysis was performed for each concentration and repeated three times. Fig. 9 shown.

[0119] Depend on Fig. 9 It can be seen that as the concentration of fetal DNA gradually increases, the RMD value also increases, with a strong correlation (R 2 >0.98), and the fetal ratio obtained by three repeated tests was relatively consistent, indicating good detection stability. However, the correlation between the fetal concentration and RMD value of Q-PCR technology was weaker than that of dd gap-PCR (R 2 The stability was poor and the differences among the three replicates were large.

[0120] ②Add αα / αα DNA to -- SEA / αα type DNA, respectively prepared to contain 15%, 12%, 9%, 6%, 3%, 0% fetal ratio of maternal plasma free DNA, according to the fetal concentration DNA ratio and the measured - SEA The linear analysis was performed for each concentration and repeated three times. Fig.10 shown.

[0121] Depend on Fig.10 It can be seen that as the concentration of fetal DNA gradually increases, the RMD value also decreases, with a strong correlation (R2 >0.98), and the fetal ratio obtained by three repeated tests was relatively consistent, indicating good detection stability. However, the correlation between the fetal concentration and RMD value of Q-PCR technology was weaker than that of dd gap-PCR (R 2 <0.93), the stability was poor, and the differences among the three replicates were large.

[0122] In summary, the detection strategy of this application is in the maternal line-- SEA Deletion and both parents carry -- SEA The non-invasive prenatal diagnosis has good feasibility, high sensitivity and good stability.

[0123] 7. Clinical application of droplet digital gap-PCR

[0124] The present invention uses dd gap-PCR detection system to detect 107 clinical pregnant women's plasma samples and compares them with clinical puncture results, including 22 paternal- SEA , 40 maternal lines -- SEA and 45 couples where both partners were -- SEA The pedigrees of the carriers were statistically analyzed using SPSS 19.0 software, and ROC curves were drawn. The detection efficiency of the detection strategy of the present invention in clinical plasma samples was verified by calculating the area under curve (AUC) and accuracy, sensitivity, and specificity;

[0125] According to Mendel's law of inheritance, 22 fetuses may inherit from their father -- SEA The maternal plasma samples of the alleles were mainly tested by non-invasive prenatal exclusion diagnosis. Based on the experimental results of the sensitivity evaluation of the previous dd gap-PCR detection method, 0.415 copies / μL was set as the minimum detectable positive copy number, that is, when - SEA When the copy number is >0.415 copies / μL, the fetus can be judged as- SEA Type α-thalassemia carriers. Prepare several reaction systems according to the optimal reaction system in (I), add 7μL of clinical plasma sample DNA to the system, then use dd H2O to make up each reaction system to 20μL, mix well, and transfer them to the sample wells of the droplet generation card respectively, then add 70μL of droplet generation oil to the droplet wells, put the raw card into the droplet generator to generate droplets, and then transfer the generated droplets to the 96PCR reaction plate, seal it with aluminum foil, and amplify it according to the optimal amplification reaction conditions in (I), and analyze the PCR amplification results on the droplet analyzer; detect the fluorescence signals of FAM and HEX read by the droplet analyzer, and the software automatically counts the proportion of PCR reaction positive droplets, and uses Poisson distribution to distribute the - SEAAnd the starting template concentration of αα gene is quantitatively calculated by calculating -- SEA and the value of αα copy number to determine the fetus's -- SEA The α-thalassemia genotype of the type was compared with the invasive puncture results. Fig.11 shown.

[0126] Depend on Fig.11 It can be seen that the detection strategy of this application is effective in 22 cases where the fetus may inherit the paternal lineage-- SEA In the clinical plasma samples of the allele, the fetuses of 19 families were accurately excluded as being -- SEA For carriers of α-thalassemia type 2, exclusion diagnosis can be performed as early as 9 weeks of pregnancy. The results show that the detection strategy of this application is effective in the paternal lineage. SEA of-- SEA In the non-invasive prenatal exclusion diagnosis of type α-thalassemia, the AUC was 0.9375, the accuracy was 86.4%, the sensitivity was 87.5%, and the specificity was 100%, showing good diagnostic efficacy.

[0127] Because in actual testing, -- SEA / α-thalassemia-- SEA The relative alleles of αα and αα are not completely equal, and their ratio is not completely equal to 1, so this application applies to 30 cases - SEA DNA from clinical plasma samples of non-pregnant women with α-thalassemia / α-α was tested to define SEA The threshold range of the relative allele ratio of αα and αα is as follows:

[0128] According to the optimal reaction system in (I), several reaction systems were prepared, 7 μL of clinical plasma sample DNA was added to the system, and then each reaction system was supplemented to 20 μL with dd H2O, mixed, and transferred to the sample wells of the droplet generation card respectively, and then 70 μL of droplet generation oil was added to the droplet wells, and the raw card was placed in the droplet generator to generate droplets, and then the generated droplets were transferred to the 96PCR reaction plate and sealed with aluminum foil, and amplified according to the optimal amplification reaction conditions in (I), and the PCR amplification results were analyzed on the droplet analyzer; the fluorescence signals of FAM and HEX read by the droplet analyzer were detected, and the proportion of PCR reaction positive droplets was automatically counted by the software, and the - in the reaction system was distributed by Poisson distribution. SEA The concentration of the starting template of the αα gene was quantitatively calculated and statistically analyzed using SPSS19.0 software to calculate its - SEA and the mean and standard deviation of the relative missing allele ratio (RMD) of α to obtain its threshold range (i.e. ), the RMD threshold measured in this application is [0.713-0.926], that is, when the RMD value is [0.713-0.926], the fetus is - SEA When the RMD value is less than 0.713, the fetus is wild type; when the RMD value is greater than 0.926, the fetus has severe α-thalassemia.

[0129] For the 40 fetuses that may inherit maternal inheritance -- SEA The maternal plasma samples of the alleles are mainly tested by dd gap-PCR combined with RMD non-invasive prenatal diagnosis method, that is, when the fetus and the mother's -- SEA When the genotype of α-thalassemia is consistent, SEA The relative allele ratio of αα to αα ranges from [0.713-0.926]. When the fetus does not carry -- SEA hour,-- SEA and the αα relative allele ratio will be <0.713.

[0130] According to the optimal reaction system in (I), several reaction systems were prepared, 7 μL of clinical plasma sample DNA was added to the system, and then each reaction system was supplemented to 20 μL with dd H2O, mixed, and transferred to the sample wells of the droplet generation card respectively, and then 70 μL of droplet generation oil was added to the droplet wells, and the raw card was placed in the droplet generator to generate droplets, and then the generated droplets were transferred to the 96PCR reaction plate and sealed with aluminum foil, and amplified according to the optimal amplification reaction conditions in (I), and the PCR amplification results were analyzed on the droplet analyzer; the fluorescence signals of FAM and HEX read by the droplet analyzer were detected, and the proportion of PCR reaction positive droplets was automatically counted by the software, and the - in the reaction system was distributed by Poisson distribution. SEA And the starting template concentration of αα gene was quantitatively calculated to obtain -- SEA and αα relative allele ratio, thus judging the fetus's -- SEA The α-thalassemia genotype of the type was compared with the invasive puncture results. Fig.12 shown.

[0131] Depend on Fig.12 It can be seen that the detection strategy of this application is in 40 cases where the fetus may inherit the maternal lineage-- SEA In clinical plasma samples with the allele, the fetuses of 33 families were accurately diagnosed as -- SEA The results show that the detection strategy of this application is in the maternal line-- SEA of-- SEAIn the non-invasive prenatal diagnosis of type α-thalassemia, the AUC value was 0.9323, the accuracy was 82.5%, the sensitivity was 83.3%, and the specificity was 93.75%, which had better sensitivity and accuracy and showed good diagnostic efficacy.

[0132] For the 45 couples both of whom were -- SEA The maternal plasma samples of the carriers are mainly tested by dd gap-PCR combined with RMD non-invasive prenatal diagnosis method, that is, when the fetus and the mother's -- SEA When the genotype of α-thalassemia is consistent, SEA The relative allele ratio of αα ranged from [0.713-0.926]; when the fetus was severe α-thalassemia (-- SEA / -- SEA )hour,-- SEA The ratio of the relative alleles to αα will be > 0.926; when the fetus does not carry -- SEA hour,-- SEA and the αα relative allele ratio will be <0.713.

[0133] According to the optimal reaction system in (I), several reaction systems were prepared, 7 μL of clinical plasma sample DNA was added to the system, and then each reaction system was supplemented to 20 μL with dd H2O, mixed, and transferred to the sample wells of the droplet generation card respectively, and then 70 μL of droplet generation oil was added to the droplet wells, and the raw card was placed in the droplet generator to generate droplets, and then the generated droplets were transferred to the 96PCR reaction plate and sealed with aluminum foil, and amplified according to the optimal amplification reaction conditions in (I), and the PCR amplification results were analyzed on the droplet analyzer; the fluorescence signals of FAM and HEX read by the droplet analyzer were detected, and the proportion of PCR reaction positive droplets was automatically counted by the software, and the - in the reaction system was distributed by Poisson distribution. SEA And the starting template concentration of αα gene was quantitatively calculated to obtain -- SEA and αα relative allele ratio, thus judging the fetus's -- SEA The α-thalassemia genotype of the type was compared with the invasive puncture results. Fig.13 shown.

[0134] Depend on Fig.13 C shows that for 45 couples, both of them are -- SEA For the clinical pedigrees of carriers, the present application accurately identified 39 fetuses with severe α-thalassemia in the differential diagnosis of whether the fetus had Hb Bart's, with an AUC value of 0.925, an accuracy rate of 86.7%, a sensitivity of 90.9%, and a specificity of 79.4%.

[0135] Whether to carry the fetusSEA Genetic differential diagnosis can also help reduce the incidence of severe α-thalassemia, so this application is for 45 couples who are both - SEA The clinical pedigree of the carrier was further analyzed. Fig.13 As shown in D.

[0136] Depend on Fig.13 D It can be seen that this application accurately identified whether 42 fetuses were - SEA The AUC value of the ROC curve for differential diagnosis of α-thalassemia type 2 was 0.9323, the accuracy was 82.5%, the sensitivity was 83.3%, and the specificity was 93.75%, showing good diagnostic efficacy.

[0137] In summary, the detection strategy of the present application (ie, the dd gap-PCR method) can accurately identify fetal SEA α-thalassemia genotype and major α-thalassemia, suitable for all possible-- SEA The test can be used to detect maternal plasma samples from clinical families with fetal risk of type α-thalassemia, with high accuracy, high sensitivity and strong specificity.

[0138] In summary, the present application designs specific primers and specific probes to effectively detect Southeast Asian deletion α-thalassemia, and screens the final concentration, annealing temperature and annealing time of the primers and probes in the reaction system. After screening, the final concentration of the primers in the reaction system is 900 nM, the final concentration of the probe in the reaction system is 250 nM, the preferred annealing temperature is 62 ° C, and the preferred annealing time is 1 min.

[0139] To verify the specificity, this application conducted a specificity test. The results showed that: SEA / -- SEA No wild-type α-specific positive droplets were found in αα / αα. SEA Type-specific positive droplets, SEA / αα can detect at the same time-- SEA The positive droplets of α wild type and α wild type were detected, and the number of positive droplets of the two genotypes was basically equal. No positive droplets appeared in the negative control, indicating that the detection strategy of the present application (ie, the dd gap-PCR method) has good specificity.

[0140] To verify the sensitivity, this application conducted linearity and sensitivity tests. The results showed that: SEA The copy number detection of the two alleles, α-type and α-wild type, had a good linear relationship, and its R 2 0.9947 and 0.9938 respectively, which can detect as low as 0.415 copies / μL -SEA type and 1.050 copies / μL of α wild-type DNA, indicating that the detection strategy of the present application (ie, the dd gap-PCR method) has excellent sensitivity.

[0141] To verify the accuracy, the present application conducted an accuracy test. The results showed that the test results of 52 puncture prenatal diagnosis samples from families with high reproductive risk of severe α-thalassemia were completely consistent with the test results of the clinical commercial kit (fluorescence PCR melting curve method), indicating that in clinical puncture prenatal diagnosis samples with confirmed fetal genotypes, the detection strategy of the present application (ie, dd gap-PCR method) can reach 100% accuracy, 100% sensitivity, and 100% specificity. Compared with the fluorescence PCR melting curve method, the detection strategy of the present application (ie, dd gap-PCR method) takes less time and has simpler data analysis, which helps to improve -- SEA Efficiency of prenatal diagnosis of α-thalassemia type 2.

[0142] To verify that in the paternal line -- SEA The feasibility of non-invasive prenatal diagnosis was investigated in this application. SEA The results showed that the measured -- SEA The ratio showed a good linear relationship with the fetal concentration ratio (R 2 =0.997), indicating that the detection strategy of the present application (ie, dd gap-PCR method) can effectively detect the minimum 2.6% paternal lineage- SEA The maternal plasma has good stability and the ratios detected three times are basically the same.

[0143] To verify that in the maternal line -- SEA Both parents carry -- SEA In order to investigate the feasibility of non-invasive prenatal diagnosis, this application conducted a NIPD feasibility verification experiment. Specifically, the feasibility of non-invasive prenatal diagnosis of genetic types ①-- SEA / ααmatrix and-- SEA / -- SEA Fetus; ②-- SEA / αα mothers and αα / αα fetuses were tested for feasibility. The results showed that for genetic type ①- SEA / ααmatrix and-- SEA / -- SEA For the fetus, as the concentration of fetal DNA gradually increases, the RMD value also increases, with a strong correlation (R 2 >0.98), and the fetal ratio obtained by three repeated tests was relatively consistent, indicating good detection stability. However, the correlation between the fetal concentration and RMD value of Q-PCR technology was weaker than that of dd gap-PCR (R2 The stability was poor and the differences between the three replicates were large. SEA For the mother with α / αα and the fetus with αα / αα, as the concentration of fetal DNA gradually increased, the RMD value also decreased, with a strong correlation (R 2 >0.98), and the fetal ratio obtained by three repeated tests was relatively consistent, indicating good detection stability. However, the correlation between the fetal concentration and RMD value of Q-PCR technology was weaker than that of dd gap-PCR (R 2 <0.93), poor stability, and large differences among three replicates. This suggests that the detection strategy of this application is in the maternal line-- SEA Deletion and both parents carry -- SEA The non-invasive prenatal diagnosis has good feasibility, high sensitivity and good stability.

[0144] In order to verify the feasibility of clinical application, this application conducted a clinical application experiment. The results showed that in 22 cases of paternal- SEA In the sample testing, this application accurately identified 19 fetuses -- SEA Genotype, AUC value was 0.9375, accuracy was 86.4%, sensitivity was 87.5%, specificity was 100%; 40 maternal- SEA 33 fetuses were accurately identified in the sample differential diagnosis SEA Genotype, AUC value is 0.9323, accuracy is 82.5%, sensitivity is 83.3%, specificity is 93.75%. For 45 cases where both parents carry -- SEA For samples, this application accurately identified 39 fetuses with severe α-thalassemia in the differential diagnosis of whether the fetus is Hb Bart's, with an AUC value of 0.925, an accuracy rate of 86.7%, a sensitivity of 90.9%, and a specificity of 79.4%, and accurately identified 42 fetuses with - SEA The AUC value was 0982, the accuracy was 93.3%, the sensitivity was 97%, and the specificity was 100%, indicating that the detection strategy of this application (ie, the dd gap-PCR method) can accurately identify fetuses in clinical applications. SEA Genotype and major α-thalassemia, and is suitable for all possible-- SEA α-thalassemia fetal risk in clinical families: maternal plasma sample testing (i.e., paternal- SEA ,maternal-- SEA And both parents are -- SEA Genetic testing for carriers) with high accuracy, high sensitivity and strong specificity.

[0145] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A primer-probe combination for detecting Southeast Asian deletion α-thalassemia, characterized in that: It includes a first primer set, a first probe, a second primer set and a second probe, the first primer set includes a first upstream primer and a first downstream primer, the second primer set includes a second upstream primer and a second downstream primer, the nucleotide sequence of the first upstream primer is shown in SEQ ID NO.1, the nucleotide sequence of the first downstream primer is shown in SEQ ID NO.2, the nucleotide sequence of the first probe is shown in SEQ ID NO.3, the nucleotide sequence of the second upstream primer is shown in SEQ ID NO.4, the nucleotide sequence of the second downstream primer is shown in SEQ ID NO.5, the nucleotide sequence of the second probe is shown in SEQ ID NO.6, the first probe is modified with a first fluorescent reporter group and a first fluorescent quencher group, and the second probe is modified with a second fluorescent reporter group and a second fluorescent quencher group.

2. The primer-probe combination for detecting Southeast Asian deletion α-thalassemia according to claim 1, characterized in that: The first primer set can specifically bind to a normal α-globin gene, and the first probe can specifically recognize the normal α-globin gene.

3. The primer-probe combination for detecting Southeast Asian deletion α-thalassemia according to claim 1, characterized in that: The second primer set can specifically bind to the Southeast Asian deletion break gene sequence, and the second probe can specifically recognize the Southeast Asian deletion break gene sequence.

4. The primer-probe combination for detecting Southeast Asian deletion α-thalassemia according to claim 1, characterized in that: The first fluorescent reporter group is selected from at least one of 6-carboxyfluorescein, phosphoramidite, (hexachlorofluorescein) active ester, sulfocyanine 5 and maleimide, the first fluorescent quenching group is selected from at least one of a minor groove binder, a black hole quencher, a phosphoamide ester and 5-tetramethylrhodamine-succinimide ester, the second fluorescent reporter group is selected from at least one of 6-carboxyfluorescein, phosphoramidite, (hexachlorofluorescein) active ester, sulfocyanine 5 and maleimide, and the second fluorescent quenching group is selected from at least one of a minor groove binder, a black hole quencher, a phosphoamide ester and 5-tetramethylrhodamine-succinimide ester.

5. Use of the primer-probe combination according to any one of claims 1 to 4 in the preparation of a reagent for detecting Southeast Asian deletion α-thalassemia.

6. A kit for detecting Southeast Asian deletion α-thalassemia, characterized in that: The invention comprises a primer-probe combination as described in any one of claims 1 to 4.

7. The kit for detecting Southeast Asian deletion α-thalassemia according to claim 6, characterized in that: In the kit, the molar ratio of the first upstream primer to the first probe is 450-1350:200-300, the molar ratio of the first downstream primer to the first probe is 450-1350:200-300, the molar ratio of the second upstream primer to the second probe is 450-1350:200-300, and the molar ratio of the second downstream primer to the second probe is 450-1350:200-300.

8. The kit for detecting Southeast Asian deletion α-thalassemia according to claim 7, characterized in that: The following components are also included: DNA polymerase, dNTP.

9. A method for detecting Southeast Asian deletion α-thalassemia for the purpose of non-disease diagnosis and / or treatment, characterized in that: The following steps are involved: Using the primer-probe combination as described in any one of claims 1 to 4, or using the kit as described in any one of claims 6 to 8, the template DNA of the sample to be tested is subjected to gap-polymerase chain reaction amplification (gap-PCR) detection to determine whether Southeast Asian deletion α-thalassemia exists.

10. The method for detecting Southeast Asian deletion α-thalassemia for the purpose of non-disease diagnosis and / or treatment according to claim 9, characterized in that: The amplification conditions are as follows: first activation treatment at 95°C for 3 minutes, followed by denaturation treatment at 98°C for 30 seconds, and then annealing and extension at 62°C for 1 minute for 40 cycles, then treatment at 98°C for 10 minutes, and finally storage at 10°C.

Images