Method for constructing thalassemia structure variation long fragment DNA template

Abstract

The invention relates to a method for constructing a long-fragment thalassemia structure variation DNA template, which comprises the following steps: respectively designing specific primers for upstream and downstream of a large fragment deletion position of a thalassemia related gene, adding uracil modified and complementarily paired connection primers, after amplification is completed, cutting by using UDG enzyme to form a viscous single-stranded terminal, and then connecting, so as to obtain the long-fragment thalassemia structure variation DNA template. Background residues are further removed through nested PCR, and finally the thalassemia structure variation long fragment DNA template is constructed. The method is simple and rapid to operate, low in cost and high in product purity, and a plurality of thalassemia structure variation long fragment DNA templates with the length larger than or equal to 15 kb and different genome complexity degrees are finally obtained by optimizing the primers and the system and can be used for evaluating the amplification performance of the primers and the like. The blank of the prior art in the aspect of efficiently constructing the specific complex long fragment template is filled, and the method has extremely high practical value and wide market application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular sequencing, and particularly relates to a method for constructing a long fragment DNA template of a thalassemia structural variation. BACKGROUND

[0002] Thalassemia is an autosomal recessive genetic disease and one of the most common monogenic genetic diseases in the world. Thalassemia is difficult to cure but can be prevented. Preventing disease occurrence through population screening and prenatal diagnosis is currently the preferred prevention measure recognized at home and abroad. Thalassemia is mainly divided into alpha-thalassemia (referred to as alpha-thalassemia) and beta-thalassemia (referred to as beta-thalassemia). Alpha-thalassemia mutations can be divided into deletion type and non-deletion type, and six alpha-gene mutations (--SEA, -α3.7, -α4.2, HBA2: c.369C>G, HBA2: c.427T>C, HBA2: c.377T>C) account for more than 98% of the total population, and the gene mutations leading to beta-thalassemia mainly include point mutations or small fragment deletions of the HBB gene, and a small number of large fragment deletions, of which eight mutations (HBB: c.124_127delTTCT, HBB: c.52A>T, HBB: c.316-197C>T, HBB: c.-78A>G, HBB: c.216_217insA, HBB: c.79G>A, HBB: c.92+1G>T, HBB: c.-79A>G) account for more than 95% of the total beta-thalassemia mutations in China. The mutation types listed above are relatively common, and the corresponding variation samples are easy to obtain, while the other mutations, including structural variations, which account for 2% of alpha-thalassemia mutations and 5% of beta-thalassemia mutations, are relatively rare and difficult to obtain samples. With the development of third-generation sequencing technology, third-generation technology has been applied to the detection of various genetic diseases, including thalassemia. In the development process of the thalassemia third-generation sequencing detection product, the amplification performance of primers needs to be evaluated when capturing the target region based on the PCR method, and for some rare structural variations, sample sources are lacking, and it is difficult to evaluate the primers using real samples, so simulated samples are needed for verification.

[0003] One of the advantages of the third-generation sequencing is the long read length. According to the product requirements, the PCR amplification product can be up to 15 kb. In PCR, there is a difference in amplification efficiency between long fragments and short fragments. For gene synthesis technology, the longer the synthesized gene fragment, the more difficult it is to synthesize, and the higher the cost. The traditional recombination construction of gene fragments is based on restriction enzymes and DNA ligase, and through a series of enzyme digestion and ligation reactions, each fragment is gradually connected. This method is time-consuming and laborious, and sometimes it is difficult to find a suitable enzyme digestion site for connecting long fragments, and short fragments increase the difficulty of connection and identification. There is also fusion PCR technology using primers with complementary ends to form PCR products with overlapping chains, and by extending the overlapping chains of PCR products, different sources of DNA fragments are connected, but this technology can only connect fragments of 1000 bp or less, and there is no reported technology for connecting long fragments of 2 kb or more. Therefore, a simple and low-cost technology is needed to construct a long fragment DNA template of a thalassemia structural variation, to truly evaluate the performance of the primer extension fragment. SUMMARY

[0004] The present application aims at the lack of related technologies for connecting long fragments in the prior art, and provides a method for efficiently and specifically constructing a long fragment DNA template of a thalassemia structural variation. The principle is as follows: specific primers are designed for the upstream and downstream of the large fragment deletion position of the gene related to thalassemia, and a connecting primer is added to the 5' end of the specific primer. The connecting primers of the two fragments to be connected are designed to be complementary to each other, and the specific primers and the connecting primers are modified by deoxyuracil. After amplification and purification of the two groups of specific primers, two DNA fragments to be connected are obtained. Then, uracil DNA glycosylase (UDG) is used to catalyze the cutting of uracil bases, so that the connecting primers on the DNA fragments form sticky single-stranded ends. Based on the base complementary pairing between the two connecting primers, the two DNA fragments are connected, and then further specific amplification by nested PCR removes the background residues, and a long fragment DNA template of a thalassemia structural variation is constructed. The method is simple and fast, low in cost and high in product purity. Through optimization of the primers, a variety of long fragment DNA templates of thalassemia structural variations with different genomic complexities and lengths of ≥15 kb are obtained.

[0005] Specifically, the application provides a method for constructing a long fragment DNA template of a thalassemia structural variation, comprising the following steps: S1, designing specific primers F1 and R1 upstream or at the 5' end of a large fragment deletion site of an alpha-globin gene or a beta-globin gene, designing specific primers F2 and R2 downstream or at the 3' end of the large fragment deletion site of the alpha-globin gene or the beta-globin gene, and adding complementary connection primers R' and F' at the 5' end of the specific primers R1 and F2, wherein uracil is contained between the specific primers and the connection primers; S2, performing PCR amplification by using the primers F1 and R1 and the primers F2 and R2 respectively to obtain an upstream target fragment 1 and a downstream target fragment 2; S3, cutting uracil bases by using a uracil DNA glycosylase to form sticky single-stranded ends of the upstream target fragment 1 and the downstream target fragment 2 respectively; S4, under the action of a DNA ligase, the upstream target fragment 1 and the downstream target fragment 2 are connected based on the principle of base complementary pairing to obtain a connected long fragment DNA; S5, designing nested PCR primers for the connected long fragment DNA sequence, removing residual upstream target fragments 1, downstream target fragments 2 and non-specific fragments by specific amplification of the nested PCR, and finally obtaining the long fragment DNA template of the thalassemia structural variation.

[0006] Further, the large fragment deletion of the alpha-globin gene in step S1 comprises Qinzhou type deletion and 5.3 kb deletion, and the large fragment deletion of the beta-globin gene comprises Yunnanese (67.128 kb beta0), delta87 (F3)-beta116 (G18), HPFH-2 / Ghanaian, Malay-2 Ggamma (Agammadeltabeta) 0-Thal.

[0007] Further, the specific primers F1 / R1 and F2 / R2 for the Qinzhou type deletion are respectively shown as SEQ ID NO. 1-4; the specific primers F1 / R1 and F2 / R2 for the 5.3 kb deletionn are respectively shown as SEQ ID NO. 5-8; the specific primers F1 / R1 and F2 / R2 for the Yunnanese (67.128 kb β0) are respectively shown as SEQ ID NO. 9-12; the specific primers F1 / R1 and F2 / R2 for the delta87(F3)-beta116(G18) are respectively shown as SEQ ID NO. 13-16; the specific primers F1 / R1 and F2 / R2 for the HPFH-2 / Ghanaian are respectively shown as SEQ ID NO. 17-20; the specific primers F1 / R1 and F2 / R2 for the Malay-2 Ggamma(Agammadeltabeta)0-Thal are respectively shown as SEQ ID NO. 21-24; and the complementary pairing connecting primers R' and F' shown as SEQ ID NO. 25-26 are added at the 5' end of each primer R1 and F2 respectively.

[0008] Further, the nested PCR primers for the Qinzhou type deletion are shown as SEQ ID NO. 27-28; the nested PCR primers for the 5.3 kb deletionn are shown as SEQ ID NO. 29-30; the nested PCR primers for the Yunnanese (67.128 kb β0) are shown as SEQ ID NO. 31-32; the nested PCR primers for the delta87(F3)-beta116(G18) are shown as SEQ ID NO. 33-34; the nested PCR primers for the HPFH-2 / Ghanaian are shown as SEQ ID NO. 35-36; the nested PCR primers for the Malay-2 Ggamma(Agammadeltabeta)0-Thal are shown as SEQ ID NO. 37-38.

[0009] Further, in step S2, the PCR amplification template is human blood gDNA, and the PCR amplification reaction procedure is: 94°C 2 min pre-denaturation; 98°C 10 s denaturation, 68°C 5 min extension, 35 cycles; 68°C 5 min extension.

[0010] Further, in step S3, the uracil base is cut for 60 min at 37°C using uracil DNA glycosylase as the catalyst.

[0011] Further, in step S4, under the action of Taq DNA ligase, the long fragment DNA is connected at 45 DEG C for 15 min.

[0012] Further, in step S5, the nest PCR amplification reaction program is as follows: pre-denaturation at 94 DEG C for 2 min, denaturation at 98 DEG C for 10 s, extension at 68 DEG C for 10 min, 15 cycles, and extension at 68 DEG C for 10 min.

[0013] Further, the method further comprises: step S6, purifying the long fragment DNA template of thalassemia structural variation, specifically: adding 1.3x PacBio SMRTbell clean beads into the template and mixing, incubating at room temperature for 10 min, then instantaneously centrifuging, placing on a magnetic stand until the liquid is clear, and discarding the supernatant. Keep the centrifuge tube on the magnetic stand, add 200 muL of 80% alcohol for rinsing, wait for 30 s, then discard the alcohol, repeat the 80% alcohol rinsing once, after discarding the alcohol, dry the magnetic beads, add 15 muL of Elution Buffer, mix and incubate at room temperature for 5 min, then place on a magnetic stand after instantaneously centrifuging until the liquid is clear, and take the supernatant into a new centrifuge tube.

[0014] The application also provides a long fragment DNA template of thalassemia structural variation constructed according to the above method.

[0015] The application also provides application of the long fragment DNA template of thalassemia structural variation in preparation of a thalassemia detection kit or screening and detection of thalassemia primers.

[0016] Beneficial effects: the application successfully overcomes the difficulties of traditional long fragment amplification and connection, develops a method for stably constructing a long fragment DNA template of thalassemia structural variation, breaks through the length limitation of conventional PCR amplification, the method is simple, fast and low in cost, and through special design and optimization of primers and optimization of the amplification system, the specificity and connection efficiency are greatly improved, further, the nest PCR is introduced to remove the background residues, the purity of the final product is significantly improved, which is convenient for direct use in downstream applications such as evaluation of the amplification performance of primers and as a sequencing standard, and has high practical value and broad market application prospect in the field of large fragment deletion diseases such as thalassemia, and provides strong tool support for precision medicine. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0018] Figure 1 Schematic diagram of primer design region for the present application; Figure 2 Schematic diagram of principle of long fragment amplification and ligation for the present application; Figure 3 Agarose gel electrophoresis diagram of target fragment 1 and target fragment 2 amplification products in the embodiments of the present application; Figure 4 Agarose gel electrophoresis diagram of target fragment 1 amplification products of reset primers in the embodiments of the present application; Figure 5 Agarose gel electrophoresis diagram of Taq enzyme ligation products in the embodiments of the present application; Figure 6 Agarose gel electrophoresis diagram of nested PCR amplification products in the embodiments of the present application; Figure 7 Agarose gel electrophoresis diagram of nested PCR amplification products of reset primers in the embodiments of the present application; Figure 8 Agarose gel electrophoresis diagram of PCR amplification products of primers to be evaluated in the embodiments of the present application; Figure 9 Template GC content distribution diagram of α-thalassemia (α1, α2) in the embodiments of the present application; Figure 10 Template GC content distribution diagram of β-thalassemia (β1, β2, β3, β4) in the embodiments of the present application. DETAILED DESCRIPTION

[0019] The following embodiments are only used to more clearly illustrate the technical solutions of the present application, and therefore are only examples, and cannot limit the protection scope of the present application. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in the present application should be understood as the usual meanings understood by the skilled in the art to which the present application belongs. Unless otherwise specified, the reagents, methods and devices used in the present application are the conventional reagents, methods and devices in the technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0020] This invention provides a method for efficiently and specifically constructing long-fragment DNA templates of thalassemia structural variants. Using optimized PCR primers and reaction systems, various long-fragment DNA templates of thalassemia structural variants with lengths ≥15kb and varying genomic complexity are obtained. These DNA templates can then be used to evaluate the amplification performance of long-fragment primers. The DNA template construction process is as follows: (1) First, design specific primers for the DNA fragment (α-globin or β-globin gene) to be recombined. The amplified region of the specific primers should cover the location of the primer to be evaluated. Taking deletion variants as an example, such as Figure 1 As shown, specific primers F1 and R1 are designed for the upstream of the large deletion site or the 5' end of the deletion site. Specifically, R1 can be designed for the upstream and downstream of the deletion start site, i.e., the 5' end. The target region amplified by specific primers F1 and R1 covers the PF position of the primer to be evaluated (corresponding to target fragment 1). Specific primers F2 and R2 are designed for the downstream of the large deletion site or the 3' end of the deletion site. Specifically, F2 can be designed for the upstream and downstream of the deletion stop site, i.e., the 3' end. The target region amplified by specific primers F2 and R2 covers the PR position of the primer to be evaluated (corresponding to target fragment 2).

[0021] (2) Amplify target fragments 1 and 2 respectively, and ligate them to obtain the target DNA fragment, as shown in the figure below. Figure 2 As shown: At one end of the target fragment ligation, a ligation primer is added to the 5' end of the specific primer. The specific primer and the ligation primer are modified with deoxyuridine. The ligation primers at the ligation ends of the two DNA fragments to be ligated are designed to be complementary. Complementary ligation primers are not limited to the sequences described in this paper. PCR is performed using the specific primers described above to obtain target fragments 1 and 2 to be ligated, respectively. After purification, the DNA fragments to be ligated are mixed and uridine DNA glycosylase (UDG) is used to catalyze the cleavage of uridine bases, causing the ligation primer portions on the DNA fragments to form sticky single-stranded ends, i.e., becoming single-stranded. Then, based on the complementary base pairing principle, the ligation primers of target fragments 1 and 2 form hydrogen bonds, and under the action of Taq DNA ligase, the two DNA fragments are ligated together.

[0022] (3) After ligation, the product contains background residues of target fragments 1 and 2 or other non-specific amplification products before ligation. To address this, nested PCR primers are designed specifically to remove the background residues through further specific amplification by nested PCR, thereby obtaining a high-purity long DNA template with rare structural variations.

[0023] The GC content of the partial region of the alpha-globin peptide chain gene is high, and the GC content of the partial region of the beta-globin peptide chain gene is low, which causes certain difficulty in primer design. If the structural variation template is to be successfully constructed, the design and screening of specific primers for the target fragment 1 and the target fragment 2 are particularly important. However, for some target regions, it is indeed difficult to design specific primers, and in the product after Taq enzyme ligation, in addition to the target product successfully ligated, there are also a large amount of background residues of the target fragment 1 and the target fragment 2. Through further specific amplification of the nested PCR, specific templates can be obtained for the difficult-to-design target region 1 or 2 specific primers and different complexity variations, while greatly reducing the background residues of the target fragment 1 and the target fragment 2. Therefore, the design and screening of specific primers for the nested PCR are also crucial.

[0024] Embodiment 1. The Qinzhou type deletion of alpha-thalassemia, the 5.3 kb deletion of the large fragment deletion, the Yunnanese (67.128 kb beta 0 ), delta87 (F3)-beta116 (G18), HPFH-2 / Ghanaian and Malay-2 Ggamma (Agammadeltabeta) 0-Thal large fragment deletion are all rare structural variations, and it is difficult to obtain positive samples, and thus it is difficult to accurately evaluate the amplification performance of the related primers. Therefore, the above structural variation templates are constructed. The structural variations and the primer information for evaluating the structural variations to be detected are shown in Table 1.

[0025] Table 1 Structural variations and primer information for evaluating the structural variations to be detected 2. According to the structural variations and the primer information for evaluating the structural variations to be detected shown in the above table, the specific primers for the corresponding target fragment 1 and the target fragment 2 and the ligation primers are designed, and the primer information is shown in Table 2.

[0026] Table 2 Specific primer information for target fragments 3. The human whole blood gDNA is extracted as the PCR amplification template, the primers shown in Table 2 are used to amplify the target fragment 1 and the target fragment 2 corresponding to the template to be constructed, and specific amplification is performed according to the system 1 and the procedure 1.

[0027] Reaction system 1: Reaction procedure 1: After amplification, use 1.3x PacBio's SMRTbell clean beads for magnetic bead purification, 80% alcohol rinsing 2 times, and finally 15 μL Elution Buffer elution to obtain the purified product. Take 50-200 ng of the purified PCR product for agarose gel electrophoresis, and the results are shown in Figure 3 As shown, except for the target fragment 1 of variant number β2, which showed a diffuse band, the other PCR products all had target bands, and some had non-specific amplification. For variant number β2, specific primers β2F1-2 and β2R1-2 (primer information is shown in Table 3) were redesigned to amplify target fragment 1, and 50-200 ng of the purified product was subjected to agarose gel electrophoresis, as shown in Figure 4 As shown, target fragment 1 of β2 was successfully amplified, and β2 used the target fragment 1 product for subsequent reactions.

[0028] Table 3 Specific primer information for resetting target fragments 4, Mix the amounts of purified target fragment 1 and target fragment 2 of different structural variations, respectively prepare the following system 2 and react according to procedure 2, and perform uracil base cutting on the PCR product to make the connection primer into a single-stranded state. After the reaction is complete, use 1.3x PacBio's SMRTbell clean beads for magnetic bead purification, 80% alcohol rinsing 2 times, and finally 15 μL Elution Buffer elution to obtain the purified product.

[0029] Reaction system 2: Reaction procedure 2: 5, prepare the following system 3 and react according to procedure 3, and perform target fragment ligation. After the reaction is complete, use 1.3x PacBio's SMRTbell clean beads for magnetic bead purification, 80% alcohol rinsing 2 times, and finally 15 μL Elution Buffer elution to obtain the purified product, and take 50-200 ng of the purified PCR product for agarose gel electrophoresis, as shown in Figure 5 As shown, target bands appeared, indicating that target fragment ligation was successful, but there were background residues in target region 1 and target region 2.

[0030] Reaction system 3: Reaction procedure 3: 6. The purified ligation product was used for nested PCR according to reaction system 4 and reaction procedure 4 to ensure that specific products were obtained. The nested PCR primer information is shown in Table 4.

[0031] Reaction system 4: Reaction procedure 4: Table 4. Nested PCR primer information Take 10 μL of the PCR product in a 200 μL centrifuge tube, add 1.3x (i.e., 13 μL) of PacBio's SMRTbell cleanbeads, mix well, incubate at room temperature for 10 minutes, then centrifuge, place on a magnetic stand until the liquid is clear, and discard the supernatant. Keep the centrifuge tube on the magnetic stand, add 200 μL of 80% alcohol for rinsing, wait for 30 seconds, then discard the alcohol, repeat the 80% alcohol rinsing once, after discarding the alcohol, dry the magnetic beads, add 15 μL of Elution Buffer, mix well, incubate at room temperature for 5 minutes, centrifuge, then place on a magnetic stand until the liquid is clear, take the supernatant to a new centrifuge tube to obtain the purified product, and take 50-200 ng of the purified product for agarose gel electrophoresis, as shown in Figure 6 As shown in Figure 2, the products of variant numbers a1, a2, b1, and b3 obtained a single band, and the products of numbers b2 and b4 appeared multiple bands, indicating non-specific amplification. Therefore, for numbers b2 and b4, the nested PCR primers C-b2-F2, C-b2-R2, and C-b4-R2 were redesigned, the primer information is shown in Table 5, and the nested PCR was performed again. After the PCR product was purified by magnetic beads, 50-200 ng of the purified product was taken for agarose gel electrophoresis, and the results are shown in Figure 7 As shown in Figure 2, the products of variant numbers a1, a2, b1, and b3 obtained a single band, and the products of numbers b2 and b4 appeared multiple bands, indicating non-specific amplification. Therefore, for numbers b2 and b4, the nested PCR primers C-b2-F2, C-b2-R2, and C-b4-R2 were redesigned, the primer information is shown in Table 5, and the nested PCR was performed again. After the PCR product was purified by magnetic beads, 50-200 ng of the purified product was taken for agarose gel electrophoresis, and the results are shown in

[0032] Table 5. Redesigned nested PCR primer information 7. To verify whether the target band is the target product, the purified nested PCR product was subjected to library construction using PacBio's SMRTbell prep kit 3.0, and sequencing was performed on the PacBio Revio platform using the Revio SPRQ reagent kit. The sequencing results were target sequences, indicating that the target band was the target product. Thus, six rare structural variant long fragment DNA templates were obtained.

[0033] 8. The purified rare structural variant templates were used to evaluate the primers for detecting structural variants. The primer information to be evaluated is shown in Table 6.

[0034] Table 6 primer information to be evaluated The above 9 primers to be evaluated are mixed into a primer pool in practical application, and the final concentration is 10 μM. According to the quality concentration and length conversion molar concentration of the rare variant template, PCR is carried out according to reaction system 5 and reaction procedure 5. As shown in Figure 8 , the target band can be successfully amplified by using the primer group to amplify the template.

[0035] Reaction system 5: Reaction procedure 5: 9、In order to verify whether the target band is the target product, the PCR product of the above step is purified by using 1.3x PacBio's SMRTbell clean beads, and 80% alcohol is rinsed twice, and finally 15 μL Elution Buffer is eluted to obtain the purified product. The purified product is used for library construction by using PacBio's SMRTbell prep kit 3.0, and sequencing is carried out on the Pacbio Revio platform by using Revio SPRQ reagent kit. The sequencing result is the target sequence, which indicates that the target band is the target product. In the primer pool composed of the primer to be evaluated, each pair of primers can effectively amplify the corresponding variant.

[0036] 10、The GC content of the construction templates of the above 6 structural variants is shown in Table 7, and the GC content range is 38.04%-59.51%. The GC distribution is shown in Figure 9 and Figure 10 As the software can only analyze sequences within 20 kb, β3 is divided into two parts for GC content distribution analysis. The above results show that the templates with different GC contents can be successfully constructed by using the method described in the application.

[0037] Table 7 GC content of structural variant construction template In conclusion, the application provides a method for stably constructing a long-fragment thalassemia structural variation DNA template, which breaks through the length limitation of conventional PCR amplification, is simple and fast to operate, and low in cost.

[0038] The above detailed embodiment describes the implementation of the application, but the application is not limited to the specific details in the above embodiment. Within the scope of the claims and technical concepts of the application, the technical solutions of the application can be modified and changed in various simple ways, and these simple modifications all belong to the protection scope of the application.

Claims

1. A method of constructing a long fragment DNA template for a structural variant of thalassemia, characterized by, Comprising: S1, designing specific primers F1, R1 upstream or 5' of the large fragment deletion site of the a-globin gene or b-globin gene, and designing specific primers F2, R2 downstream or 3' of the large fragment deletion site of the a-globin gene or b-globin gene, and adding complementary ligation primers R' and F' at the 5' end of specific primers R1 and F2 respectively, containing uracil between the specific primers and the ligation primers; S2, respectively using primers F1, R1 and primers F2, R2 for PCR amplification to obtain upstream target fragment 1 and downstream target fragment 2; S3, using uracil DNA glycosylase to catalyze uracil base cleavage to form sticky single-stranded ends of the upstream target fragment 1 and the downstream target fragment 2 respectively; S4, under the action of DNA ligase, the upstream target fragment 1 and the downstream target fragment 2 are connected based on the principle of base complementary pairing to obtain a long fragment DNA; S5, designing nested PCR primers for the long fragment DNA sequence, removing residual upstream target fragment 1, downstream target fragment 2 and non-specific fragments by specific amplification of nested PCR, and finally obtaining the long fragment DNA template of the thalassemia structural variation.

2. The method of claim 1, wherein, The large fragment deletion of the a-globin gene in step S1 includes Qinzhou type deletion and 5.3 kb deletion; the large fragment deletion of the b-globin gene includes Yunnanese (67.128 kb β0), delta87(F3)-beta116(G18), HPFH-2 / Ghanaian, Malay-2 Ggamma(Agammadeltabeta)0-Thal.

3. The method of claim 2, wherein, The specific primers F1 / R1 and F2 / R2 for Qinzhou type deletion are shown in SEQ ID NO. 1-4 respectively; the specific primers F1 / R1 and F2 / R2 for the 5.3 kb deletion are shown in SEQ ID NO. 5-8 respectively; the specific primers F1 / R1 and F2 / R2 for the Yunnanese (67.128 kb β0) are shown in SEQ ID NO. 9-12 respectively; the specific primers F1 / R1 and F2 / R2 for the delta87(F3)-beta116(G18) are shown in SEQ ID NO. 13-16 respectively; the specific primers F1 / R1 and F2 / R2 for the HPFH-2 / Ghanaian are shown in SEQ ID NO. 17-20 respectively; the specific primers F1 / R1 and F2 / R2 for the Malay-2 Ggamma(Agammadeltabeta)0-Thal are shown in SEQ ID NO. 21-24 respectively; The specific primers F1 / R1 and F2 / R2 for Qinzhou type deletion are shown in SEQ ID NO. 1-4 respectively; the specific primers F1 / R1 and F2 / R2 for the 5.3 kb deletion are shown in SEQ ID NO. 5-8 respectively; the specific primers F1 / R1 and F2 / R2 for the Yunnanese (67.128 kb β0) are shown in SEQ ID NO. 9-12 respectively; the specific primers F1 / R1 and F2 / R2 for the delta87(F3)-beta116(G18) are shown in SEQ ID NO. 13-16 respectively; the specific primers F1 / R1 and F2 / R2 for the HPFH-2 / Ghanaian are shown in SEQ ID NO. 17-20 respectively; the specific primers F1 / R1 and F2 / R2 for the Malay-2 Ggamma(Agammadeltabeta)0-Thal are shown in SEQ ID NO. 21-24 respectively; and the complementary pairing connecting primers R' and F' are added at the 5' end of each primer R1 and F2, respectively, as shown in SEQ ID NO. 25-26.

4. The method of claim 2, wherein, The nested PCR primers for the Qinzhou type deletion are shown in SEQ ID NO. 27-28; the nested PCR primers for the 5.3 kb deletion are shown in SEQ ID NO. 29-30; the nested PCR primers for the Yunnanese (67.128 kb β0) are shown in SEQ ID NO. 31-32; the nested PCR primers for the delta87 (F3)-beta116 (G18) are shown in SEQ ID NO. 33-34; the nested PCR primers for the HPFH-2 / Ghanaian are shown in SEQ ID NO. 35-36; the nested PCR primers for the Malay-2 Ggamma (Agammadeltabeta)0-Thal are shown in SEQ ID NO. 37-38.

5. The method of claim 1, wherein, In step S2, the PCR amplification template is human blood gDNA, and the PCR amplification reaction procedure is: 94°C 2 min pre-denaturation; 98°C 10 s denaturation, 68°C 5 min extension, 35 cycles; 68°C 5 min extension.

6. The method of claim 1, wherein, In step S3, the uracil base is cleaved by using uracil DNA glycosylase as a catalyst at 37°C for 60 min.

7. The method of claim 1, wherein, In step S4, the long fragment DNA is obtained by connecting under the action of Taq DNA ligase at 45°C for 15 min.

8. The method of claim 1, wherein, In step S5, the nested PCR amplification reaction procedure is: 94°C 2 min pre-denaturation; 98°C 10 s denaturation, 68°C 10 min extension, 15 cycles; 68°C 10 min extension.

9. A thalassemia structural variation long fragment DNA template constructed according to the method of any one of claims 1-8.

10. Use of the thalassemia structural variation long fragment DNA template of claim 9 in the preparation of a thalassemia detection kit or in the screening and detection of thalassemia primers.

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