WO2022206573A1 - 同时检测hba1/2和hbb基因位点多种突变的方法和试剂盒 - Google Patents

同时检测hba1/2和hbb基因位点多种突变的方法和试剂盒 Download PDF

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WO2022206573A1
WO2022206573A1 PCT/CN2022/082906 CN2022082906W WO2022206573A1 WO 2022206573 A1 WO2022206573 A1 WO 2022206573A1 CN 2022082906 W CN2022082906 W CN 2022082906W WO 2022206573 A1 WO2022206573 A1 WO 2022206573A1
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hba
hbb
hba1
mutations
primers
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French (fr)
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毛爱平
张晓杰
张文琦
张建光
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北京贝瑞和康生物技术有限公司
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6869—Methods for sequencing
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    • C12Q2600/156—Polymorphic or mutational markers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to a combination of primers and a method for simultaneously detecting multiple mutations of HBA1/2 and HBB genes using a three-generation long-read sequencing platform, and a kit suitable for the method.
  • Hemoglobin is a special protein that transports oxygen in red blood cells.
  • Adult hemoglobin (HbA) is a tetramer composed of two pairs of ⁇ -globin and two pairs of ⁇ -globin. Absence or insufficiency of alpha-globin or beta-globin synthesis can lead to dysthalassemia, also known as thalassemia.
  • the deletion or deficiency of ⁇ -globin caused by mutation of ⁇ -globin gene is called ⁇ -thalassemia (abbreviated as ⁇ -thalassaemia); the deletion or deficiency of ⁇ -globin caused by mutation of ⁇ -globin gene is called ⁇ -thalassemia (referred to as ⁇ -thalassemia).
  • Thalassaemia is the most common single-gene genetic disease in the world, and it is an autosomal recessive inheritance. The high incidence areas include southern China, Southeast Asia, the Mediterranean region, India, the Middle East and Africa [1,2] .
  • the human alpha-globin gene cluster is located on chromosome 16 and contains 7 loci: 5'-zeta-pseudozeta-mu-psedudoalpha-1-alpha-2-alpha-1-theta-3 '.
  • HBA1 alpha-1
  • HBA2 alpha-2
  • HBA1 and HBA2 genes include deletion and non-deletion mutations.
  • HBA1 and HBA2 genes in my country are - ⁇ 3.7, - ⁇ 4.2, and --SEA, and the non-deletion mutations are HBA2: c.369C>G, HBA2: c.377T>C, HBA2 :c.427T>C.
  • HBA2:c.369C>G HBA2: c.377T>C
  • HBA2 :c.427T>C HBA2 c.427T>C.
  • These six gene mutations account for 98% of the total number of ⁇ -thalassemias in the Chinese population, so the existing clinical routine molecular diagnosis mainly targets the above mutations [3,4] . With the continuous elucidation of the pathogenic mechanism of ⁇ -thalassaemia, more and more mutation types have been discovered.
  • HK ⁇ is due to the deletion of - ⁇ 3.7 and the recombination of ⁇ anti4.2
  • antiHK ⁇ is due to the deletion of - ⁇ 4.2 and the recombination of ⁇ anti3.7.
  • the traditional Gap-PCR method cannot distinguish between HK ⁇ and - ⁇ 3.7, and cannot distinguish between antiHK ⁇ and - ⁇ 4.2, which will lead to misdiagnosis during screening [9] .
  • different PCR systems are needed to accurately distinguish these genotypes, and simultaneous detection cannot be achieved in the same system.
  • the human beta-globin gene cluster is located on chromosome 11 and contains five loci: 5'-epsilon-gamma-G-gamma-A-delta-beta-3'.
  • HBB(beta) gene encodes globin in adults, and the other four genes are all expressed during embryonic development or at very low levels in adults.
  • the genetic variation leading to ⁇ -thalassemia is mainly point mutation or small fragment deletion of HBB gene, and a few are large fragment deletion [6,7] .
  • Combining Ithanet, HbVar, LOVD and LOVD-China thalassaemia database more than 1000 non-deletion mutations of HBB gene have been found worldwide.
  • kits for the detection of HBA1/2 gene deletion mutations are based on the multiplex Gap-PCR method, such as ⁇ -Mediterranean developed by Yaneng Biotechnology (Shenzhen) Co., Ltd, Shenzhen Yishengtang Biotechnology Co., Ltd.
  • Anemia detection kits these products can only achieve 3 to 4 common deletional thalassaemia mutations (- ⁇ 3.7, - ⁇ 4.2, --SEA and --THAI).
  • the detection kits for the diagnosis of non-deletion ⁇ - and ⁇ -thalassemia are based on PCR-RDB method, which mainly detects the common 3 HBA2 and 17 HBB gene mutation sites.
  • the present invention provides a method based on multiplex PCR amplification and third-generation sequencing to simultaneously detect multiple mutations in HBA1/2 and HBB gene regions.
  • Multiplex PCR amplification can achieve simultaneous amplification of HBA1/2 and HBB gene deletion and non-deletion mutations in a single reaction tube, and the third-generation sequencing platform has the characteristics of read length measurement, high calibration accuracy and high throughput, which can Achieve accurate, rapid and high-throughput detection of HBA1/2 and HBB gene mutations.
  • the method involved in the invention is easy to operate, the multiplex PCR and the third-generation library have reliable quality and strong repeatability, and are favorable for the application of the third-generation sequencing technology in clinical detection.
  • the purpose of the present invention is to solve the problem that the current HBA1/2 and HBB gene mutation detection has low accuracy, cannot detect multiple deletion and non-deletion mutations at the same time, cannot determine whether the mutations are linked, cannot detect uncommon mutations, and clinically miss detection and misdetection, etc.
  • multiplex PCR amplification of HBA1/2 and HBB gene mutation fragments and preparation of third-generation sequencing libraries the goal of accurate and rapid detection of multiple HBA1/2 and HBB gene mutations in multiple samples is achieved.
  • the present invention relates to a primer set for simultaneously amplifying HBA1/2 and HBB gene mutations, the primer set comprising one or more primer pairs (positions of the following 8 primers) As shown in Figure 1):
  • HBB-F HBB-R.
  • the HBA-F1 primers are located between the genome hg38chr16:165401-169817; the HBA-F2 primers are located between the genome hg38chr16:163801-165400; the HBA-F3 primers are located in the genome hg38chr16:158801- Between 163800; Described HBA-F4 primer is located at the upstream of genome hg38 chr16:149801; Described HBA-R1 primer is located between genome hg38 chr16:178388-186641; Described HBA-R2 primer is located at genome hg38 chr16: 184801 downstream; and the HBB-F and HBB-R primers are located upstream and downstream of the genome hg38 chr11:5224302-5228938, respectively.
  • the primers can amplify the complete and complete sequences of the HBA1/2 and HBB genes, including any type of mutated sequence within the scope of the primers.
  • the amplification product of each primer is less than 15Kb.
  • degenerate base primers are used if there are SNPs at the primer positions.
  • the primer set includes the following primer pairs: HBA-F1 and HBA-R1 primer pair; and HBB-F and HBB-R primer pair.
  • Primer sequence number primer name Primer sequence (5'-3') SEQ ID NO: 1 HBA-F1 ACCCAGGCAACATCAGGGAGAGCTTT SEQ ID NO: 2 HBA-F2 CGGAGCGATCTGGGCTCTGTGTTCTCAG SEQ ID NO: 3 HBA-F3 CATACCCTTTGCAAGCACACGTACTAAC SEQ ID NO: 4 HBA-F4 CACGAGTAAAACATCAAGTACACTCCAGC SEQ ID NO: 5 HBA-R1 CTGAAGCAGCAGGARTGGAGAAGGAAAT SEQ ID NO: 6 HBA-R2 ATTCCTCCCGTGTCCGTATTCCTTAC SEQ ID NO: 7 HBB-F CRTGACTGAATTTTACCTTCACACCTAA SEQ ID NO: 8 HBB-R TTGACACCAYGGCCCACTTAATGAGG
  • the primer set that can simultaneously amplify HBA1/2 and HBB gene mutations can simultaneously detect at least 903 known point mutations and 33 structures on the HBA1/2 gene locus Variations (including --SEA, - ⁇ 3.7, - ⁇ 4.2, --THAI, --FIL, ⁇ anti3.7, ⁇ anti4.2, HK ⁇ , antiHK ⁇ , --MED-I, --MED-II, - ⁇ 6.3, - ⁇ 5.6, -11.1, - ⁇ MAL3.5, - ⁇ 3.8, - ⁇ 2.7, - ⁇ 2.4, - ⁇ 2.8, - ⁇ 1.2, - ⁇ 0.8, -9.7 , Qinzhou type deletion, --BRIT, - ⁇ 3.5, --SA, - ⁇ 20.5, --NOR, --CANT, --SPAN, --GEO, 5.3kb deletion, and - ⁇ 5.2), HBB 1135 point mutations and 2 structural variants (including 3.5kb deletion and Taiwanese) known at the locus (see Tables 9-12).
  • 903 point mutations and 33 structural variants at the HBA1/2 gene locus described herein, and 1135 point mutations and 2 structural variants at the HBB gene locus are all known point mutations in the prior art Or structural variants, which can be queried in LOVD-China, HbVar, Ithanet and LOVD Thalassaemia databases; see Table 9-12 for detailed mutation information.
  • the primer set of the present invention can simultaneously detect and distinguish three mutation types - ⁇ 3.7, ⁇ anti4.2 and HK ⁇ , and can also simultaneously detect and distinguish three types of - ⁇ 4.2, ⁇ anti3.7 and antiHK ⁇ type of mutation.
  • DNA with different sequences of 5-50 nt can be added to the 5' end of the primer, that is, DNA barcode (Barcode), to distinguish different samples; preferably, the 5' end Barcode of the F and R primers They can be the same or different, and those skilled in the art can choose according to their needs.
  • DNA barcode Barcode
  • the primer set is used for multiplex PCR amplification of HBA1/2 and HBB gene fragments; it can also be used to detect whether different mutations of HBA1/2 and HBB genes are linked.
  • HBA-F1, HBA-F2, HBA-F3 and HBA-F4 the four HBA forward primers (HBA-F1, HBA-F2, HBA-F3 and HBA-F4) and the two HBA reverse primers (HBA-R1 and HBA-R2) can be prepared by different The combined form of detection of different HBA1/2 gene mutation types.
  • the primer pairs for detecting different HBA1/2 gene mutation types by different combinations are: four HBA forward primers (HBA-F1, HBA-F2, HBA-F3 and HBA- Different primer pairs consisting of F4) and two HBA reverse primers (HBA-R1 and HBA-R2), selected from one or more of the following primer pairs:
  • the primer pair is HBA-F1 and HBA-R1 primer pair; the HBA-F1 and HBA-R1 primer pair can detect 903 HBA1/2 gene site mutations and 17 structures Variations (including - ⁇ 3.7, - ⁇ 4.2, ⁇ anti3.7, ⁇ anti4.2, HK ⁇ , antiHK ⁇ , - ⁇ 6.3, - ⁇ 5.6, - ⁇ MAL3.5, - ⁇ 3.8, - ⁇ 2.7, - ⁇ 2.4, - ⁇ 2.8, - ⁇ 1.2, - ⁇ 0.8, 5.3kb deletion and - ⁇ 5.2).
  • the HBA-F1 and HBA-R1 primer pairs of the present invention can simultaneously detect and distinguish three mutation types - ⁇ 3.7, ⁇ anti4.2 and HK ⁇ , and can also simultaneously detect and distinguish - ⁇ 4.2 , ⁇ anti3.7 and antiHK ⁇ three mutation types.
  • the primer set of the present invention can be used for multiplex primer PCR amplification of HBA1/2 and HBB gene fragments including mutation types within the scope of all primers. Combined with the subsequent PacBio sequencing platform, the mutation types of all HBA1/2 and HBB gene fragments within the primer range can be detected.
  • kits for simultaneously detecting multiple mutations of HBA1/2 and HBB genes including the following reagents:
  • the reagents for multiplex PCR amplification include a DNA polymerase, a reaction buffer, and a primer set.
  • the primer set in the kit is selected from one or more primer pairs among the following 8 primers (positions are shown in Figure 1):
  • HBB-F HBB-R.
  • the HBA-F1 primers are located between the genome hg38 chr16:165401-169817; the HBA-F2 primers are located between the genome hg38 chr16:163801-165400; the HBA-F3 primers are located in the genome hg38 chr16: 158801 -163800; the HBA-F4 primer is located in the upstream of the genome hg38 chr16:149801; the HBA-R1 primer is located between the genome hg38 chr16: 178388-186641; the HBA-R2 primer is located in the genome hg38 chr16 : downstream of 184801; and the HBB-F and HBB-R primers are located upstream and downstream of genome hg38 chr11:5224302-5228938, respectively.
  • the primers can amplify the complete and complete sequences of the HBA1/2 and HBB genes, including any type of mutated sequence within the scope of the primers.
  • the amplification product of each primer is less than 15Kb.
  • degenerate base primers are used if there are SNPs at the primer positions.
  • the primers HBA-F1, HBA-F2, HBA-F3, HBA-F4, HBA-R1, HBA-R2, HBB-F and HBB-R of the primer set in the kit Its sequences are shown in SEQ ID NOs: 1-8 in Table 1.
  • the primer set of the kit includes the following primer pairs: HBA-F1 and HBA-R1 primer pair; and HBB-F and HBB-R primer pair.
  • DNA with different sequences of 5-50 nt can be added to the 5' end of the primers in the kit to distinguish different samples; preferably, the 5' ends of the F and R primers
  • the terminal Barcodes can be the same or different, and those skilled in the art can choose according to their needs.
  • HBA forward primers HBA-F1, HBA-F2, HBA-F3 and HBA-F4, and two HBA reverse primers HBA-R1 and HBA -R2 can detect different HBA1/2 gene mutation types through different combinations.
  • the PCR amplification product can be purified or not purified before the next reaction, which can be selected by those skilled in the art as needed.
  • the reagents for constructing the third-generation sequencing library include end repair enzymes, adapters, ligases, DNA purification magnetic beads, reaction buffers and exonuclease.
  • the kit can simultaneously detect 903 point mutations and 33 structural variations (including --SEA, - ⁇ 3.7, - ⁇ 4.2, --THAI, --FIL, ⁇ anti3.7, ⁇ anti4.2,HK ⁇ ,antiHK ⁇ ,--MED-I,--MED-II,- ⁇ 6.3,- ⁇ 5.6,--11.1,- ⁇ MAL3.5,- ⁇ 3. 8. - ⁇ 2.7, - ⁇ 2.4, - ⁇ 2.8, - ⁇ 1.2, - ⁇ 0.8, -9.7, Qinzhou type deletion, --BRIT, - ⁇ 3.5, --SA, - ⁇ 20. 5. --NOR, --CANT, --SPAN, --GEO, 5.3kb deletion and - ⁇ 5.2), 1135 point mutations and 2 structural variants at the HBB locus (including 3.5kb deletion and Taiwanese) (See Tables 9-12).
  • the kit can simultaneously detect and distinguish three mutation types - ⁇ 3.7, ⁇ anti4.2 and HK ⁇ , and can also simultaneously detect and distinguish three types of - ⁇ 4.2, ⁇ anti3.7 and antiHK ⁇ type of mutation.
  • the primer set in the kit is used for multiplex PCR amplification of HBA1/2 and HBB gene fragments; further, the primer set can be used to detect whether different mutations of HBA1/2 and HBB genes are chain.
  • multiplex PCR amplification is accomplished in a single reaction tube.
  • the third-generation sequencing is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.
  • PacBio library adapter ligation can use blunt end ligation or TA ligation.
  • the PacBio universal blunt-ended linker sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3' (SEQ ID NO: 9), which forms a blunt-ended stem-loop linker adapter by annealing.
  • 5-50nt DNA (Barcode) with different sequences can be added to the stem to form different adapters with Barcode.
  • PacBio libraries with different Barcodes can be pooled and sequenced.
  • the PacBio universal TA linker sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 10), annealed to form a blunt-ended stem-loop linker adapter.
  • 5-50nt DNA (Barcode) with different sequences can be added to the stem to form different adapters with Barcode.
  • PacBio libraries with different Barcodes can be pooled and sequenced.
  • the PacBio linker can be with or without Barcode.
  • the PacBio linker has a Barcode designed by PacBio company or a self-designed Barcode, which can be selected by those skilled in the art as required.
  • the PacBio library is matched to a Pacific Biosciences sequencing platform.
  • the reagents for constructing the third-generation Nanopore library include end repair enzymes, linkers, ligases, DNA purification magnetic beads, 80% ethanol and reaction buffer.
  • Nanopore library adapter ligation can use blunt end ligation or TA ligation.
  • Nanopore adapters can be with or without Barcode.
  • the Nanopore connector has a Barcode designed by ONT Company or a Barcode designed by itself, which can be selected by those skilled in the art as required.
  • the Nanopore library is matched with ONT's sequencing platform.
  • a third aspect of the present invention provides a method for detecting or simultaneously detecting multiple mutations of HBA1/2 and HBB genes in a subject, comprising the following steps:
  • the multiplex PCR primer set used in the method of the present invention is selected from primers as described above.
  • DNA Barcode
  • DNA with different sequences of 5-50 nt can be added to the 5' end of the primers described above to distinguish different samples.
  • the 5'-end Barcodes of the F and R primers can be the same or different, and those skilled in the art can choose as needed.
  • the method can detect, and can detect simultaneously, multiple mutations at the HBA1/2 and HBB gene loci, including one or more of the following:
  • HBA1/2 gene locus including --SEA, - ⁇ 3.7, - ⁇ 4.2, --THAI, --FIL, ⁇ anti3.7, ⁇ anti4.2, HK ⁇ , antiHK ⁇ ,--MED-I,--MED-II,- ⁇ 6.3,- ⁇ 5.6,--11.1,- ⁇ MAL3.5,- ⁇ 3.8,- ⁇ 2.7,- ⁇ 2.4,- ⁇ 2 .8, - ⁇ 1.2, - ⁇ 0.8, -9.7, Qinzhou type deletion, --BRIT, - ⁇ 3.5, --SA, - ⁇ 20.5, --NOR, --CANT, --SPAN, --GEO, 5.3kb deletion and - ⁇ 5.2).
  • the method can simultaneously detect and distinguish three mutation types - ⁇ 3.7, ⁇ anti4.2 and HK ⁇ , and can also simultaneously detect and distinguish three types of - ⁇ 4.2, ⁇ anti3.7 and antiHK ⁇ type of mutation.
  • multiplex PCR amplification is accomplished in a single reaction tube.
  • the sample is selected from a biological sample or gDNA extracted from the sample.
  • the biological sample is selected from cultured cell lines, blood, amniotic fluid, villi, gametes, blastocyst cells, synovial fluid, urine, sweat, saliva, feces, cerebrospinal fluid, ascites, pleural fluid, bile or pancreatic fluid, etc.
  • the third-generation sequencing of the method is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.
  • PacBio library adaptor ligation can use blunt end ligation or TA ligation.
  • the PacBio universal blunt-end linker sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAT-3' (SEQ ID NO: 9), annealed to form a blunt-ended stem-loop linker adapter.
  • 5-50nt DNA (Barcode) with different sequences can be added to the stem to form different adapters with Barcode.
  • PacBio libraries with different Barcodes can be pooled and sequenced.
  • the PacBio universal TA linker sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 10), annealed to form a blunt-ended stem-loop linker adapter.
  • 5-50nt DNA (Barcode) with different sequences can be added to the stem to form adapters with different Barcodes, and PacBio libraries with different Barcodes can be mixed together for sequencing.
  • the PacBio linker can be with or without Barcode.
  • the PacBio linker has a Barcode designed by PacBio company or a self-designed Barcode. Those skilled in the art can choose according to needs.
  • the PacBio library is matched to a Pacific Biosciences sequencing platform.
  • the reagents for constructing the third-generation Nanopore library include end repair enzymes, linkers, ligases, DNA purification magnetic beads, 80% ethanol and reaction buffer.
  • Nanopore library adapter ligation can use blunt end ligation or TA ligation.
  • Nanopore adapters can be with or without Barcode, which can be selected by those skilled in the art as needed.
  • the Nanopore connector has a Barcode designed by ONT Company or a Barcode designed by itself, which can be selected by those skilled in the art as required.
  • the Nanopore library is matched with ONT's sequencing platform.
  • the method described in the present invention can realize the simultaneous detection of multiple mutations in HBA1/2 and HBB genes in multiple samples with high specificity, accuracy and speed.
  • the detection range is wide.
  • the invention can simultaneously detect 903 kinds of point mutations and 33 kinds of structural variations on the HBA1/2 gene locus, and 1135 kinds of point mutations and 2 kinds of structural variations on the HBB gene locus.
  • the traditional method needs to set up a detection system for each mutation type, while the present invention simultaneously detects multiple deletion and non-deletion thalassaemia mutations in one reaction primer system, including rare structural variations, such as HK ⁇ , antiHK ⁇ , ⁇ anti3.7 and ⁇ anti4.2 and so on.
  • Templates for PCR can be extracted genomic DNA, human cell lines or specific tissues.
  • Three-generation sequencing can realize 384 kinds of Barcode adapters, and in fact, more kinds of Barcode adapters can be designed according to needs. Or use the dual Barcode system of primers with Barcodes and adapters with Barcodes to achieve more Barcode combinations.
  • the high-throughput characteristics of the third-generation sequencing platform determine that high-throughput sample detection can be achieved.
  • PacBio's dumbbell-shaped library can perform multiple rounds of interpretation during sequencing, and the base accuracy of the corrected sequencing result is greater than 99%. Moreover, PacBio sequencing errors are random, and the accuracy of bases corrected by sequencing depth is greater than 99.9%. Therefore, the deletion and non-deletion HBA1/2 and HBB gene mutations within the detection range of the primers can be accurately interpreted. Meanwhile, due to the characteristic of PacBio read length measurement, the method of the present invention can also detect whether different mutations are linked.
  • the detection time is flexible.
  • the Nanopore platform can generate data in minutes, and data analysis can be started in minutes or hours depending on the actual data volume requirements.
  • the detection time requirement is relatively high, the Nanopore platform has a time advantage.
  • Figure 1 is a schematic diagram of the design of multiplex PCR primers, wherein Figure 1A represents the HBA1/2 gene mutation, and Figure 1B represents the HBB gene mutation.
  • FIG. 2 is a DNA gel image of amplifying samples with different HBA1/2 gene mutations according to the multiplex PCR method in Example 1.
  • FIG. 2 is a DNA gel image of amplifying samples with different HBA1/2 gene mutations according to the multiplex PCR method in Example 1.
  • Figure 3 is a graph of PacBio sequencing results of representative HBA1/2 and HBB gene mutation samples.
  • the left of A is the ⁇ / ⁇ anti3.7 sample
  • the right of A is the ⁇ /HK ⁇ sample
  • the left of B is the HBA1: ⁇ .95+1 ⁇ >A heterozygous mutant sample
  • the middle of B is HBA2:c .123delG heterozygous mutation sample
  • B right is the HBB:c.91A>G heterozygous mutation sample.
  • FIG. 4 is a result verification diagram that is inconsistent with the traditional detection method due to the wider or more accurate detection range of the present invention.
  • A refers to the primer design method of reference [9] to verify and distinguish ⁇ , - ⁇ 3.7, - ⁇ 4.2, ⁇ anti3.7, ⁇ anti4.2, and HK ⁇ .
  • B is the Sanger sequencing validation map of three representative samples.
  • Example 1 Amplification of different HBA1/2 and HBB gene mutations using the multiplex PCR method of the present invention
  • Step 1 Multiplex PCR Amplification
  • the amplified product was placed in a centrifuge at 10,000 rpm for 20 min. After centrifugation, it was placed horizontally, and 4 ⁇ L of supernatant was added to a new tube.
  • the reaction system was prepared according to Table 6 below:
  • the reaction was carried out according to the following conditions: 37°C for 20min; 25°C for 15min; 65°C for 10min. After the reaction was completed, 0.5 ⁇ L of Exonuclease III (NEB, Cat#M0206L) and 0.5 ⁇ L of Exonuclease VII (NEB, Cat#M0379L) were added, and the reaction was continued at 37° C. for 1 hour.
  • the DNA was purified twice with 0.6x Ampure PB magnetic beads (PacBio, Cat# 100-265-900) according to the manufacturer's instructions and finally eluted with 10 uL of Elution Buffer. The resulting DNA eluate is the target DNAPacBio sequencing library.
  • DNA concentration was determined using Qubit dsDNA HS reagent (ThermoFisher, Cat# Q32851) on a Qubit 3 Fluoromter (ThermoFisher, Cat# Q33216). When there are multiple sample PacBio sequencing libraries, equal amounts of the libraries can be mixed together to prepare a mixed library.
  • the binding reagent PacBio, Cat# 101-820-200
  • primers PacBio, Cat# 100-970-100
  • the representative sequencing results are shown in Figure 3.
  • the detection results of the two samples in Figure A by the method of the present invention are ⁇ / ⁇ anti3.7 and ⁇ /HK ⁇ respectively, and the detection results of the three samples in Figure B by the method of the present invention are HBA1:c .95+1G>A heterozygous mutation, HBA2:c.123delG heterozygous mutation and HBB:c.91A>G heterozygous mutation, consistent with Sanger sequencing results.
  • the ⁇ -thalassemia gene detection kit (Gap-PCR method) of Yaneng Biotechnology (Shenzhen) Co., Ltd. was used to detect three deletion mutations of HBA1/2 gene - ⁇ 3.7, - ⁇ 4.2 and --SEA.
  • Non-deletion ⁇ -thalassemia gene detection kit (PCR-reverse dot blot method) to detect three point mutations in HBA2: c.369C>G, HBA2: c.377T>C, HBA2: c.427T>C
  • ⁇ - Thalassemia gene detection kit (PCR-reverse dot blot method) detects 19 mutations in 17 loci of HBB gene. The results are shown in Table 7 and Table 8.
  • the method of the present invention detects HK ⁇ mutation; seven samples (AA160, AD044, AD125, AF048, AI129, AJ034) can not detect structural variation by the Gap-PCR-based method, while the method of the present invention detects the presence of ⁇ anti3.7, ⁇ anti4 .2 or --THAI structural variation; in the other 33 samples, the method of the present invention detected the HBA1/2 and HBB gene point mutations that were not included in the detection range of the sub-energy PCR-reverse dot blot method. 33 inconsistent samples were verified by PCR or PCR-Sanger sequencing, as shown in Figure 4, and the results were all consistent with the method of the present invention.
  • the results detected by the method of the present invention are compared with a control kit and verified by PCR or PCR-Sanger sequencing, and the specificity and sensitivity both reach 100%. And compared with traditional Gap-PCR and PCR-DRB detection techniques, the detection accuracy was improved by 1.88% (33/1759). Therefore, the present invention utilizes the multiplex PCR method combined with PacBio sequencing, which can accurately and efficiently detect multiple mutations of HBA1/2 and HBB genes at the same time.
  • Tables 9-12 show the point mutations and structural variations that can be detected by the primer sets, kits and methods of the present invention.

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Abstract

本发明公开一种同时检测HBA1/2和HBB基因位点多种突变的引物组、试剂盒和方法。其中所述试剂盒包括以下试剂：(1)用于多重PCR扩增的试剂；和(2)用于构建三代测序文库的试剂。其中所述方法包括以下步骤：(1)制备受试者样本；(2)多重PCR同时扩增所述样本中HBA1/2和HBB基因片段；(3)构建三代测序文库；(4)测序并分析HBA1/2和HBB基因突变类型。

Description

同时检测HBA1/2和HBB基因位点多种突变的方法和试剂盒 技术领域
本发明涉及一种利用三代长读长测序平台同时检测HBA1/2和HBB基因多种突变的引物组合和方法，以及适用于此方法的试剂盒。
背景技术
血红蛋白(Hemoglobin，简称Hb)是红细胞内运输氧的特殊蛋白质。成年人血红蛋白(Adult Hemoglobin，简称HbA)是由两对α-珠蛋白和两对β-珠蛋白组成的四聚体。α-珠蛋白或β-珠蛋白的合成缺失或不足会导致珠蛋白生成障碍性贫血，又称地中海贫血。由α-珠蛋白基因突变引起的α珠蛋白缺失或不足称为α-地中海贫血(简称α-地贫)；由β-珠蛋白基因突变引起的β珠蛋白缺失或不足称为β-地中海贫血(简称β-地贫)。地贫是世界上最常见的单基因遗传病，属常染色体隐性遗传，高发地区包括中国南方、东南亚、地中海地区、印度、中东和非洲等地区 [1,2]。
人源α-珠蛋白(alpha-globin)基因簇位于16号染色体，共包含7个基因位点：5’-zeta-pseudozeta-mu-psedudoalpha-1-alpha-2-alpha-1-theta-3’。其中只有HBA1(alpha-1)和HBA2(alpha-2)两个基因在胚胎和成年人中具有编码珠蛋白的能力，其他均为假基因或预测基因，或只在胚胎发育早期翻译表达珠蛋白。HBA1和HBA2基因的突变包括缺失型突变和非缺失型突变。我国最常见的缺失型HBA1和HBA2基因的突变为-α3.7，-α4.2，和--SEA， 非缺失型突变为HBA2:c.369C>G，HBA2:c.377T>C，HBA2:c.427T>C。这六种基因突变占中国人群α-地贫总数的98％，因此现有的临床常规分子诊断主要针对上述突变 [3,4]。随着α-地贫致病机制的不断阐明，越来越多的突变类型被发现。综合Ithanet、HbVar、LOVD和LOVD-China地贫数据库，目前在世界范围已经发现约50种α-珠蛋白基因簇区域的缺失，和超过900种HBA1和HBA2基因的非缺失型突变可导致α-珠蛋白表达的减低或缺失。迄今为止在中国人群中发现了104种α-珠蛋白基因突变，其中包括28种缺失型突变和76种非缺失型突变 [5]。除此之外还有些罕见的结构变异，如3.7三联体型αααanti3.7，4.2三联体型αααanti4.2，HKαα和antiHKαα等。HKαα的产生是由于-α3.7缺失和αααanti4.2重组导致，而antiHKαα的产生是由于-α4.2缺失和αααanti3.7重组导致。传统的Gap-PCR方法无法区分HKαα和-α3.7，也无法区分antiHKαα和-α4.2，在筛选时会导致误诊 [9]。而准确区分这些基因型需要借助不同的PCR体系，无法在同一体系内实现同时检测。这些说明α-地贫有较广的基因突变谱，增加目前α-地贫基因缺陷的筛查范围，将会有效避免异常基因型的漏检。
人源β-珠蛋白(beta-globin)基因簇位于11号染色体，共包含5个基因位点：5’-epsilon-gamma-G-gamma-A-delta-beta-3’。其中只有HBB(beta)基因在成年人中编码珠蛋白，其他四个基因均在胚胎发育过程中表达，或者在成年人中表达量非常低。导致β-地贫的遗传变异主要为HBB基因的点突变或小片段缺失，少数为大片段缺失 [6,7]。综合Ithanet、HbVar、LOVD和LOVD-China地贫数据库，目前在世界范围已经发现超过1000种HBB基因的非缺失型突变。迄今为止在中国人群中发现了129种β-珠蛋白基因非缺失型突变，但目前主要检测的主要是已知常见的17个位点19种突变，包括c.-82C>A、c.-80T>C、c.-79A>G、 c.-78A>G、c.-78A>C、c.-11_-8delAAAC、c.79G>A、c.92+1G>T、c.92+5G>C、c.316-197C>T、c.2T>G、c.45_46insG、c.84_85insG、c.52A>T、c.94delC、c.126_129delCTTT、c.130G>T、c.216_217insA、c.216_217insT [8]。目前缺乏可行的方法实现一次性检测所有的HBB基因突变。
目前检测HBA1/2基因缺失型突变的试剂盒大多基于多重Gap-PCR的方法，如亚能生物技术(深圳)有限公司、深圳益生堂生物科技有限公司和广州达安基因各自开发的α-地中海贫血检测试剂盒，这些产品均只能实现3～4种常见的缺失型地贫突变(-α3.7、-α4.2、--SEA和--THAI)。目前诊断非缺失型α-和β-地中海贫血检测试剂盒均是基于PCR-RDB法，主要检测常见的3个HBA2和17个HBB基因突变位点。这些检测试剂盒主要有以下几个方面局限：
1、无法实现在同一体系内同时检测缺失型和非缺失型α-和β-地中海贫血相关突变检测；
2、只检测常见的地贫突变，覆盖范围有限，容易造成漏检，比如罕见的HBA1/2和HBB基因点突变，以及αααanti3.7和αααanti4.2等结构变异；
3、常规的Gap-PCR无法区分α-地贫的-α3.7缺失和ΗKαα结构变异，也无法区分-α4.2缺失和antiΗKαα结构变异，会造成一定程度的误检 [9]；
4、当HBA1/2或HBB基因位点上同时存在两个或多个突变时，现有的方法无法区分顺式还是反式突变。
发明内容
鉴于此，本发明提供了一种基于多重PCR扩增和三代测序的方法来同时检测HBA1/2和HBB基因区域多种突变。多重PCR扩增可在单反应管中实现同时扩增HBA1/2和HBB基因缺失型和非缺失型突变，而三代测序平台具有读长测长、 校准精确度高和高通量等特点，可以实现准确、快速并高通量地检测HBA1/2和HBB基因突变。本发明涉及的方法操作简便，多重PCR和三代文库质量可靠且重复性强，有利于三代测序技术在临床检测上的应用。
本发明的目的在于解决现阶段HBA1/2和HBB基因突变检测存在精确度低和不能同时检测多种缺失和非缺失突变，无法确定突变是否连锁，无法检测非常见突变，临床上存在漏检和误检等问题。通过多重PCR同时扩增HBA1/2和HBB基因突变片段及制备三代测序文库，实现精确和快速检测多个样品HBA1/2和HBB基因多种突变的目标。
首先，根据本发明的第一方面，本发明涉及一种用于同时扩增HBA1/2和HBB基因突变的引物组，所述引物组包含如下8条引物中的一个或多个引物对(位置如图1所示)：
四条正向引物HBA-F1、HBA-F2、HBA-F3、HBA-F4；
两条反向引物HBA-R1、HBA-R2；和
HBB-F、HBB-R。
其中所述的HBA-F1引物位于基因组hg38 chr16:165401-169817之间；所述的HBA-F2引物位于基因组hg38chr16:163801-165400之间；所述的HBA-F3引物位于基因组hg38 chr16：158801-163800之间；所述的HBA-F4引物位于基因组hg38 chr16:149801的上游；所述的HBA-R1引物位于基因组hg38 chr16:178388-186641之间；所述的HBA-R2引物位于基因组hg38 chr16:184801的下游；和所述的HBB-F和HBB-R引物分别位于基因组hg38 chr11:5224302-5228938上下游。
所述引物可以扩增HBA1/2和HBB基因的完整全部序列，包括引物范围内任何类型的突变序列。优选地，每个引物的扩增产物小于15Kb。优选地，如果引物位置有SNP，则使用简并碱基引物。
在一个优选的实施方案中，所述引物组包括以下的引物对：HBA-F1和HBA-R1引物对；以及HBB-F和HBB-R引物对。
在一个具体的实施方案中，其中所述引物序列如表1中SEQ ID NO:1-8所示。
表1：检测引物序列
引物序列号 引物名称 引物序列(5'-3')
SEQ ID NO:1 HBA-F1 ACCCAGGCAACATCAGGGAGAGCTTT
SEQ ID NO:2 HBA-F2 CGGAGCGATCTGGGCTCTGTGTTCTCAG
SEQ ID NO:3 HBA-F3 CATACCCTTTGCAAGCACACGTACTAAC
SEQ ID NO:4 HBA-F4 CACGAGTAAAACATCAAGTACACTCCAGC
SEQ ID NO:5 HBA-R1 CTGAAGCAGCAGGARTGGAGAAGGAAAT
SEQ ID NO:6 HBA-R2 ATTCCTCCCGTGTCCGTATTCCTTAC
SEQ ID NO:7 HBB-F CRTGACTGAATTTTACCTTCACACCTAA
SEQ ID NO:8 HBB-R TTGACACCAYGGCCCACTTAATGAGG
在一个优选的实施方案中，其中所述的可以同时扩增HBA1/2和HBB基因突变的引物组，其至少可以同时检测HBA1/2基因位点上已知的903种点突变和33种结构变异(包括--SEA、-α3.7、-α4.2、--THAI、--FIL、αααanti3.7、αααanti4.2、HKαα、antiHKαα、--MED-I、--MED-II、-α6.3、-α5.6、--11.1、-αMAL3.5、-α3.8、-α2.7、-α2.4、-α2.8、-α1.2、-α0.8、-9.7、Qinzhou type deletion、--BRIT、-α3.5、--SA、-α20.5、--NOR、--CANT、--SPAN、--GEO、5.3kb deletion和-α5.2)，HBB基因位点上已知的1135种点突变和2种结构变异(包括3.5kb deletion和Taiwanese)(参见表9-12)。
其中，本文所述的HBA1/2基因位点上的903种点突变和33种结构变异，以及HBB基因位点上的1135种点突变和 2种结构变异均为现有技术已知的点突变或结构变异，可在LOVD-China、HbVar、Ithanet和LOVD地贫数据库中查询；详细突变信息参见表9-12。
在一个优选的实施方案中，本发明的引物组可以同时检测并区分-α3.7、αααanti4.2和HKαα三种突变类型，还可以同时检测并区分-α4.2、αααanti3.7和antiHKαα三种突变类型。
在一个实施方案中，可以在所述引物的5’端加入5-50nt不同序列的DNA，即DNA条形码(Barcode)，用于区分不同的样本；优选地，F和R引物的5’端Barcode可以相同或者不同，本领域技术人员可以根据需要选择。
在一个优选的实验方案中，其中所述引物组用于多重PCR扩增HBA1/2和HBB基因片段；还可用于检测HBA1/2和HBB基因的不同突变是否连锁。
在一个实验方案中，所述的四条HBA正向引物(HBA-F1，HBA-F2，HBA-F3和HBA-F4)和两条HBA反向引物(HBA-R1和HBA-R2)可以通过不同的组合形式检测不同的HBA1/2基因突变类型。
在一个优选的实施方案中，所述的通过不同的组合形式检测不同的HBA1/2基因突变类型的引物对为：四条HBA正向引物(HBA-F1，HBA-F2，HBA-F3和HBA-F4)和两条HBA反向引物(HBA-R1和HBA-R2)组成的不同引物对，选自以下的一种或多种引物对：
HBA-F1和HBA-R1引物对；
HBA-F1和HBA-R2引物对；
HBA-F2和HBA-R1引物对；
HBA-F2和HBA-R2引物对；
HBA-F3和HBA-R1引物对；
HBA-F3和HBA-R2引物对；
HBA-F4和HBA-R1引物对；以及
HBA-F4和HBA-R2引物对；
在一个具体的实验方案中，其中所述的引物对为HBA-F1和HBA-R1引物对；该HBA-F1和HBA-R1引物对可以检测903种HBA1/2基因位点突变和17种结构变异(包括-α3.7、-α4.2、αααanti3.7、αααanti4.2、HKαα、antiHKαα、-α6.3、-α5.6、-αMAL3.5、-α3.8、-α2.7、-α2.4、-α2.8、-α1.2、-α0.8、5.3kb deletion和-α5.2)。
在一个具体的实施方案中，本发明的HBA-F1和HBA-R1引物对可以同时检测并区分-α3.7、αααanti4.2和HKαα三种突变类型，还可以同时检测并区分-α4.2、αααanti3.7和antiHKαα三种突变类型。
本发明的引物组可以用于多重引物PCR扩增包括所有引物范围内突变类型的HBA1/2和HBB基因片段。再结合后续的PacBio测序平台，可以检测引物范围内所有的HBA1/2和HBB基因片段的突变类型。
根据本发明第二方面，提供了一种同时检测HBA1/2和HBB基因多种突变的试剂盒，包括以下试剂：
(1)用于多重PCR扩增HBA1/2和HBB基因片段的试剂；和
(2)用于构建三代测序文库的试剂。
在一个实施方案中，其中所述用于多重PCR扩增的试剂包括DNA聚合酶、反应缓冲液和引物组。
在一个优选的实施方案中，所述试剂盒中的引物组选自以下的8条(位置如图1所示)引物中的一个或多个引物对：
四条正向引物HBA-F1、HBA-F2、HBA-F3、HBA-F4；
两条反向引物HBA-R1、HBA-R2；和
HBB-F、HBB-R。
其中所述的HBA-F1引物位于基因组hg38 chr16:165401-169817之间；所述的HBA-F2引物位于基因组hg38  chr16:163801-165400之间；所述的HBA-F3引物位于基因组hg38 chr16：158801-163800之间；所述的HBA-F4引物位于基因组hg38 chr16:149801的上游；所述的HBA-R1引物位于基因组hg38 chr16:178388-186641之间；所述的HBA-R2引物位于基因组hg38 chr16:184801的下游；和所述的HBB-F和HBB-R引物分别位于基因组hg38 chr11:5224302-5228938上下游。所述引物可以扩增HBA1/2和HBB基因的完整全部序列，包括引物范围内任何类型的突变序列。
优选地，每个引物的扩增产物小于15Kb。优选地，如果引物位置有SNP，则使用简并碱基引物。
在一个优选的实施方案中，其中所述试剂盒中引物组的引物HBA-F1、HBA-F2、HBA-F3、HBA-F4、HBA-R1、HBA-R2、HBB-F和HBB-R，其序列如表1中SEQ ID NO:1-8所示。
在一个优选的实施方案中，所述试剂盒的引物组包括以下的引物对：HBA-F1和HBA-R1引物对；以及HBB-F和HBB-R引物对。
在一个优选的实施方案中，可以在所述试剂盒中的引物的5’端加入5-50nt不同序列的DNA(Barcode)，用于区分不同的样本；优选地，F和R引物的5’端Barcode可以一样或者不一样，本领域技术人员可以根据需要选择。
在一个优选的实施方案中，其中对于所述试剂盒中引物，四条HBA正向引物HBA-F1、HBA-F2、HBA-F3和HBA-F4，以及两条HBA反向引物HBA-R1和HBA-R2，可通过不同的组合检测不同的HBA1/2基因突变类型。
在一个实施方案中，对于所述试剂盒，PCR扩增产物在进行下一步反应前，可以纯化或者不纯化，本领域技术人员可以根据需要选择。
在另一个实施方案中，其中所述试剂盒中，用于构建三代测序文库的试剂包括末端修复酶、接头、连接酶、DNA纯 化磁珠、反应缓冲液和外切酶。
在一个实施方案中，其中所述试剂盒可以同时检测HBA1/2基因位点上903种点突变和33种结构变异(包括--SEA、-α3.7、-α4.2、--THAI、--FIL、αααanti3.7、αααanti4.2、HKαα、antiHKαα、--MED-I、--MED-II、-α6.3、-α5.6、--11.1、-αMAL3.5、-α3.8、-α2.7、-α2.4、-α2.8、-α1.2、-α0.8、-9.7、Qinzhou type deletion、--BRIT、-α3.5、--SA、-α20.5、--NOR、--CANT、--SPAN、--GEO、5.3kb deletion和-α5.2)，HBB基因位点上1135种点突变和2种结构变异(包括3.5kb deletion和Taiwanese)(参见表9-12)。
在一个优选的实施方案中，其中所述试剂盒可以同时检测并区分-α3.7、αααanti4.2和HKαα三种突变类型，还可以同时检测并区分-α4.2、αααanti3.7和antiHKαα三种突变类型。
在一个具体的实施方案中，其中试剂盒中所述引物组用于多重PCR扩增HBA1/2和HBB基因片段；进一步地，所述引物组可用于检测HBA1/2和HBB基因的不同突变是否连锁。
在一个具体的实施方案中，其中对于所述试剂盒，多重PCR扩增在单反应管中完成。
在一个优选的实施方案中，三代测序选自Pacific Biosciences公司的PacBio测序或ONT公司的Nanopore测序。
在一个具体的实施方案中，PacBio文库接头连接可以使用平末端连接或TA连接的方式。
在一个具体的实施方案中，PacBio通用平末端接头序列为5’-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3’(SEQ ID NO:9)，通过退火形成平末端茎环状结构接头适配子。可以在茎部加上5-50nt不同序列的DNA(Barcode)形成不同带Barcode的接头适配子。带有不同Barcode的PacBio文库可以混合在一起测序。
在一个具体的实施方案中，PacBio通用TA接头序列为5’-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3’(SEQ ID NO:10)，通过退火形成平末端茎环状结构接头适配子。可以在茎部加上5-50nt不同序列的DNA(Barcode)形成不同带Barcode的接头适配子。带有不同Barcode的PacBio文库可以混合在一起测序。
在一个实施方案中，PacBio接头可以带或者不带Barcode。优选地，PacBio接头带PacBio公司设计的Barcode或者自行设计的Barcode，本领域技术人员可以根据需要选择。
在一个优选的实施方案中，所述PacBio文库与Pacific Biosciences公司测序平台匹配。
在一个优选的实施方案，其中所述用于构建三代Nanopore文库的试剂包括末端修复酶、接头、连接酶、DNA纯化磁珠、80％乙醇和反应缓冲液。
在一个实施方案中，Nanopore文库接头连接可以使用平末端连接或TA连接的方式。
在一个实施方案中，Nanopore接头可以带或者不带Barcode。优选地，Nanopore接头带ONT公司设计的Barcode或者自行设计的Barcode，本领域技术人员可以根据需要选择。
在一个优选的实施方案中，所述Nanopore文库与ONT公司测序平台匹配。
本发明第三方面提供了一种检测或同时检测受试者HBA1/2和HBB基因多种突变的方法，包括以下步骤：
(1)制备受试者样本；
(2)多重PCR同时扩增样本中HBA1/2和HBB基因突变片段；
(3)构建三代测序文库；
(4)测序并分析HBA1/2和HBB基因突变类型。
在一个实施方案中，本发明方法使用的多重PCR引物组，选自如上文所述的引物。优选地，可以在上文所述的引物的5’端加入5-50nt不同序列的DNA(Barcode)，用于区分不同的样本。
在一个优选的实施方案中，F和R引物的5’端Barcode可以一样或者不一样，本领域技术人员可以根据需要选择。
在一个优选的实施方案中，其中所述方法可以检测，且可以同时检测HBA1/2和HBB基因位点多种突变，包括以下的一种或多种：
HBA1/2基因位点上903种点突变和33种结构变异(包括--SEA、-α3.7、-α4.2、--THAI、--FIL、αααanti3.7、αααanti4.2、HKαα、antiHKαα、--MED-I、--MED-II、-α6.3、-α5.6、--11.1、-αMAL3.5、-α3.8、-α2.7、-α2.4、-α2.8、-α1.2、-α0.8、-9.7、Qinzhou type deletion、--BRIT、-α3.5、--SA、-α20.5、--NOR、--CANT、--SPAN、--GEO、5.3kb deletion和-α5.2)。
HBB基因位点上1135种点突变和2种结构变异(包括3.5kb deletion和Taiwanese)(表9-12)。
在一个优选的实施方案中，其中所述方法可以同时检测并区分-α3.7、αααanti4.2和HKαα三种突变类型，还可以同时检测并区分-α4.2、αααanti3.7和antiHKαα三种突变类型。
在一个优选的实施方案中，其中所述方法用于检测HBA1/2和HBB基因的不同突变是否连锁。
在一个优选的实施方案中，其中所述方法中，多重PCR扩增在单反应管中完成。
在一个实施方案中，其中所述样本选自生物样本或样本提取的gDNA。其中生物样本选自培养的细胞系、血液、羊水、绒毛、配子、囊胚细胞、关节液、尿液、汗液、唾液、粪便、脑脊液、腹水、胸水、胆汁或胰腺液等。
在一个具体的实施方案中，其中所述方法的三代测序选自Pacific Biosciences公司的PacBio测序或ONT公司的Nanopore测序。
在一个实施方案中，PacBio文库接头连接可以使用平末端连接或TA连接的方式。
在一个实施方案中，PacBio通用平末端接头序列为5’-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3’(SEQ ID NO:9)，通过退火形成平末端茎环状结构接头适配子。可以在茎部加上5-50nt不同序列的DNA(Barcode)形成不同带Barcode的接头适配子。带有不同Barcode的PacBio文库可以混合在一起测序。
在一个实施方案中，PacBio通用TA接头序列为5’-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3’(SEQ ID NO:10)，通过退火形成平末端茎环状结构接头适配子。可以在茎部加上5-50nt不同序列的DNA(Barcode)形成不同带Barcode的接头适配子，带有不同Barcode的PacBio文库可以混合在一起测序。
在一个实施方案中，PacBio接头可以带或者不带Barcode。在优选的实施方案中，PacBio接头带PacBio公司设计的Barcode或者自行设计的Barcode。本领域技术人员可以根据需要选择。
在一个优选的实施方案中，所述PacBio文库与Pacific Biosciences公司测序平台匹配。
在一个优选的实施方案中，其中所述用于构建三代Nanopore文库的试剂包括末端修复酶、接头、连接酶、DNA纯化磁珠、80％乙醇和反应缓冲液。
在一个实施方案中，Nanopore文库接头连接可以使用平末端连接或TA连接的方式。
在一个实施方案中，Nanopore接头可以带或者不带 Barcode，本领域技术人员可以根据需要选择。优选地，Nanopore接头带ONT公司设计的Barcode或者自行设计的Barcode，本领域技术人员可以根据需要选择。
在一个优选的实施方案中，所述Nanopore文库与ONT公司测序平台匹配。
基于PCR扩增和第三代高通量测序特定组合的本发明所述的方法可高特异性地、准确和快速地实现同时检测多个样品HBA1/2和HBB基因多种突变。
本发明所述方法和试剂盒的优异技术效果主要在于以下几个方面：
(1)检测范围广。本发明可以同时检测HBA1/2基因位点上903种点突变和33种结构变异，HBB基因位点上1135种点突变和2种结构变异。
(2)多种突变类型单管检测。传统的方法针对每种突变类型都需要设置一种检测体系，而本发明在一个反应引物体系里同时检测多种缺失型和非缺失型地贫突变，包括罕见的结构变异，如HKαα、antiHKαα、αααanti3.7和αααanti4.2等。
(3)检测误检率低。常规的Gap-PCR无法区分α-地贫的-α3.7缺失和HKαα结构变异，也无法区分-α4.2缺失和αντιHKαα结构变异，会造成一定程度的误检。而本发明可以很好区分这些缺失突变和罕见结构变异。
(4)样本多样化。用于PCR的模板可以是经提取的基因组DNA，也可以是人源细胞系或特定的组织。
(5)高通量检测。三代测序可实现384种Barcode接头，实际还可以根据需要设计更多种Barcode接头。或者利用引物带Barcode和接头带Barcode的双Barcode系统实现更多种Barcode组合。三代测序平台的高通量特性决定可以实现高通量样品检测。
(6)精确度高。PacBio的哑铃状文库在测序时可进行多轮解读，矫正后测序结果碱基精确度大于99％。而且PacBio测序错误是随机的，再通过测序深度矫正碱基精确度大于99.9％。因此可以精确解读引物检测范围内的缺失型和非缺失型HBA1/2和HBB基因突变。同时，由于PacBio读长测长的特性，本发明的方法还可以检测不同突变是否连锁。
(7)检测时间灵活。Nanopore平台可在数分钟内产生数据，可根据实际数据量需求在数分钟或数小时内开启数据分析。当对检测时效要求比较高时，Nanopore平台具有时间优势。
附图说明
图1是多重PCR引物设计示意图，其中图1A表示HBA1/2基因突变，和图1B表示HBB基因突变。
图2是根据实施例1中的多重PCR方法扩增不同HBA1/2基因突变样本的DNA凝胶图。
图3是代表性的HBA1/2和HBB基因突变样本PacBio测序结果图。其中图3中，A左是αα/αααanti3.7样本，A右是αα/ΗKαα样本；图3中，Β左是ΗΒΑ1：χ.95+1Γ＞Α杂合突变样本，B中间是HBA2:c.123delG杂合突变样本，B右是HBB:c.91A>G杂合突变样本。
图4是由于本发明检测范围更广或更精确引起的，与传统检测方法不一致的结果验证图。其中图4中，A是参照参考文献 [9]的引物设计方法验证和区分αα，-α3.7，-α4.2，αααanti3.7，αααanti4.2，和HKαα。图4中，B是三个代表性样本Sanger测序验证图。
具体实施方式
实施例1：利用本发明的多重PCR方法扩增不同HBA1/2 和HBB基因突变
按照下表2制备反应体系，扩增不同类型HBA1/2和HBB基因突变的外周血样本：
表2：
Figure PCTCN2022082906-appb-000001
在PCR仪上，按如下表3所示条件进行预扩增：
表3：
Figure PCTCN2022082906-appb-000002
扩增完成后，每个样本取20ul，在1％的DNA凝胶上检测，结果如图2所示，HBA1/2基因的不同缺失型突变和HBB 基因均能有效扩增。
实施例2：利用本发明涉及的多重PCR方法构建PacBio测序文库
步骤1：多重PCR扩增
按照下表4制备反应体系，扩增不同类型HBA1/2和HBB基因突变的外周血样本：
表4：
Figure PCTCN2022082906-appb-000003
在PCR仪上，按如下表5所示条件进行预扩增：
表5：
Figure PCTCN2022082906-appb-000004
Figure PCTCN2022082906-appb-000005
扩增完成后，将扩增产物放入离心机中，10000rpm，离心20min。离心结束后水平静置放置，取4μL上清加入新的管内。
步骤2：构建PacBio测序文库
按照下表6制备反应体系：
表6：
Figure PCTCN2022082906-appb-000006
在PCR仪上，按如下条件进行反应：37℃ 20min；25℃15min；65℃ 10min。反应完成后，加入0.5μL Exonuclease III(NEB,Cat#M0206L)和0.5μL Exonuclease VII(NEB,Cat#M0379L)，继续在37℃反应1小时。用0.6x Ampure PB磁珠(PacBio,Cat#100-265-900)依照制造商的说明书纯化两次，最后用10uL Elution Buffer洗脱DNA。所得DNA洗脱液即是目标DNAPacBio测序文库。用Qubit dsDNA HS试剂 (ThermoFisher,Cat# Q32851)在Qubit 3 Fluoromter(ThermoFisher,Cat#Q33216)上测定DNA浓度。当有多个样本PacBio测序文库时，可以取等量的文库混合在一起，制备成混合文库。
步骤3：PacBio上机测序和分析
根据文库的总浓度与摩尔浓度，将适当体积的文库与结合试剂(PacBio，Cat#101-820-200)和引物(PacBio,Cat#100-970-100)反应，制备成最终可上机文库。代表性测序结果如图3所示，图A中两个样本经本发明方法检测结果分别为αα/αααanti3.7和αα/HKαα，图B中三个样本经本发明方法检测结果为HBA1:c.95+1G>A杂合突变，HBA2:c.123delG杂合突变和HBB:c.91A>G杂和突变，与Sanger测序结果一致。
实施例3：HBA1/2和HBB基因突变的检测和验证
从长沙市妇幼保健医院、重庆医科大学第一附属医院、广西医科大学第一附属医院、广西藏族自治区人民医院、广州医科大学附属第三医院、贵州省人民医院、海南妇女儿童医学中心、湖南家辉遗传专科医院、江西省妇幼保健院、遂宁市中心医院、厦门妇幼保健院、和云南妇幼保健院收集1759个受试者的外周血作为验证样品1759例，参照实施例2，利用本发明方法(和试剂盒)同时检测HBA1/2和HBB基因位点多种突变。同时用亚能生物技术(深圳)有限公司的α-地中海贫血基因检测试剂盒(Gap-PCR法)检测HBA1/2基因-α3.7、-α4.2和--SEA三种缺失型突变，非缺失型α-地中海贫血基因检测试剂盒(PCR-反向点杂交法)检测HBA2:c.369C>G,HBA2:c.377T>C，HBA2:c.427T>C三种点突变，β-地中海贫血基因检测试剂盒(PCR-反向点杂交法)检测HBB基因的17个位点的19种突变。结果如表7、表8所 示。
利用本发明得到的结果和对照结果，1759例样本中，1726例样本结果完全一致(表7)，另外33例样本不一致(表8)。其中三例样本(AC077、AD020、AK166)，亚能基于Gap-PCR的方法检测为-α3.7突变，而传统的Gap-PCR方法无法区分-α3.7和HKαα突变，这三例样本经本发明方法检测为HKαα突变；七例样本(AA160、AD044、AD125、AF048、AI129、AJ034)亚能基于Gap-PCR的方法没有检测出结构变异，而本发明方法检测出含有αααanti3.7、αααanti4.2或--THAI结构变异；另外33例样本，本发明方法检测到了亚能PCR-反向点杂交法检测范围不包括的HBA1/2和HBB基因点突变。对33例不一致的样本进行了PCR或PCR-Sanger测序验证，如图4所示，结果均与本发明方法一致。
表7：
Figure PCTCN2022082906-appb-000007
Figure PCTCN2022082906-appb-000008
Figure PCTCN2022082906-appb-000009
表8：
Figure PCTCN2022082906-appb-000010
Figure PCTCN2022082906-appb-000011
因此，利用本发明方法检测的结果，经过对照试剂盒对 比和PCR或PCR-Sanger测序验证，特异性和灵敏度均达到100％。而且与传统的Gap-PCR和PCR-DRB检测技术相比，检测精确度提高了1.88％(33/1759)。因此，本发明利用多重PCR方法结合PacBio测序，能够准确高效地同时检测HBA1/2和HBB基因多种突变。
以下表9-12显示了本发明引物组、试剂盒以及方法能够检测的点突变和结构变异。
表9：可检测的HBA1点突变
Figure PCTCN2022082906-appb-000012
Figure PCTCN2022082906-appb-000013
Figure PCTCN2022082906-appb-000014
表10：可检测的HBA2点突变
Figure PCTCN2022082906-appb-000015
Figure PCTCN2022082906-appb-000016
Figure PCTCN2022082906-appb-000017
表11：可检测的HBB点突变
Figure PCTCN2022082906-appb-000018
Figure PCTCN2022082906-appb-000019
Figure PCTCN2022082906-appb-000020
Figure PCTCN2022082906-appb-000021
Figure PCTCN2022082906-appb-000022
Figure PCTCN2022082906-appb-000023
Figure PCTCN2022082906-appb-000024
表12：HBA1/2和HBB的部分结构变异
Figure PCTCN2022082906-appb-000025
Figure PCTCN2022082906-appb-000026
需要说明的是，虽然已通过以上实施例阐明了本发明的一些特征，但不能用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。多重PCR反应和三代测序文库构建中所涉及的反应试剂、反应条件等等可以根据具体的需要进行相应的调整和改变。因此对于本领域技术人员来说，在不脱离本发明的构思和原则之内，还可做出若干简单替换，这些均应包含在本发明的保护范围之内。
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Claims (25)

  1. 一种用于同时扩增HBA1/2和HBB基因突变的引物组，所述引物组包含如下的一个或多个引物：
    四条HBA正向引物HBA-F1、HBA-F2、HBA-F3、HBA-F4；
    两条HBA反向引物HBA-R1、HBA-R2；和
    HBB引物HBB-F、HBB-R；
    其中，所述的HBA-F1引物位于基因组hg38 chr16:165401-169817之间；所述的HBA-F2引物位于基因组hg38 chr16:163801-165400之间；所述的HBA-F3引物位于基因组hg38 chr16：158801-163800之间；所述的HBA-F4引物位于基因组hg38 chr16:149801的上游；所述的HBA-R1引物位于基因组hg38 chr16:178388-186641之间；所述的HBA-R2引物位于基因组hg38 chr16:184801的下游；以及所述的HBB-F和HBB-R引物分别位于基因组hg38 chr11:5224302-5228938上下游。
  2. 根据权利要求1所述的引物组，其特征在于，所述引物组可同时检测HBA1/2和HBB基因位点上多种突变，所述突变至少包括：
    如表9和表10所示的HBA1/2基因位点上903种点突变和33种结构变异，其中结构变异包括--SEA、-α3.7、-α4.2、--THAI、--FIL、αααanti3.7、αααanti4.2、HKαα、antiHKαα、--MED-I、--MED-II、-α6.3、-α5.6、--11.1、-α MAL3.5、-α3.8、-α2.7、-α2.4、-α2.8、-α1.2、-α0.8、-9.7、Qinzhou type deletion、--BRIT、-α3.5、--SA、-α20.5、--NOR、--CANT、--SPAN、--GEO、5.3kb deletion和-α5.2；以及
    如表11所示的HBB基因位点上1135种点突变和2种结构变异，其中结构变异包括3.5kb deletion和Taiwanese。
  3. 根据权利要求1所述的引物组，其中所述引物 HBA-F1、HBA-F2、HBA-F3、HBA-F4、HBA-R1、HBA-R2、HBB-F和HBB-R的序列分别如SEQ ID NO:1-8所示。
  4. 根据权利要求1-3任一项所述的引物组，其中所述的四条HBA正向引物HBA-F1、HBA-F2、HBA-F3和HBA-F4，以及两条HBA反向引物HBA-R1和HBA-R2，通过不同的组合检测不同的HBA1/2基因突变类型。
  5. 根据权利要求1-3任一项所述的引物组，其中所述引物在5’端加上5-50nt不同序列的DNA，即DNA条形码，用于区分不同样本。
  6. 根据权利要求1-3任一项所述的引物组，其中所述引物组用于多重PCR扩增HBA1/2和HBB基因片段。
  7. 根据权利要求1-3任一项所述的引物组，其中所述引物组可用于检测HBA1/2和HBB基因的不同突变是否连锁。
  8. 一种可同时检测HBA1/2和HBB基因的多种突变的试剂盒，包括以下试剂：
    1)用于多重PCR扩增HBA1/2和HBB基因片段的试剂；和
    2)用于构建三代测序文库的试剂。
  9. 根据权利要求8所述的试剂盒，其中所述用于多重PCR扩增的试剂包括DNA聚合酶、反应缓冲液和引物组。
  10. 根据权利要求9所述的试剂盒，其中所述引物组为权利要求1-7中任一项所述的引物组。
  11. 根据权利要求8所述的试剂盒，其中所述试剂盒用于同时检测HBA1/2和HBB基因的突变或常见HBA1/2和HBB基因的不同突变是否连锁。
  12. 根据权利要求8所述的试剂盒，其中所述用于构建三代测序文库的试剂包括接头、连接酶、DNA纯化磁珠、反应缓冲液和外切酶。
  13. 根据权利要求8所述的试剂盒，其中所述HBA1/2 和HBB基因的多种突变至少包括：
    如表9和表10所示的HBA1/2基因位点上903种点突变和33种结构变异，其中结构变异包括--SEA、-α3.7、-α4.2、--THAI、--FIL、αααanti3.7、αααanti4.2、HKαα、antiHKαα、--MED-I、--MED-II、-α6.3、-α5.6、--11.1、-αMAL3.5、-α3.8、-α2.7、-α2.4、-α2.8、-α1.2、-α0.8、-9.7、Qinzhou type deletion、--BRIT、-α3.5、--SA、-α20.5、--NOR、--CANT、--SPAN、--GEO、5.3kb deletion、和-α5.2；以及
    如表11所示的HBB基因位点上1135种点突变和2种结构变异，其中结构变异包括3.5kb deletion和Taiwanese。
  14. 根据权利要求13所述的试剂盒，其中所述试剂盒可以同时检测并区分-α3.7、αααanti4.2和HKαα三种突变类型；和/或可以同时检测并区分-α4.2、αααanti3.7和antiHKαα三种突变类型。
  15. 根据权利要求8所述的试剂盒，其中所述多重PCR扩增在单反应管中完成。
  16. 根据权利要求8所述的试剂盒，其中所述三代测序选自Pacific Biosciences公司的PacBio测序或Oxford Nanopore Technologies(ONT)的Nanopore测序。
  17. 一种同时检测受试者HBA1/2和HBB基因突变的方法，包括以下步骤：
    1)制备受试者样本；
    2)多重PCR同时扩增所述样本中HBA1/2和HBB基因片段；
    3)构建三代测序文库；
    4)测序并分析HBA1/2和HBB基因突变类型。
  18. 根据权利要求17所述的方法，其中所述多重PCR的引物组包括如权利要求1-7任一项所述的引物组。
  19. 根据权利要求17所述的方法，其中所述HBA1/2和 HBB基因突变至少包括以下突变中的一种或以上：
    如表9和表10所示的HBA1/2基因位点上903种点突变和33种结构变异，其中结构变异包括--SEA、-α3.7、-α4.2、--THAI、--FIL、αααanti3.7、αααanti4.2、HKαα、antiHKαα、--MED-I、--MED-II、-α6.3、-α5.6、--11.1、-αMAL3.5、-α3.8、-α2.7、-α2.4、-α2.8、-α1.2、-α0.8、-9.7、Qinzhou type deletion、--BRIT、-α3.5、--SA、-α20.5、--NOR、--CANT、--SPAN、--GEO、5.3kb deletion和-α5.2；和
    如表11所示的HBB基因位点上1135种点突变和2种结构变异，其中结构变异包括3.5kb deletion和Taiwanese。
  20. 根据权利要求17所述的方法，其中所述方法可以同时检测并区分-α3.7、αααanti4.2和HKαα三种突变类型；以及可以同时检测并区分-α4.2、αααanti3.7和antiHKαα三种突变类型。
  21. 根据权利要求17所述的方法，其中所述方法用于检测HBA1/2和HBB基因的不同突变是否连锁。
  22. 根据权利要求17所述的方法，其中所述样本选自生物样本或样本提取的gDNA。
  23. 根据权利要求22所述的方法，其中生物样本选自培养的细胞系、血液、羊水、绒毛、配子、囊胚细胞、关节液、尿液、汗液、唾液、粪便、脑脊液、腹水、胸水、胆汁或胰腺液。
  24. 根据权利要17所述的方法，其中所述多重PCR扩增在单反应管中完成。
  25. 根据权利要求17所述的方法，其中所述三代测序选自Pacific Biosciences公司的PacBio测序或Oxford Nanopore Technologies的Nanopore测序。
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