CN112708674A - Method and kit for simultaneously detecting multiple mutations of HBA1/2 and HBB gene locus - Google Patents
Method and kit for simultaneously detecting multiple mutations of HBA1/2 and HBB gene locus Download PDFInfo
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Abstract
本发明涉及一种同时检测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。The present invention relates to a primer set, kit and method for simultaneously detecting multiple mutations of HBA1/2 and HBB gene sites. The kit includes the following reagents: (1) reagents for multiplex PCR amplification; and (2) reagents for constructing a third-generation sequencing library. The method includes the following steps: (1) preparing a subject sample; (2) amplifying HBA1/2 and HBB gene fragments in the sample simultaneously by multiplex PCR; (3) constructing a third-generation sequencing library; (4) sequencing And analyze HBA1/2 and HBB gene mutation types.
Description
Technical Field
The invention relates to simultaneous detection by utilizing a third-generation long-read sequencing platformHBA1/2AndHBBprimer combination and method for multiple mutations of gene, and kit suitable for the method.
Background
Hemoglobin (Hb) is a specific protein for transporting oxygen in erythrocytes. Adult human Hemoglobin (HbA) is a tetramer composed of two pairs of α -globin and two pairs of β -globin. The loss or deficiency of alpha-globin or beta-globin synthesis leads to globin dysgenesisAnemia is also known as thalassemia. Alpha globin deficiency or deficiency caused by alpha globin gene mutation is called alpha thalassemia (short for alpha thalassemia); the loss or deficiency of beta-globin caused by mutation of beta-globin gene is called beta-thalassemia (short for beta-thalassemia). The thalassemia is the most common monogenic genetic disease in the world and belongs to autosomal recessive inheritance, and high-incidence areas comprise southern China, southeast Asia, Mediterranean areas, India, middle east, Africa and other areas[1,2]。
The human alpha-globin (alpha-globin) gene cluster is located on chromosome 16 and comprises 7 gene loci: 5 '-zeta-pseudozeta-mu-pseudoalpha-1-alpha-2-alpha-1-theta-3'. Therein is only provided withHBA1 (alpha-1) andHBA2(alpha-2) two genes have the ability to encode globin in both embryonic and adult, the others are either pseudogenes or predisposing genes, or express globin translationally only early in embryonic development.HBA1AndHBA2mutations of genes include deletion-type mutations and non-deletion-type mutations. The most common deletion type in ChinaHBA1AndHBA2the mutation of the gene is-alpha 3.7, -alpha 4.2, and-SEA, and the non-deletion mutation is HBA2: c.369C>G，HBA2: c.377T>C，HBA2: c.427T>C. The six gene mutations account for 98 percent of the total alpha-thalassemia of Chinese population, so the existing clinical routine molecular diagnosis mainly aims at the mutations[3,4]. With the continuous elucidation of the mechanisms underlying α -thalassemia, more and more types of mutations are discovered. Combining the Ithanet, HbVar, LOVD and LOVD-China DieBignt databases, a deletion of about 50 alpha-globin gene cluster regions has been found worldwide, and over 900HBA1AndHBA2non-deletion mutations in the gene may result in reduced or absent expression of alpha-globin. 104 mutations of alpha-globin gene have been found in Chinese population, including 28 deletion-type mutations and 76 non-deletion-type mutations[5]. In addition, there are some rare structural variations, such as 3.7 triplet α α anti3.7, 4.2 triplet α α anti4.2, HK α α and anti HK α α α. The production of HK α α is due to- α 3.7 deletion and α α anti4.2 recombination, while the production of anti HK α α is due to- α 4.2 deletion and α α α anti3.7 recombination. Conventional Gap-PCThe R method cannot distinguish HK alpha and-alpha 3.7 and anti HK alpha and-alpha 4.2, and can cause misdiagnosis in screening[9]. And different PCR systems are needed to accurately distinguish the genotypes, and the simultaneous detection cannot be realized in the same system. These show that the alpha-thalassemia has a wider gene mutation spectrum, the screening range of the existing alpha-thalassemia gene defect is enlarged, and the omission of abnormal genotypes is effectively avoided.
The human beta-globin (beta-globin) gene cluster is located on chromosome 11 and comprises 5 gene loci: 5 '-epsilon-gamma-G-gamma-A-delta-beta-3'. Therein is only provided withHBBThe (beta) gene encodes globin in adults, and all four other genes are expressed during embryonic development or are expressed in very low amounts in adults. The genetic variation leading to beta-thalassemia is mainlyHBBPoint mutation or small fragment deletion of gene, few large fragment deletion[6,7]. The comprehensive Ithanet, HbVar, LOVD and LOVD-China database has been found to be more than 1000 in the worldHBBNon-deletion mutations of genes. 129 non-deletion type mutations of beta-globin genes are found in Chinese population so far, but the main detection at present is mainly known and common 17-site 19 mutations comprising 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]. At present, the feasible method for realizing one-time detection is not availableHBBAnd (3) gene mutation.
Current detectionHBA1/2The gene deletion type mutation kit is mostly based on a multiple Gap-PCR method, such as alpha-thalassemia detection kits developed by respectively Shenzhen biotechnology (Shenzhen) Limited, Shenzhen probiotic biotechnology Limited and Guangzhou Daan gene, and the products can only realize 3-4 common deletion type thalassemia mutations (-alpha 3.7, -alpha 4.2, - -SEA and-THAI). The existing detection kit for diagnosing non-deletion alpha-thalassemia and beta-thalassemia is based on a PCR-RDB method and mainly detects 3 common alpha-thalassemia and beta-thalassemiaHBA2And 17HBBThe site of gene mutation. These detection kits are mainly limited in several ways:
1. the detection of the related mutation of deletion type alpha-thalassemia and non-deletion type alpha-thalassemia can not be simultaneously detected in the same system;
2. only common thalassemia mutations are detected, the coverage range is limited, and omission is easily caused, such as rare thalassemia mutationsHBA1/2AndHBBgene point mutation, structural variation of alpha anti3.7, alpha anti4.2 and the like;
3. the conventional Gap-PCR cannot distinguish between the alpha-thalassemia-alpha 3.7 deletion and the HK alpha structure variation, and cannot distinguish between the-alpha 4.2 deletion and the anti HK alpha structure variation, which can cause a certain degree of false detection[9]；
4. When in useHBA1/2OrHBBWhen two or more mutations exist on a gene locus at the same time, the existing method cannot distinguish cis-mutation from trans-mutation.
Disclosure of Invention
In view of this, the present invention provides a method based on multiplex PCR amplification and third-generation sequencing for simultaneous detectionHBA1/ 2AndHBBmultiple mutations in a gene region. The multiple PCR amplification can realize simultaneous amplification in a single reaction tubeHBA1/2AndHBBthe third generation sequencing platform has the characteristics of reading length, measuring length, high calibration accuracy, high flux and the like, and can realize accurate, rapid and high-flux detectionHBA1/2AndHBBand (3) gene mutation. The method provided by the invention is simple and convenient to operate, the multiple PCR and third-generation library are reliable in quality and strong in repeatability, and the application of the third-generation sequencing technology to clinical detection is facilitated.
The invention aims to solve the current stageHBA1/2AndHBBthe gene mutation detection has the problems of low precision, incapability of simultaneously detecting various deletion and non-deletion mutations, incapability of determining whether the mutations are linked, incapability of detecting unusual mutations, omission, false detection and the like in clinic. Simultaneous amplification by multiplex PCRHBA1/2AndHBBgene mutation fragment and preparation of third-generation sequencing library to realize accurate and rapid detection of multiple samplesHBA1/2AndHBBmultiple mutations of the gene are targeted.
First, according to a first aspect of the present invention, the present invention relates to a method for simultaneous amplificationHBA1/2AndHBBa primer set for gene mutation, the primer set comprising one or more primer pairs (positions are shown in figure 1) of the following 8 primers:
four forward primers HBA-F1, HBA-F2, HBA-F3 and HBA-F4;
two reverse primers HBA-R1 and HBA-R2; and
HBB-F、HBB-R。
wherein the HBA-F1 primer is positioned between the genome hg38 chr16: 165401-169817; the HBA-F2 primer is positioned between the genome hg38 chr16: 163801-165400; the HBA-F3 primer is positioned in a genome hg38 chr16: 158801-; the HBA-F4 primer is positioned at the upstream of the genome hg38 chr16: 149801; the HBA-R1 primer is positioned between the genome hg38 chr16: 178388-186641; the HBA-R2 primer is positioned at the downstream of the hg38 chr16:184801 genome; and the HBB-F and HBB-R primers are respectively positioned at the upstream and downstream of the genome hg38 chr11: 5224302-5228938.
The primer can amplifyHBA1/2AndHBBthe complete entire sequence of the gene, including any type of mutated sequence within the primer. Preferably, the amplification product of each primer is less than 15 Kb. Preferably, degenerate base primers are used if the primers have SNPs placed therein.
In a preferred embodiment, the primer set comprises the following primer pairs: HBA-F1 and HBA-R1 primer pairs; and HBB-F and HBB-R primer pairs.
In a specific embodiment, wherein the primer sequences are shown as SEQ ID NOS: 1-8 in Table 1.
In a preferred embodiment, wherein said amplification can be performed simultaneouslyHBA1/2AndHBBprimer set for gene mutation capable of at least simultaneous detectionHBA1/2903 point mutations and 33 structural variations (including-SEA-α 3.7, - - α 4.2, - -THAI, - - -FIL, - - -. alpha.0.1.2 anti3.7,. alpha.3.4.5 anti4.2, HK.alpha.6. alpha.7, anti HK. alpha.alpha. - - - -MED-I, -. MED-II, - -. alpha.6.3, - -. alpha.5.6, -. 11.1, - -. alpha.MAL 3.5, - -. alpha.3.8, - -. alpha.2.7, - -. alpha.2.8, - -alpha.0.8, - -9.7, Qinzhou type deletion, -. BRIT, - -. alpha.3.5, -. SA, - -. alpha.20.5, -. NOR, -. CANT, -. SPAN, -. GEO, 5.3kb deletion and-. alpha.5.2),HBBknown 1135 point mutations and 2 structural variations (including 3.5 kb deletion and Taiwanese) at the gene locus (see tables 9-12).
Wherein, as described hereinHBA1/2903 point mutations and 33 structural mutations at the gene locus, andHBBthe 1135 point mutations and the 2 structural mutations on the gene locus are known point mutations or structural mutations in the prior art and can be inquired in LoVD-China, HbVar, Ithanet and LOVD local anemial databases; see tables 9-12 for detailed mutation information.
In a preferred embodiment, the primer set of the present invention can simultaneously detect and distinguish three types of mutations- α 3.7, α α α anti4.2 and HK α α, and can also simultaneously detect and distinguish three types of mutations- α 4.2, α α α anti3.7 and anti HK α.
In one embodiment, 5 to 50nt of DNA with different sequences, i.e., DNA Barcode (Barcode), can be added to the 5' end of the primer for distinguishing different samples; preferably, the 5' end Barcode of the F and R primers may be the same or different, and may be selected by those skilled in the art as desired.
In a preferred embodiment, the primer set is used for multiplex PCR amplificationHBA1/2AndHBBa gene fragment; can also be used for detectionHBA1/2AndHBBwhether different mutations of a gene are linked.
In one experimental protocol, 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 detect different combinationsHBA1/2The type of gene mutation.
In a preferred embodiment, said detecting the difference by different combinationsHBA1/2The primer pairs of the gene mutation types are as follows: four stripsDifferent primer pairs consisting of an HBA forward primer (HBA-F1, HBA-F2, HBA-F3 and HBA-F4) and two HBA reverse primers (HBA-R1 and HBA-R2), one or more primer pairs selected from:
HBA-F1 and HBA-R1 primer pairs;
HBA-F1 and HBA-R2 primer pairs;
HBA-F2 and HBA-R1 primer pairs;
HBA-F2 and HBA-R2 primer pairs;
HBA-F3 and HBA-R1 primer pairs;
HBA-F3 and HBA-R2 primer pairs;
HBA-F4 and HBA-R1 primer pairs; and
HBA-F4 and HBA-R2 primer pairs;
in a specific embodiment, the primer pair is HBA-F1 primer pair and HBA-R1 primer pair; the HBA-F1 and HBA-R1 primer pair can detect 903 HBA1/2 gene site mutations and 17 structural variations (including-alpha 3.7, -alpha 4.2, alpha anti3.7, alpha anti4.2, HK alpha, anti HK alpha, alpha 6.3, -alpha 5.6, -alpha MAL3.5, -alpha 3.8, -alpha 2.7, -alpha 2.4, -alpha 2.8, -alpha 1.2, -alpha 0.8, 5.3kb deletion and-alpha 5.2).
In a specific embodiment, the HBA-F1 and HBA-R1 primer pairs of the present invention can simultaneously detect and distinguish three types of mutations-alpha 3.7, alpha anti4.2 and HK alpha, and can also simultaneously detect and distinguish three types of mutations-alpha 4.2, alpha anti3.7 and anti HK alpha.
The primer set of the present invention can be used for multiplex primer PCR amplification including all types of mutations within the primer rangeHBA1/2AndHBBa gene fragment. By combining with the subsequent PacBio sequencing platform, all primers in the range of the primers can be detectedHBA1/2AndHBBmutation type of gene fragment.
According to a second aspect of the invention, there is provided a simultaneous detectionHBA1/2AndHBBa kit for multiple mutations in a gene comprising the following reagents:
(1) for multiplex PCR amplificationHBA1/2AndHBBan agent for a gene fragment; and
(2) reagents for constructing a third generation sequencing library.
In one embodiment, wherein the reagents for multiplex PCR amplification comprise a DNA polymerase, a reaction buffer, and a primer set.
In a preferred embodiment, the primer set in the kit is selected from one or more primer pairs of the following 8 (positions shown in figure 1):
four forward primers HBA-F1, HBA-F2, HBA-F3 and HBA-F4;
two reverse primers HBA-R1 and HBA-R2; and
HBB-F、HBB-R。
wherein the HBA-F1 primer is positioned between the genome hg38 chr16: 165401-169817; the HBA-F2 primer is positioned between the genome hg38 chr16: 163801-165400; the HBA-F3 primer is positioned in a genome hg38 chr16: 158801-; the HBA-F4 primer is positioned at the upstream of the genome hg38 chr16: 149801; the HBA-R1 primer is positioned between the genome hg38 chr16: 178388-186641; the HBA-R2 primer is positioned at the downstream of the hg38 chr16:184801 genome; and the HBB-F and HBB-R primers are respectively positioned at the upstream and downstream of the genome hg38 chr11: 5224302-5228938. The primer can amplifyHBA1/2AndHBBthe complete entire sequence of the gene, including any type of mutated sequence within the primer.
Preferably, the amplification product of each primer is less than 15 Kb. Preferably, degenerate base primers are used if the primers have SNPs placed therein.
In a preferred embodiment, 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 have the sequences shown in SEQ ID NO. 1-8 in Table 1.
In a preferred embodiment, the primer set of the kit comprises the following primer pairs: HBA-F1 and HBA-R1 primer pairs; and HBB-F and HBB-R primer pairs.
In a preferred embodiment, 5 to 50nt of DNA (Barcode) of different sequences may be added to the 5' end of the primers in the kit for distinguishing between different samples; preferably, the 5' end Barcode of the F and R primers may be the same or different, and may be selected by those skilled in the art as desired.
In a preferred embodiment, wherein four HBA forward primers HBA-F1, HBA-F2, HBA-F3 and HBA-F4, and two HBA reverse primers HBA-R1 and HBA-R2 are used as primers in the kit, different combinations can be used to detect different HBA forward primers HBA-F1, HBA-F2, HBA-F3 and HBA-F4, and different HBA reverse primers HBA-R3838HBA1/2The type of gene mutation.
In one embodiment, for the kit, the PCR amplification product may or may not be purified before proceeding to the next reaction, and may be selected as desired by one skilled in the art.
In another embodiment, wherein in the kit, the reagents for constructing the third generation sequencing library include a terminal repair enzyme, a linker, a ligase, a DNA purification magnetic bead, a reaction buffer, and an exonuclease.
In one embodiment, wherein the kit can detect simultaneouslyHBA1/2903 point mutations and 33 structural mutations at the gene site (including- -SEA, - - α 3.7, - - α 4.2, - -THAI, - -FIL,. alpha.0. alpha.1. alpha.2 anti3.7,. alpha.3. alpha.4. alpha.5 anti4.2,. alpha.6. alpha.7,. alpha.HK. alpha.α,. MED-I,. alpha.6.3, - - α 5.6,. alpha.11.1, - -. alpha.MAL 3.5, - -. alpha.3.8, - - α 2.7, - - α 2.4, - - α 2.8, - - α 1.2, - - α 0.8, - -9.7, Qinzhou type deletion,. beta.3, - - α 3.5,. alpha.SA, - - α 20.5,. alpha.NOR,. alpha.1,. SPAN,. beta.O, 5.3kb and. alpha.5.2. delta. 5. delta. beta.5. gene site and. delta. 5. structural mutations at the gene site (see. 12. delta. 12).
In a preferred embodiment, the kit can simultaneously detect and distinguish three mutation types of-alpha 3.7, alpha anti4.2 and HK alpha, and can also simultaneously detect and distinguish three mutation types of-alpha 4.2, alpha anti3.7 and anti HK alpha.
In a specific embodiment, wherein the primer sets in the kit are used for multiplex PCR amplification of HBA1/2 and HBB gene fragments; further, the primer set can be used for detectionHBA1/2AndHBBwhether different mutations of a gene are linked.
In a specific embodiment, wherein for said kit, multiplex PCR amplification is done in a single reaction tube.
In a preferred embodiment, the third generation sequencing is selected from PacBio sequencing by Pacific Biosciences or Nanopore sequencing by ONT.
In a specific embodiment, PacBio library adaptor ligation may use blunt end ligation or TA ligation.
In a specific embodiment, the PacBio universal blunt-ended linker sequence is 5 '-pATCTCTCTCTTCCTCCCTCCCTCCGTTGTTGTTGAGAGAGAT-3' (SEQ ID NO: 9), and a blunt-ended stem-loop structure linker aptamer is formed by annealing. Different linker aptamers with Barcode can be formed by adding DNA (Barcode) with 5-50nt different sequences to the stem. The PacBio libraries with different barcodes can be sequenced mixed together.
In a specific embodiment, the PacBio universal TA linker sequence is 5 '-pATCTCTCTCTTTTCCTCCCTCCCTCTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 10), and a blunt-ended stem-loop structure linker aptamer is formed by annealing. Different linker aptamers with Barcode can be formed by adding DNA (Barcode) with 5-50nt different sequences to the stem. The PacBio libraries with different barcodes can be sequenced mixed together.
In one embodiment, the PacBio linker may or may not be Barcode. Preferably, the PacBio linker is a Barcode designed by PacBio corporation or a Barcode designed by itself, which can be selected by those skilled in the art as desired.
In a preferred embodiment, the PacBio library is matched to the Pacific Biosciences sequencing platform.
In a preferred embodiment, wherein the reagents for constructing the three-generation Nanopore library include a terminal repair enzyme, a linker, a ligase, a DNA purification magnetic bead, 80% ethanol, and a reaction buffer.
In one embodiment, Nanopore library adaptor ligation may be by blunt end ligation or TA ligation.
In one embodiment, the Nanopore linker may or may not be Barcode. Preferably, the Nanopore linker is either Barcode, available from ONT corporation, or Barcode, available from self-designed sources, and can be selected by those skilled in the art as desired.
In a preferred embodiment, the Nanopore library is matched to the ONT corporation sequencing platform.
In a third aspect of the invention there is provided a method of detecting or simultaneously detecting a subjectHBA1/2AndHBBa method of multiple mutations in a gene comprising the steps of:
(1) preparing a sample of a subject;
(2) multiplex PCR simultaneous amplification in samplesHBA1/2AndHBBa mutated segment of the gene;
(3) constructing a third generation sequencing library;
(4) sequencing and analysisHBA1/2AndHBBthe type of gene mutation.
In one embodiment, the multiplex PCR primer set used in the method of the invention is selected from the primers as described above. Preferably, 5 to 50nt of DNA (Barcode) of different sequences may be added to the 5' end of the primers described above for distinguishing between different samples.
In a preferred embodiment, the 5' end of the F and R primers, Barcode, may be the same or different and may be selected by one skilled in the art as desired.
In a preferred embodiment, wherein the method is detectable, and can detect simultaneouslyHBA1/2AndHBBa plurality of mutations at a locus of a gene, including one or more of:
HBA1/2903 point mutations and 33 structural variations at the gene site (including- -SEA, - - α 3.7, - - α 4.2, - -THAI, - -FIL,. alpha.0. alpha.1. alpha.2 anti3.7,. alpha.3. alpha.4. alpha.5 anti4.2,. alpha.6. alpha.7, anti HK. alpha.alpha., - -MED-I, - -MED-II, - - α 6.3, - - α 5.6,. alpha.11.1, - - α MAL3.5, - - α 3.8, - - α 2.7, - - α 2.4, - - α 2.8, - - α 1.2, - - α 0.8, - -9.7, Qinzhou type deletion,. beta.3, - - α 3.5,. SA, - - α 20.5,. alpha.NOR,. alpha.1, SPAN,. beta.O, 5.3kb, and. alpha.5.2).
1135 point mutations and 2 structural variations (including 3.5 kb deletion and Taiwanese) were present in the HBB gene locus (tables 9-12).
In a preferred embodiment, wherein the method can simultaneously detect and distinguish three types of mutations-alpha 3.7, alpha anti4.2 and HK alpha, the method can also simultaneously detect and distinguish three types of mutations-alpha 4.2, alpha anti3.7 and anti HK alpha.
In a preferred embodiment, wherein the method is for detectingHBA1/2AndHBBwhether different mutations of a gene are linked.
In a preferred embodiment, wherein the method, multiplex PCR amplification is performed in a single reaction tube.
In one embodiment, wherein the sample is selected from a biological sample or gDNA extracted from a sample. Wherein the biological sample is selected from cultured cell lines, blood, amniotic fluid, villi, gametes, blastocytes, synovial fluid, urine, sweat, saliva, stool, cerebrospinal fluid, ascites, pleural fluid, bile or pancreatic fluid.
In a specific embodiment, wherein the third generation sequencing of the method is selected from PacBio sequencing by Pacific Biosciences or Nanopore sequencing by ONT.
In one embodiment, PacBio library adaptor ligation may use blunt-end ligation or TA ligation.
In one embodiment, the PacBio universal blunt-ended linker sequence is 5 '-pATCTCTCTCTTCCTCCCTCCCTCTGTTGTTGTTGAGAGAGAGAT-3' (SEQ ID NO: 9), and a blunt-ended stem-loop structure linker aptamer is formed by annealing. Different linker aptamers with Barcode can be formed by adding DNA (Barcode) with 5-50nt different sequences to the stem. The PacBio libraries with different barcodes can be sequenced mixed together.
In one embodiment, the PacBio universal TA linker sequence is 5 '-pATCTCTCTCTTTTCCTCCCTCCCTCTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 10), and a blunt-ended stem-loop structure linker aptamer is formed by annealing. Different Barcode-bearing linker aptamers can be formed by adding 5-50nt of DNA (Barcode) with different sequences to the stems, and PacBio libraries with different Barcode can be mixed together for sequencing.
In one embodiment, the PacBio linker may or may not be Barcode. In a preferred embodiment, the PacBio linker is a Barcode designed by PacBio Inc. or a Barcode designed by itself. One skilled in the art can select as desired.
In a preferred embodiment, the PacBio library is matched to the Pacific Biosciences sequencing platform.
In a preferred embodiment, wherein the reagents for constructing the three-generation Nanopore library comprise a terminal repair enzyme, a linker, a ligase, a DNA purification magnetic bead, 80% ethanol and a reaction buffer.
In one embodiment, Nanopore library adaptor ligation may be by blunt end ligation or TA ligation.
In one embodiment, the Nanopore linker may or may not be a Barcode, and may be selected by one skilled in the art as desired. Preferably, the Nanopore linker is either Barcode, available from ONT corporation, or Barcode, available from self-designed sources, and can be selected by those skilled in the art as desired.
In a preferred embodiment, the Nanopore library is matched to the ONT corporation sequencing platform.
The method based on the specific combination of PCR amplification and third-generation high-throughput sequencing can realize the simultaneous detection of a plurality of samples with high specificity, accuracy and rapidnessHBA1/2AndHBBmultiple mutations in the gene.
The excellent technical effects of the method and the kit mainly lie in the following aspects:
(1) the detection range is wide. The invention can detect simultaneouslyHBA1/2903 point mutations and 33 structural mutations on the gene locus, and 1135 point mutations and 2 structural mutations on the HBB gene locus.
(2) Single tube detection of multiple mutation types. The traditional method needs to arrange a detection system for each mutation type, and the invention simultaneously detects multiple deletion type and non-deletion type poor mutations in a reaction primer system, including rare structural variations, such as HK alpha, anti HK alpha, alpha anti3.7, alpha anti4.2 and the like.
(3) The detection error rate is low. Conventional Gap-PCR cannot distinguish between the-alpha 3.7 deletion of alpha-thalassemia and the HK alpha structural variation, and cannot distinguish between the-alpha 4.2 deletion and the alpha v tau iota HK alpha structural variation, which can cause a certain degree of false detection. The present invention can distinguish these deletion mutations from rare structural variations well.
(4) The samples were diversified. The template for PCR may be extracted genomic DNA, or may be a human cell line or a specific tissue.
(5) High-throughput detection. The third generation sequencing can realize 384 Barcode linkers, and actually more Barcode linkers can be designed according to needs. Or a double Barcode system of primer strip Barcode and linker strip Barcode is utilized to realize more Barcode combinations. The high throughput characteristics of the third generation sequencing platform determine that high throughput sample detection can be achieved.
(6) The accuracy is high. The dumbbell library of PacBio can be subjected to multiple rounds of reading during sequencing, and the base accuracy of the corrected sequencing result is more than 99%. And PacBio sequencing errors were random and corrected for base accuracy by sequencing depth to greater than 99.9%. Therefore, the deletion type and non-deletion type HBA1/2 andHBBand (3) gene mutation. Meanwhile, due to the characteristic of reading length and measuring length by PacBio, the method disclosed by the invention can also be used for detecting whether different mutations are linked.
(7) The detection time is flexible. The Nanopore platform can generate data in minutes, and can start data analysis in minutes or hours according to actual data volume requirements. The Nanopore platform has time advantages when the requirement for detection time efficiency is high.
Drawings
FIG. 1 is a schematic diagram of multiplex PCR primer design, in which FIG. 1A showsHBA1/2Gene mutation, and FIG. 1B showsHBBAnd (3) gene mutation.
FIG. 2 is a DNA gel diagram of samples amplified with different HBA1/2 gene mutations according to the multiplex PCR method in example 1.
FIG. 3 is representativeHBA1/2AndHBBgene mutation sample PacBio sequencing result graph. Wherein in FIG. 3, to the left A is the sample α α/α α α anti3.7 and to the right A is the sample α/HK α; in FIG. 3, B is HBA1 χ.95+1 Γ>A heterozygous mutant sample, B middle HBA2: c.123delG heterozygous mutant sample, B right HBB: c.91A>G heterozygous mutant samples。
FIG. 4 is a graph of the verification of results inconsistent with the conventional detection method due to the broader or more accurate detection range of the present invention. Wherein in FIG. 4, A is a reference[9]The primer design method of (1) verifies and distinguishes between α α α, - α 3.7, - α 4.2, α α α α anti3.7, α α α anti4.2, and HK α α. In fig. 4, B is a Sanger sequencing validation chart of three representative samples.
Detailed Description
Example 1: amplification of differences Using the multiplex PCR method of the inventionHBA1/2AndHBBgene mutation
The reaction systems were prepared to amplify different types according to Table 2 belowHBA1/2AndHBBgene mutated peripheral blood sample:
on a PCR instrument, pre-amplification was performed under the conditions shown in Table 3 below:
after amplification was complete, 20ul of each sample was tested on a 1% DNA gel, the results are shown in FIG. 2,HBA1/2different deletion type mutations of genes andHBBthe genes can be effectively amplified.
Example 2: construction of PacBio sequencing library Using the multiplex PCR method of the present invention
Step 1: multiplex PCR amplification
The reaction system was prepared according to the following table 4 to amplify peripheral blood samples with different types of HBA1/2 and HBB gene mutations:
on a PCR instrument, pre-amplification was performed under the conditions shown in Table 5 below:
after amplification, the amplification product was put into a centrifuge at 10000rpm for 20 min. After the centrifugation, the mixture was left standing horizontally, and 4. mu.L of the supernatant was added to a new tube.
Step 2: construction of PacBio sequencing library
The reaction system was prepared as follows in table 6:
on a PCR instrument, the reaction is carried out according to the following conditions: 37 ℃ for 20min, 25 ℃ for 15 min and 65 ℃ for 10 min. After the reaction was completed, 0.5. mu.L of Exonase III (NEB, Cat # M0206L) and 0.5. mu.L of Exonase VII (NEB, Cat # M0379L) were added and the reaction was continued at 37 ℃ for 1 hour. The DNA was purified twice using 0.6X Ampure PB beads (PacBio, Cat # 100-. The resulting DNA eluate was the target DNACBio sequencing library. The DNA concentration was determined on a Qubit 3 Fluorometer (ThermoFisher, Cat # Q33216) using a Qubit dsDNA HS reagent (ThermoFisher, Cat # Q32851). When there are multiple samples of the PacBio sequencing library, equal amounts of the library can be mixed together to prepare a mixed library.
And step 3: sequencing and analysis on a PacBio machine
According to the total concentration and molar concentration of the library, the library with an appropriate volume is reacted with a binding reagent (PacBio, Cat #101-820-200) and a primer (PacBio, Cat # 100-970-100) to prepare the final operable library. Representative sequencing results are shown in FIG. 3, in which two samples in panel A are detected by the method of the invention to respectively obtain alpha/alpha anti3.7 and alpha/HK alpha, and three samples in panel B are detected by the method of the invention to obtain HBA1: c.95+1G > A heterozygous mutation, HBA2: c.123delG heterozygous mutation and HBB: c.91A > G heterozygous mutation, which are consistent with Sanger sequencing results.
Example 3:HBA1/2andHBBdetection and validation of Gene mutations
From Changsha, women and children health Hospital, Chongqing medical universityFirst subsidiary hospital, first subsidiary hospital of Guangxi medical university, Guangxi Tibetan nationality autonomous region people hospital, Guangzi medical university subsidiary third hospital, Guizhou province people hospital, Hainan women's medical center, Hunan Jiahui genetic specialty hospital, Jiangxi province women's health care hospital, Xinning city center hospital, Xiamen women's health care hospital, and Yunnan women's health care hospital, 1759 examples of peripheral blood samples as validation samples were collected, and referring to example 2, simultaneous detection was performed by the method (and kit) of the present inventionHBA1/2AndHBBmultiple mutations at the gene locus. Simultaneously, alpha-thalassemia gene detection kit (Gap-PCR method) of the Shenzhen Limited company is used for detectingHBA1/2Detection of HBA2: c.369C by gene-alpha 3.7, -alpha 4.2 and-SEA three deletion type mutation and non-deletion type alpha-thalassemia gene detection kit (PCR-reverse dot hybridization method)>G, HBA2:c.377T>C，HBA2:c.427T>C three point mutation, beta-thalassemia gene detection kit (PCR-reverse dot hybridization method) detects 19 mutations of 17 sites of HBB gene. The results are shown in tables 7 and 8.
Using the results obtained by the present invention and the control results, 1726 of the 1759 samples were completely consistent (table 7), and the other 33 samples were inconsistent (table 8). Three samples (AC 077, AD020 and AK 166) can be detected as-alpha 3.7 mutation by a Gap-PCR method, but the traditional Gap-PCR method can not distinguish-alpha 3.7 mutation from HK alpha mutation, and the three samples are detected as HK alpha mutation by the method; seven samples (AA 160, AD044, AD125, AF048, AI129, AJ 034) were sub-energetic and no structural variation was detected by the Gap-PCR-based method, whereas the method of the invention detected structural variations containing alpha anti3.7, alpha anti4.2 or- -THAI; in addition, in 33 samples, the method of the invention detects HBA1/2 and HBB gene point mutation which are not included in the detection range of the sub-energy PCR-reverse dot hybridization method. The results of PCR or PCR-Sanger sequencing verification on 33 samples with different conditions, as shown in FIG. 4, are consistent with the method of the present invention.
Therefore, the detection result obtained by the method of the invention is verified by comparison of a control kit and PCR or PCR-Sanger sequencing, and the specificity and the sensitivity reach 100 percent. And compared with the traditional Gap-PCR and PCR-DRB detection technology, the detection precision is improved by 1.88 percent (33/1759). Therefore, the invention combines the multiple PCR method with PacBio sequencing, and can accurately and efficiently detect simultaneouslyHBA1/2AndHBBmultiple mutations in the gene.
The following tables 9 to 12 show point mutations and structural variations that can be detected by the primer set, kit and method of the present invention.
It should be noted that although some features of the present invention have been illustrated by the above embodiments, they are not intended to limit the present invention, and various modifications and changes can be made by those skilled in the art. The reagents, reaction conditions, etc. involved in the multiplex PCR reaction and the third-generation sequencing library construction can be adjusted and changed according to specific needs. It will thus be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims.
Reference to the literature
[1] Modell B, Darlison M. Bull World Health Organ. Global epidemiology of haemoglobin disorders and derived service indicators. 2008 Jun; 86(6): 480-7. Doi:10.2471/blt.06.036673.
[2] Chui DH. Alpha-thalassaemia and population health in Southeast Asia. Ann Hum Biol. 2005 Mar-Apr; 32(2): 123-30. doi: 10.1080/03014460500075084.
[3] Xuxiangmin. Mediterranean anemia prevention and control guidelines [ M ]. Beijing: civil and military medical publishers, 2011.
[4] Zhang L, Zhang Q, Tang Y, Cong P, Ye Y, Chen S, Zhang X, Chen Y, Zhu B, Cai W, Chen S, Cai R, Guo X, Zhang C, Zhou Y, Zou J, Liu Y, Chen B, Yan S, Chen Y, Zhou Y, Ding H, Li X, Chen D, Zhong J, Shang X, Liu X, Qi M, Xu X. Hum Mutat. 2019 Dec;40(12):2221-2229. LOVD-DASH: A comprehensive LOVD database coupled with diagnosis and an at-risk assessment system for hemoglobinopathies. doi: 10.1002/humu.23863. Epub 2019 Sep 11.
[5] The Chinese medical society medical genetics division hereditary diseases clinical practice guidelines are written. Holding the pen: spin, Zhanxinhua, Yang Fang, Xuxiangmin. Clinical practice guidelines for alpha-thalassemia. Journal of Chinese medical genetics. March 2020, Vol.37, No. 3.
[6] Thein SL. Molecular basis of β thalassemia and potential therapeutic targets. Blood Cells Mol Dis. 2018 May;70:54-65. doi: 10.1016/j.bcmd.2017.06.001. Epub 2017 Jun 20.
[7] Shang X, Xu X. Update in the genetics of thalassemia: What clinicians need to know. Best Pract Res Clin Obstet Gynaecol. 2017 Feb;39:3-15. doi: 10.1016/j.bpobgyn.2016.10.012. Epub 2016 Oct 26.
[8] The Chinese medical society medical genetics division hereditary diseases clinical practice guidelines are written. Holding the pen: jade, Wu scholarly, Zhang Xinhua, Von Xiao Du, Xunxiang. Clinical practice guidelines for beta-thalassemia. Journal of Chinese medical genetics. March 2020, Vol.37, No. 3.
[9] Shang X, Li Q, Cai R, Huang J, Wei X, Xu X. Molecular characterization and clinical presentation of HKαα and anti-HKαα alleles in southern Chinese subjects. Clin Genet. 2013 May; 83(5): 472-6. doi: 10.1111/cge.12021. Epub 2012 Oct 10.
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| CN112080558A (en) * | 2019-06-13 | 2020-12-15 | 北京贝瑞和康生物技术有限公司 | Kit and method for simultaneously detecting HBA1/2 and HBB gene mutation |
| CN113564248A (en) * | 2021-09-26 | 2021-10-29 | 北京贝瑞和康生物技术有限公司 | Method and kit for simultaneous detection of multiple mutations at HBA1/2, HBB and HBD gene loci |
| CN114752668A (en) * | 2022-05-13 | 2022-07-15 | 深圳市优圣康生物科技有限公司 | Anemia screening kit and method for CRISPR and CAS9 targeted capture of long DNA fragments |
| WO2022206573A1 (en) * | 2021-03-29 | 2022-10-06 | 北京贝瑞和康生物技术有限公司 | Method and kit for simultaneously detecting a plurality of mutations on hba1/2 and hbb loci |
| CN118853875A (en) * | 2024-09-25 | 2024-10-29 | 北京贝瑞和康生物技术有限公司 | Primer set, kit and application thereof for detecting multiple mutations of human embryonic α-thalassemia |
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| CN111379032A (en) * | 2018-12-28 | 2020-07-07 | 北京贝瑞和康生物技术有限公司 | A method and kit for constructing a sequencing library that simultaneously realizes genome copy number variation detection and gene mutation detection |
| CN112080558A (en) * | 2019-06-13 | 2020-12-15 | 北京贝瑞和康生物技术有限公司 | Kit and method for simultaneously detecting HBA1/2 and HBB gene mutation |
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| CN108796061A (en) * | 2017-11-07 | 2018-11-13 | 深圳华大临床检验中心有限公司 | For the primer sets of thalassaemia mutations type genetic test, kit, its application and library constructing method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112080558A (en) * | 2019-06-13 | 2020-12-15 | 北京贝瑞和康生物技术有限公司 | Kit and method for simultaneously detecting HBA1/2 and HBB gene mutation |
| CN112080558B (en) * | 2019-06-13 | 2024-03-12 | 杭州贝瑞和康基因诊断技术有限公司 | Kit and method for simultaneously detecting HBA1/2 and HBB gene mutation |
| WO2022206573A1 (en) * | 2021-03-29 | 2022-10-06 | 北京贝瑞和康生物技术有限公司 | Method and kit for simultaneously detecting a plurality of mutations on hba1/2 and hbb loci |
| CN113564248A (en) * | 2021-09-26 | 2021-10-29 | 北京贝瑞和康生物技术有限公司 | Method and kit for simultaneous detection of multiple mutations at HBA1/2, HBB and HBD gene loci |
| CN114752668A (en) * | 2022-05-13 | 2022-07-15 | 深圳市优圣康生物科技有限公司 | Anemia screening kit and method for CRISPR and CAS9 targeted capture of long DNA fragments |
| CN118853875A (en) * | 2024-09-25 | 2024-10-29 | 北京贝瑞和康生物技术有限公司 | Primer set, kit and application thereof for detecting multiple mutations of human embryonic α-thalassemia |
| CN118853875B (en) * | 2024-09-25 | 2024-12-20 | 北京贝瑞和康生物技术有限公司 | Primer set, kit and application thereof for detecting multiple mutations of human embryonic α-thalassemia |
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| WO2022206573A1 (en) | 2022-10-06 |
| CN112708674B (en) | 2021-06-29 |
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