CN114381515A - Kit for detecting abnormal hemoglobin gene mutation - Google Patents

Abstract

The application discloses a kit for detecting human abnormal hemoglobin gene mutation. The kit for detecting the abnormal hemoglobin gene mutation comprises a reagent A for detecting 8 abnormal alpha-globin gene point mutations and a reagent B for detecting 6 abnormal beta-globin gene point mutations; the reagent A comprises a group of alpha globin specific primer pairs and eight alpha globin mutation site specific probes; reagent B comprises two groups of beta globin specific primer pairs and five beta globin mutation site specific probes. The kit for detecting the abnormal hemoglobin gene mutation can detect 14 abnormal hemoglobin gene mutations simultaneously, and has the advantages of more comprehensive detection sites, higher efficiency, high accuracy and good repeatability.

Description

Kit for detecting abnormal hemoglobin gene mutation

Technical Field

The application relates to the technical field of human abnormal hemoglobin gene detection, in particular to a kit for detecting abnormal hemoglobin gene mutation.

Background

The pathogenesis of abnormal hemoglobin is a group of hereditary hemoglobinopathies in which the molecular structure of hemoglobin is altered due to changes in the primary structure of globin. The amino acids in the normal hemoglobin are nonpolar amino acids, and constitute the sites of mutual contact between subunits, contact between heme and globin chain, and contact between globin chain spiral segments; if the amino acid abnormality occurs in the molecule, the spatial conformation and the function of the hemoglobin are greatly influenced, and the clinical manifestation is obvious. Certain abnormal hemoglobins can cause thalassemia phenotype, and the common pathological mechanism is that the expression of the affected globin chain is reduced due to structural change of peptide chains or gene mutation. However, if this abnormal change in amino acids occurs outside the hemoglobin molecule, the effect on hemoglobin is small and often there is no apparent clinical manifestation. Most of the abnormal hemoglobins discovered at present belong to the substitution of amino acids outside the hemoglobin molecule, the substitution does not affect the stability of the hemoglobin molecule or the function of the hemoglobin molecule, and patients carrying such mutant genes usually have no obvious clinical manifestation.

Hemoglobinopathies pose a serious physical, psychological and economic burden on the relevant families and society. Hemoglobinopathies can be divided into thalassemia (abbreviated as thalassemia) and abnormal hemoglobin. Most abnormal hemoglobins do not cause clinical phenotype, but a few abnormal hemoglobins such as Hb S, Hb E and the like can exist alone or compound thalassemia to cause obvious clinical manifestations; also, asymptomatic hemoglobinopathies may cause an increase in symptoms with age.

There have been 1300 more aberrant hemoglobin variants globally, at least 44 of which are responsible for non-deleted alpha thalassemia. The main common symptoms are Hb Westmead, Hb CS, Hb QS and Hb E, and the 4 abnormal hemoglobins are Hb variants which can cause alpha-or beta-thalassemia. Other common abnormal hemoglobins include Hb New York, Hb Q-Thailand, Hb J-Bangkok, and the like; wherein Hb Q-Thailand is a variation occurring in the α 1 gene and linked to the- α 4.2 deletion. Hb Q-Thailand complex alpha 0-thalassemia heterozygotes can cause Hb H disease, and abnormal hemoglobins such as Hb New York and Hb J-Bangkok generally do not cause obvious phenotypes. In addition, there are other rare abnormal hemoglobins, which are located in the α -globin gene cluster and the β -globin gene cluster. The main types of hemoglobinopathies are alpha and beta thalassemia, and the incidence rate of the high-incidence area can reach 4.4% -15.2%. The existing research has more researches on the carrying condition of the thalassemia gene, but relatively few researches on abnormal hemoglobin. Most of the abnormal hemoglobins in more than 80 types found in the present environment have mild or even no clinical symptoms; however, moderate to severe anemia may manifest when combined poverty; however, the prior art lacks an effective treatment means, and patients can only rely on long-term blood transfusion for treatment, thereby causing heavy burden to families and society.

High performance liquid chromatography and capillary electrophoresis methods are commonly used clinically, and abnormal hemoglobin is grouped according to the position of an abnormal band appearing during electrophoresis. However, since a plurality of hemoglobins can be contained in the same electrophoretic zone, the electrophoretic technique cannot distinguish the specific types of hemoglobins, and the identification by the globin gene sequence analysis is still required. In particular, most of the abnormal hemoglobin does not affect the function of the hemoglobin, or has little effect on the function, and only a small part of the abnormal hemoglobin can cause corresponding functional abnormality, so that the method has important practical significance for accurately typing the abnormal hemoglobin generated by the electrophoresis analysis to judge the specific property of the abnormal hemoglobin. Therefore, the genotyping of the abnormal hemoglobin is beneficial to enhancing and improving the cognition and attention of the abnormal hemoglobin, solving the practical problem and also has important reference value for genetic counseling and clinical treatment.

At present, the number of kits for detecting abnormal hemoglobin genes in the market is small; existing kits are also only capable of detecting a few types of partially abnormal hemoglobin genes, such as the three alpha globin genes Hb Westmead (WS), Hb ConstantSpring (CS) and Hb Qungsze (QS), or the beta globin gene Hb E (CD 26). With the intensive research and development of abnormal hemoglobin genes and more abnormal hemoglobin bands discovered by the popularization of hemoglobin electrophoresis detection, the existing detection method and products cannot meet the use requirements.

In addition, studies have been reported on the detection of point mutations in the α and β globin genes, such as amplification-inhibited mutation system PCR (ARMS-PCR), allele-specific oligonucleotide hybridization (ASO), PCR-Reverse Dot hybridization (PCR-Reverse Dot, PCR-RDB), and DNA sequencing. Wherein, the amplification hampering mutation system PCR (ARMS-PCR) adopts two PCR reaction tubes to detect one by one aiming at each mutation site, the operation is complicated, the time and the labor are wasted, the detection condition of the technology is difficult to control, and false negative and false positive results are easy to appear. The allele specific oligonucleotide hybridization (ASO) method can reduce false negative and false positive results, but still needs to detect each mutation site one by one, has low detection flux and wastes time and labor. A PCR-Reverse Dot hybridization (PCR-Reverse Dot Blot, PCR-RDB) method has the advantages of high sensitivity, good specificity, high flux and the like, can simultaneously detect three mutation types of WS, QS and CS, is widely applied to alpha-thalassemia gene detection at present, but is complicated in manual operation, firstly carries out PCR amplification on alpha 2 globin gene, and then observes a detection result through a series of operations such as denaturation, hybridization, membrane washing, color development and the like, is long in detection time, and reduces the working efficiency. Meanwhile, the uncovering operation of the PCR product greatly increases the risk of carrying pollution in a laboratory. The DNA sequencing technology comprises the steps of firstly carrying out PCR amplification, then purifying PCR products, constructing a sequencing library, carrying out computer sequencing, carrying out data analysis on sequencing results and the like; the problems of complicated operation, long time consumption, carrying pollution and the like also exist.

In general, the existing method for detecting the abnormal hemoglobin gene point mutation has the disadvantages of high detection cost, large workload, complex operation, small detection flux, difficulty in realizing automation and standardization and incapability of meeting the requirements of large-scale population screening and clinical routine detection at present; in addition, no kit capable of detecting abnormal hemoglobin more comprehensively exists in the current market. Therefore, the development of a technical scheme for genotyping a plurality of common abnormal hemoglobins which can meet the requirements of large-scale population screening and clinical routine detection is urgently needed.

Disclosure of Invention

The purpose of the application is to provide a novel kit for detecting abnormal hemoglobin gene mutation.

The following technical scheme is adopted in the application:

one aspect of the application discloses a kit for detecting abnormal hemoglobin gene mutation, which comprises a reagent A for detecting 8 abnormal alpha-globin gene point mutations and a reagent B for detecting 6 abnormal beta-globin gene point mutations; wherein, the reagent A comprises a group of alpha globin specific primer pairs and eight alpha globin mutation site specific probes; the upstream primer of the group of alpha globin specific primer pairs is a sequence shown by Seq ID No.1, the downstream primer is a sequence shown by Seq ID No.2, and the eight alpha globin mutation site specific probes are sequences shown by Seq ID No.3 to Seq ID No.10 in sequence; the reagent B comprises two groups of beta globin specific primer pairs and five beta globin mutation site specific probes; two groups of beta globin specific primer pairs, wherein the upstream primer of one group of beta globin specific primer pairs is a sequence shown in Seq ID No.11, and the downstream primer is a sequence shown in Seq ID No. 12; the upstream primer of the other group of beta globin specific primer pairs is a sequence shown in Seq ID No.13, and the downstream primer is a sequence shown in Seq ID No. 14; the five beta globin mutation site specific probes are shown as Seq ID No.15 to Seq ID No.19 in sequence;

Seq ID No.1：5’-CGGCACTCTTCTGGTCCCCACAG-3’；

Seq ID No.2：5’-CCACTCAGACTTTATTCAAAGAC-3’；

Seq ID No.3：5’-CCGACCTTACGCCAGGCGGCC-3’；

Seq ID No.4：5’-GCCTCACCTCTCCAGGGCCTC-3’；

Seq ID No.5：5’-AAGGACAGGAACATCCTGC-3’；

Seq ID No.6：5’-GGCATGCCNNNNNNNNNCACGTGCGC-3’；

Seq ID No.7：5’-TTGGGCATNNNNNNNNNGTCCACGTGCGC-3’；

Seq ID No.8：5’-CACCCGAAGCTTGTGCGC-3’；

Seq ID No.9：5’-GGAGGCGTGCACCGCAGGG-3’；

Seq ID No.10：5’-GGAGGTCAGCACGGCGCTCACA-3’；

Seq ID No.11：5’-GGAGCAGGGAGGGCAGGAGCCAGGG-3’；

Seq ID No.12：5’-AGGGTCCCATAGACTCACCCT-3’；

Seq ID No.13：5’-GGCCCTTTTGCTAATCATGTT-3’；

Seq ID No.14：5’-AAGGCCCTTCATAATATCCCC-3’；

Seq ID No.15：5’-AACGTGGATGAAGTTGGTGGT-3’；

Seq ID No.16：5’-CTGTTATGGACAACCCNNNNNNNNNGTGAAG-3’；

Seq ID No.17：5’-CTGTTATNNNNNNNNNACCCTAAGGTGA-3’；

Seq ID No.18：5’-ACGTGGATCCTCAGAACTTCA-3’；

Seq ID No.19：5’-TGGGCCAG CTCACAGACC-3’；

wherein, the probes of the sequences shown in Seq ID No.6, Seq ID No.7, Seq ID No.16 and Seq ID No.17 are arch bridge probes, and the 'NNNNNNNNN' in the four probes represents a sequence which is not matched with a target sequence and has the length of 5-15bp, so that the four probes are in an arch bridge shape after being combined with the target; the 5 'ends of the eight alpha globin mutation site specific probes and the five beta globin mutation site specific probes are all marked with the same or different fluorescent groups, and the 3' ends are all marked with the same or different fluorescent quenching groups.

It should be noted that the kit of the present application is mainly composed of an α globin specific primer pair and eight α globin mutation site specific probes for detecting 8 abnormal α -globin gene point mutations, and a β globin specific primer pair and five β globin mutation site specific probes for detecting 6 abnormal β -globin gene point mutations. In an implementation manner of the application, by combining the kit and the fluorescent probe melting curve technology of the application, eight kinds of alpha-abnormal hemoglobin gene point mutations of CD15, CD30, CD34, CD74, CD75, CD90, CD121 and CD134 and six kinds of beta-abnormal hemoglobin gene point mutations of CD22-1, CD22-2, CD56, CD59, CD101 and CD113 can be rapidly detected at one time, that is, 14 kinds of abnormal hemoglobin gene mutations can be simultaneously detected. The kit can detect abnormal hemoglobin more comprehensively, and can meet the use requirements of large-scale crowd screening and clinical routine gene detection.

It should be noted that, in 14 kinds of abnormal hemoglobin gene mutations detected by the present application, some sites are closely spaced, and in order to better distinguish two mutation sites which are closely spaced, the present application designs arch bridge probes for some sites, such as probes having sequences shown in Seq ID No.6, Seq ID No.7, Seq ID No.16 and Seq ID No. 17. The main principle of the arch bridge probe is that a segment of sequence which is not matched with a target sequence is connected between the probes, and when the probes are combined with the target sequence, the unmatched part of the probes can form a bag-shaped structure, namely the arch bridge shape; the capsular structure entraps one of two adjacent mutation sites therein; therefore, when another unincorporated mutation site is detected through the Tm of the melting peak of the target gene, the interference of the adjacent mutation site can be effectively avoided, and the accurate interpretation of the mutation site with a relatively short distance is realized. The specific base number of the sequence which is connected in the arch bridge probe and does not match with the target sequence is determined according to the distance between two adjacent mutation sites, and the length of the unmatched sequence is about 5-15bp generally. It can be understood that if the length of the mismatch sequence is less than 5bp, it indicates that the positions of two adjacent mutation sites are too close to each other, and it is difficult to distinguish them by the design of the arch bridge probe; if the length of the mismatching sequence is more than 15bp, the interval between two adjacent mutation sites is larger, and a conventional detection probe can be directly designed aiming at the two mutation sites without adopting an arch bridge probe. Also, too long a length of mismatched sequence in the arch bridge probe will affect the normal binding of the arch bridge probe to the target sequence.

In one implementation mode of the application, the kit further comprises an internal reference specific primer pair and an internal reference specific probe for detecting an internal reference gene beta-actin, wherein an upstream primer of the internal reference specific primer pair is a sequence shown by Seq ID No.20, a downstream primer of the internal reference specific primer pair is a sequence shown by Seq ID No.21, and the internal reference specific probe is a sequence shown by Seq ID No. 22;

Seq ID No.20：5’-GCTCCATCCTGGCCTCGC-3’；

Seq ID No.21：5’-CGTCCACCGCAAATGCTTCT-3’；

Seq ID No.22：5’-CGTCAACACCCCAGCCA-3’。

it should be noted that the internal reference specific primer pairs of the sequences shown in Seq ID nos. 20 and 21 are only specific amplification primer pairs of the internal reference gene β -actin used in one specific implementation manner of the present application, and it is not excluded that other internal reference gene β -actin specific primer pairs may also be used, and even other internal reference genes and detection primers thereof may also be used. Of course, in an implementation manner of the present application, specifically, the primer pair of the internal reference gene is added to the reagent a for detecting 8 kinds of α -abnormal hemoglobin gene point mutations and/or added to the reagent B for detecting 6 kinds of β -abnormal hemoglobin gene point mutations for multiplex PCR amplification, so the selection of the internal reference gene and its specific primer pair also needs to consider the influence on the multiplex PCR amplification.

In one implementation mode of the application, the 5 'end of the probe with the sequence shown in Seq ID No.3 is marked with a fluorescent group CY5, and the 3' end is marked with a fluorescence quenching group BQ 3; the 5 'end of the probe with the sequence shown in Seq ID No.4 is marked with a fluorescent group CY5, and the 3' end is marked with a fluorescence quenching group BQ 3; the 5 'end of the probe with the sequence shown in Seq ID No.5 is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.6 is marked with a fluorescent group FAM, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.7 is marked with a fluorescent group FAM, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.8 is marked with a fluorescent group ROX, and the 3' end is marked with a fluorescence quenching group BQ 2; the 5 'end of the probe with the sequence shown in Seq ID No.9 is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.10 is marked with a fluorescent group ROX, and the 3' end is marked with a fluorescence quenching group BQ 2; the 5 'end of the probe with the sequence shown in Seq ID No.15 is marked with a fluorescent group FAM, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.16 is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.17 is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.18 is marked with a fluorescent group ROX, and the 3' end is marked with a fluorescence quenching group BQ 2; the 5 'end of the probe with the sequence shown in Seq ID No.19 is marked with a fluorescent group CY5, and the 3' end is marked with a fluorescence quenching group BQ 3; the 5 'end of the probe having the sequence shown in Seq ID No.22 is labeled with a fluorescent group FAM, and the 3' end is labeled with a fluorescence quenching group BQ 1.

In one implementation of the present application, the reagent a for detecting 8 α -aberrant hemoglobin gene point mutations is reacted in one PCR reaction tube, and the reagent B for detecting 6 β -aberrant hemoglobin gene point mutations is reacted in another PCR reaction tube; thus, in principle, the fluorophores labeled by each probe in the same PCR reaction tube need to be different to be distinguished from each other, and the specific fluorophores defined above are only fluorophores specifically used in one implementation manner of the present application, and do not exclude that other fluorophores, such as TET, JOE, CY3, etc., can also be used, and are selected according to the real-time fluorescent PCR amplification instrument used. The fluorescence quenching group may be selected according to the requirement, and is not particularly limited herein.

It should be further noted that, in the same tube reaction, if the difference between the melting curve peak values of the two targets is large, the detection probes of the two targets may also use the same fluorophore; this reduces the number of fluorescence detection channels required and requires less instrumentation. At this time, two or more melting curve peaks can be obtained for the same fluorescence detection channel. For example, in the eight alpha globin mutation site specific probes, the probes of Seq ID No.3 and Seq ID No.4 are both labeled fluorescent groups CY5, and the melting curve peak values of the two targets can be obtained in the detection channel of the CY5 fluorescent signal.

The application also discloses application of the kit in preparing a reagent for detecting abnormal hemoglobin gene mutation or diagnosing hemoglobinopathy.

It can be understood that the kit of the present application is a specific detection primer and probe designed for 8 kinds of alpha-abnormal hemoglobin gene point mutations and 6 kinds of beta-abnormal hemoglobin gene point mutations, and therefore, the kit can be used for preparing a reagent for detecting abnormal hemoglobin gene mutation, and further can be used for diagnosing hemoglobinopathy. The primers and the probes can be independently packaged according to requirements, for example, dry powder forms of the primers and the probes are used as products, and are diluted when used, and a reaction system is prepared according to requirements; or according to the preferred scheme given in an implementation manner of the present application, the primers and the probes in the reagent a for detecting 8 kinds of alpha-abnormal hemoglobin gene point mutations are combined together to form a group of reaction system, the primers and the probes in the reagent B for detecting 6 kinds of beta-abnormal hemoglobin gene point mutations are combined together to form another group of reaction system, and when in use, the reaction system is directly subpackaged into a PCR tube, and the DNA to be detected and the DNA polymerase are added.

The application also discloses another kit for detecting abnormal hemoglobin gene mutation, which comprises a reaction solution I and a reaction solution II; the reaction solution I comprises a group of alpha globin specific primer pairs, eight alpha globin mutation site specific probes, a group of internal reference specific primer pairs, an internal reference specific probe, dNTPs and PCR reaction buffer solution; the upstream primer of the group of alpha globin specificity primer pairs is a sequence shown by Seq ID No.1, the downstream primer is a sequence shown by Seq ID No.2, the eight alpha globin mutation site specificity probes are sequences shown by Seq ID No.3 to Seq ID No.10 in sequence, the upstream primer of the group of internal reference specificity primer pairs is a sequence shown by Seq ID No.20, the downstream primer is a sequence shown by Seq ID No.21, and the internal reference specificity probe is a sequence shown by Seq ID No. 22; the reaction solution II comprises two groups of beta globin specific primer pairs, five beta globin mutation site specific probes, dNTPs and PCR reaction buffer solution; two groups of beta globin specific primer pairs, wherein the upstream primer of one group of beta globin specific primer pairs is a sequence shown in Seq ID No.11, and the downstream primer is a sequence shown in Seq ID No. 12; the upstream primer of the other group of beta globin specific primer pairs is a sequence shown in Seq ID No.13, and the downstream primer is a sequence shown in Seq ID No. 14; the five beta globin mutation site specific probes are shown as Seq ID No.15 to Seq ID No.19 in sequence; wherein "NNNNNNNNN" in the probes of the sequences shown in Seq ID No.6, Seq ID No.7, Seq ID No.16 and Seq ID No.17 represents a sequence of 5 to 15bp in length which does not match the target sequence; eight alpha globin mutation site specific probes, one internal reference specific probe and five beta globin mutation site specific probes have the same or different fluorescent groups marked at the 5 'ends and the same or different fluorescent quenching groups marked at the 3' ends.

In the kit for detecting abnormal hemoglobin gene mutation of the present invention, the reagent for detecting abnormal hemoglobin gene mutation of the present invention is actually assembled into the reaction solution I and the reaction solution II according to a reaction system optimized in advance. Wherein, the reaction liquid I is a reaction system for detecting 8 kinds of alpha-abnormal hemoglobin gene point mutation, and the reaction liquid II is a reaction system for detecting 6 kinds of beta-abnormal hemoglobin gene point mutation. Therefore, the specific definition of the primers and probes in reaction solutions I and II, and the fluorescent group and the fluorescence quenching group of the probes can be referred to the reagent of the present application.

In one implementation manner of the present application, the kit of the present application further includes a reaction solution III, and the reaction solution III is DNA polymerase.

It is understood that DNA polymerase can be selectively added to the kit of the present application as desired for ease of use; of course, the DNA polymer may be a product purchased from a commercial supplier, and is not particularly limited thereto.

In one implementation manner of the present application, the kit of the present application includes a reagent a positive quality control substance and a reagent B positive quality control substance; the positive quality control product of the reagent A is a positive plasmid containing an amplification target sequence of the group of alpha globin specific primer pairs, and the positive plasmid respectively contains a segment with known content and homozygous CD15 genotype and a segment with known content and wild genotype; the positive quality control product of the reagent B is a positive plasmid containing the amplification target sequences of the two groups of beta globin specificity primer pairs, and respectively contains a segment with known content and homozygous CD113 genotype and a segment with known content and wild genotype.

The CD15 homozygous genotype used in the reagent a positive quality control product of the present application may be replaced with a homozygous genotype for another mutation among the 8 α -aberrant hemoglobin gene point mutations, for example, a CD30 homozygous genotype, a CD34 homozygous genotype, a CD74 homozygous genotype, a CD75 homozygous genotype, a CD90 homozygous genotype, a CD121 homozygous genotype, or a CD134 homozygous genotype, or any combination of the 8 homozygous genotypes. Similarly, the CD113 homozygous genotype used in the reagent B positive quality control may be replaced with a purified genotype of another mutation among the 6 β -aberrant hemoglobin gene point mutations, such as CD22-1 homozygous genotype, CD22-2 homozygous genotype, CD56 homozygous genotype, CD59 homozygous genotype, or CD101 homozygous genotype, or any combination of these 6 homozygous genotypes.

In one implementation manner of the present application, the kit of the present application further comprises a reagent a negative quality control product and a reagent B negative quality control product, wherein the reagent a negative quality control product is a plasmid containing the amplification target sequence of the group of alpha globin specific primer pairs, and the genotype of the amplification target sequence is wild type; the negative quality control product of the reagent B is a plasmid which contains amplification target sequences of the two groups of beta globin specific primer pairs and the genotype of which is wild type.

The positive quality control material and the negative quality control material are mainly used for controlling the quality of a reaction system and ensuring the normal operation of the reaction and the accuracy and effectiveness of result judgment; in principle, in the actual use process, artificially synthesized nucleic acid or PCR amplified fragment with known concentration and genotype can be used as a positive control or a negative control to achieve the purpose of quality control; plasmids are primarily for the convenience of long-term storage. Therefore, the positive quality control material and the negative quality control material can be selectively added into the kit according to the requirement.

In one implementation of the present application, the kit of the present application further comprises a blank control, which is sterilized deionized water.

It is understood that the blank of sterilized deionized water can be selectively added to the kit according to the requirement, and the blank of sterilized deionized water conventionally used in laboratories can also be used, and is not particularly limited herein.

The beneficial effect of this application lies in:

the kit for detecting the abnormal hemoglobin gene mutation can detect 14 abnormal hemoglobin gene mutations simultaneously, has more comprehensive detection sites, higher efficiency and high automation degree, and improves the detection efficiency of clinical samples; the arch bridge probe design is utilized to distinguish two point mutation sites with close distance, and the distinguishing degree is high, the accuracy is high and the repeatability is good. In an application mode of the application, a PCR melting curve technology is combined, and mutation site interpretation is directly carried out through melting peaks appearing in different channels and corresponding Tm values, so that the method is more intuitive; and the method can be directly carried out after the real-time fluorescent PCR amplification reaction, and the operation of opening the tube is not needed, so that the probability of sample pollution is reduced. The reagent and the kit can detect abnormal hemoglobin more comprehensively, and can meet the use requirements of large-scale crowd screening and clinical routine molecular diagnosis.

Drawings

FIG. 1 is a schematic illustration of an arch bridge probe in an embodiment of the present application;

FIG. 2 shows the results of the detection of CD74 heterozygous and CD75 heterozygous mutant samples by the A-tube reagent in the examples of the present application;

FIG. 3 shows the results of the detection of CD34 heterozygous mutation samples and CD121 heterozygous mutation samples by the A-tube reagent in the examples of the present application;

FIG. 4 shows the results of the detection of CD90 heterozygous and CD134 heterozygous mutant samples by the A-tube reagent in the examples of the present application;

FIG. 5 shows the results of the detection of CD15 heterozygous and CD30 heterozygous mutant samples by the A-tube reagent in the examples of the present application;

FIG. 6 shows the results of the detection of CD22-1 heterozygous and CD22-2 heterozygous mutant samples by the B-tube reagent in the examples of the present application;

FIG. 7 shows the results of the detection of CD56 heterozygous mutation samples and CD59 heterozygous mutation samples by the B-tube reagent in the examples of the present application;

FIG. 8 shows the result of detecting CD101 heterozygous mutation sample by B-tube reagent in the example of the present application;

FIG. 9 shows the result of detecting CD113 heterozygous mutation sample by B-tube reagent in the example of the present application.

Detailed Description

The existing abnormal hemoglobin gene mutation detection technology has the defects of high operation requirement conditions, tedious method, long time consumption, difficulty in realizing automation and the like, and cannot meet the requirements of simple, convenient and quick clinical diagnosis. In addition, no product capable of comprehensively detecting abnormal hemoglobin exists in the current market. In the existing technical scheme of the melting curve of the fluorescent probe, the detection area graduation of adjacent single-base mutation sites is poor, the difference of the change of melting point values (Tm values) caused by mutation of different sites is small, and the mutation conditions that misjudgment is easy to occur or adjacent different sites cannot be distinguished are caused during result interpretation. More importantly, no research or report about the search technology of the abnormal hemoglobin gene mutation melting curve technology exists at present.

Therefore, the reagent and the kit for detecting the abnormal hemoglobin gene are developed rapidly and stably by adopting a fluorescent probe melting curve technology, and 8 alpha-globin gene point mutations and 6 beta-globin gene point mutations can be detected simultaneously through one test. Meanwhile, the arch bridge probe is used, so that the Tm value of a melting peak of a target gene can be prevented from being interfered by SNP (single nucleotide polymorphism) or other detection sites, and the accurate interpretation of a mutation site with a short distance can be realized.

Specifically, the reagent of the present application comprises a reagent A for detecting 8 kinds of alpha-abnormal hemoglobin gene point mutations, and a reagent B for detecting 6 kinds of beta-abnormal hemoglobin gene point mutations; wherein, the reagent A comprises a group of alpha globin specific primer pairs and eight alpha globin mutation site specific probes; the upstream primer of the group of alpha globin specific primer pairs is a sequence shown by Seq ID No.1, the downstream primer is a sequence shown by Seq ID No.2, and the eight alpha globin mutation site specific probes are sequences shown by Seq ID No.3 to Seq ID No.10 in sequence; a group of internal reference specific primer pairs for detecting internal reference gene beta-actin and an internal reference specific probe for detecting internal reference gene beta-actin, wherein the upstream primer of the group of specific internal reference primer pairs is a sequence shown by Seq ID No.20, the downstream primer is a sequence shown by Seq ID No.21, and the specific internal reference probe is a sequence shown by Seq ID No. 22; the reagent B comprises two groups of beta globin specific primer pairs and five beta globin mutation site specific probes; two groups of beta globin specific primer pairs, wherein the upstream primer of one group of beta globin specific primer pairs is a sequence shown in Seq ID No.11, and the downstream primer is a sequence shown in Seq ID No. 12; the upstream primer of the other group of beta globin specific primer pairs is a sequence shown in Seq ID No.13, and the downstream primer is a sequence shown in Seq ID No. 14; the five beta globin mutation site specific probes are shown as Seq ID No.15 to Seq ID No.19 in sequence; wherein "NNNNNNNNN" in the probe represents a sequence of 5-15bp in length that does not match the target sequence; the 5 'ends of the eight alpha globin mutation site specific probes, the one internal reference specific probe and the five beta globin mutation site specific probes are all marked with the same or different fluorescent groups, and the 3' ends are all marked with the same or different fluorescent quenching groups.

The main principle of the arch bridge probe is that a section of base which is not matched with a target sequence is connected in the middle of the probe, and when the probe is combined with the target sequence, the unmatched part of the base can form a saccular structure; the saccular structure can envelop mutation sites adjacent to the target, does not hybridize with the target sequence, and does not influence the detection of the target; the number of the basic groups of the arch bridge probe is determined according to the distance between two mutation sites, and is generally about 5-15 bp. The specific principle is shown in fig. 1.

The principle of the melting curve technology of the application is as follows: the probes for detecting alpha-abnormal hemoglobin and beta-abnormal hemoglobin in the application comprise hybridization probes and arch bridge probes. The principle of the melting curve technology is to analyze a melting curve and a melting point value (Tm) formed by hybridizing a specific probe with a target sequence after PCR amplification is completed, and detect whether a base mutation and a specific mutation type exist in a probe coverage area. The method specifically comprises PCR amplification and melting curve analysis, wherein asymmetric PCR is firstly used for amplification, a single-chain target sequence is enriched, so that a probe can be hybridized with the target sequence in the melting curve analysis process, specifically, a corresponding probe is designed in a region needing to be detected, an upstream primer and a downstream primer are designed at the periphery of the designed probe, and a segment containing a region to be detected is amplified by using the upstream primer and the downstream primer; and (4) after the PCR amplification is finished, performing melting curve analysis.

The reagent and the kit can simultaneously detect 8 kinds of alpha-abnormal hemoglobin gene point mutations and 6 kinds of beta-abnormal hemoglobin gene point mutations, are combined with a fluorescence PCR-melting curve method, and have the advantages of comprehensive detection sites, simplicity in operation, rapidness in detection and the like; the method makes up the defects of the traditional detection method, not only increases the detection types of the abnormal hemoglobin genes, but also simplifies the operation steps, shortens the time for detecting the abnormal hemoglobin genes, provides a better platform for developing efficient and powerful genetic disease detection, and can better meet the use requirements of large-scale population screening and clinical routine molecular diagnosis.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Example one

Materials and methods

1. Detection material

In this example, 80 whole blood samples were tested and tested; of these, 60 were positive samples, and 20 were negative samples.

The whole blood sample collection and storage method of this example is as follows:

(1) all sample sources were anticoagulated whole blood, and the anticoagulant used was sodium citrate or EDTA.

(2) Collecting samples: and (4) extracting 1-5 mL of venous blood into a tube containing an anticoagulant, and marking sample information.

(3) Blood sample preservation: standing the anticoagulated whole blood at room temperature for no more than 24 hours, storing at 2-8 ℃ for no more than one month, storing at-30 to-15 ℃ for no more than two years, storing at-85 to-75 ℃ for a long time, and avoiding repeated freezing and thawing during freezing storage.

(4) Blood sample transportation: when the anticoagulated whole blood is transported, an ice kettle or a foam box is required to be sealed with an ice bag, the ice bag is ensured not to be thawed, and the time limit in transit is not more than 72 hours.

2. Nucleic acid extraction

In this example, genomic DNA of 80 whole blood samples was extracted using "nucleic acid extraction reagent" available from Yaerg corporation (Yuetshen apparatus No. 20150099, Yuetshen apparatus No. 20200318). The specific operation refers to the kit use instruction.

The DNA concentration and purity of the extracted genome DNA of 80 whole blood samples are detected, and a nucleic acid quantifier or an ultraviolet spectrophotometer is particularly adopted, so that the results show that the concentration of the 80 whole blood samples in the test is 20-100 ng/mu L, the purity A260/280 is 1.6-2.2, and the subsequent detection requirements can be met.

3. Design and screening of primers and probes

In this example, human globin gene sequences were obtained from GenBank database, and Primer Premier 5 and Oligo6.0 were used to design corresponding Primer and probe sequences based on different deletion or mutation types. Both primers and probes were artificially synthesized oligonucleotides, and were synthesized by Shanghai Yingjun Biotechnology Ltd. In this example, specific detection primers and probes were designed for eight α -aberrant hemoglobin gene point mutations of CD15, CD30, CD34, CD74, CD75, CD90, CD121 and CD134, and six β -aberrant hemoglobin gene point mutations of CD22-1, CD22-2, CD56, CD59, CD101 and CD113, and the specific primer, probe sequence and mutation site to be detected are shown in table 1.

TABLE 1 primer Probe sequences that detect the site of mutation

In Table 1, "NNNNNNNNN" indicates a sequence of 5-15bp in length that does not match the target sequence, and this example specifically uses a random sequence of 9 bases that repeats four bases selected from A, T, C, and G.

The detailed information of the 8 α -aberrant hemoglobin gene point mutations and the 6 β -aberrant hemoglobin gene point mutations are shown in table 2.

TABLE 2 mutation site information

4. Construction of positive quality control product and negative quality control product plasmid

The plasmid is constructed by synthetic bases, specifically, by Jinzhi Biotechnology, Inc., Suzhou.

And selecting an alpha globin specific primer pair to amplify homozygous mutation positive plasmid with the genotype of a target sequence of CD15 and plasmid with the genotype of wild type, and mixing to prepare the positive quality control product of the reagent A.

Selecting a homozygous mutation positive plasmid with the genotype of the amplified target sequence of the beta globin specificity primer pair as CD113 and a plasmid with the genotype of the wild type to be mixed to prepare the positive quality control product of the reagent B.

And selecting a plasmid with the genotype of the amplified target sequence of the alpha globin specific primer pair as a wild type as a negative quality control product of the reagent A.

And selecting the plasmid with the genotype of the amplification target sequence of the beta globin specific primer pair as the wild type as the negative quality control product of the reagent B.

PCR amplification and melting curve analysis

In the embodiment, 8 primers and probes for alpha-abnormal hemoglobin gene point mutation and internal reference specific primers and probes for internal reference gene beta-actin are added into the same reaction tube for multi-target detection, and are marked as tube A reagents; 6 primers and probes of beta-abnormal hemoglobin gene point mutation are added into the same reaction tube for multi-target detection, and the reaction tube is marked as a B tube reagent. The PCR reaction solution system of the two-tube reagent is shown in Table 3.

TABLE 3 PCR reaction System

According to the PCR reaction system prepared in Table 3, the prepared A tube reagent was dispensed into a 96-well plate, and the amount of each well was 20. mu.L; the B-tube reagent was dispensed into another 96-well plate in an amount of 20. mu.L per well. Extracting the genome DNA of 80 whole blood samples, wherein each sample DNA corresponds to one hole, and adding 5 mu L of DNA into a 96-well plate filled with a tube A reagent; similarly, 80 samples of genomic DNA from whole blood were prepared in a single well, and 5. mu.L of the DNA was dispensed into a 96-well plate containing the reagent for B-tube. Namely, the genomic DNA of 80 whole blood samples was detected by using the A-tube reagent and the B-tube reagent, respectively. Each 96-well plate of each tube of reagent is provided with at least one positive quality control product, one negative quality control product and one water blank control.

Two 96-well plates were placed on a real-time fluorescent PCR amplification instrument Bio-Rad CFX96 for reaction and melting curve analysis, with the reaction conditions: pre-denaturation at 95 ℃ for 5 min; then 50 cycles were entered: 30sec at 95 ℃, 20s at 62 ℃ and 30s at 72 ℃; after the circulation is finished, denaturation is carried out for 5min at 95 ℃; renaturation at 40 deg.C for 10 min; melting analysis and collecting FAM, HEX, ROX, Cy5 channel fluorescence signals at 40-90 ℃.

6. Sensitivity test

Sensitivity analysis is carried out on detection sites of 8 alpha-point mutations (CD15, CD30, CD34, CD74, CD75, CD90, CD121 and CD134) and 6 beta-point mutations (CD22-1, CD22-2, CD56, CD59, CD101 and CD113) by adopting the reaction system. The genomic DNA samples containing the respective genotypes were diluted to the concentrations of 100 ng/. mu.L, 50 ng/. mu.L, 30 ng/. mu.L, 10 ng/. mu.L, 5 ng/. mu.L, 2 ng/. mu.L, and 1 ng/. mu.L. And (3) detecting and analyzing the melting curve of 7 diluted samples by adopting a reaction system in the step 5 of PCR amplification and melting curve analysis.

7. Specificity test

The following samples were subjected to nucleic acid extraction according to the method "2. nucleic acid extraction", respectively: (1) a whole blood sample of clinically normal measurement of sodium citrate and EDTA, (2) a blood sample of a patient who had been administered deferoxamine, (3) a hemolytic sample, (4) a blood sample in which the concentrations of triglyceride and total bilirubin in a lipemia sample were 13.8mmol/L and 359.28. mu. mol/L, respectively, (5)3 samples of G-6-PD blood, (6)1 sample of iron deficiency anemia, (7)1 sample of Toxoplasma infected whole blood, and (8)1 sample of hepatitis B virus DNA. And 5.PCR amplification and melting curve analysis, wherein the reaction system in the step 5 is adopted to detect the nucleic acid extracted from the sample and analyze the melting curve.

Second, the detection result

Test results of 1.80 examples of Whole blood samples

The detection results of 80 samples in the embodiment show that 20 negative samples only have the detection signal of the reference gene beta-actin and no other signals; the genotype of 60 positive samples was consistent with expectations with an accuracy of 100%. It is demonstrated that the primers and probes of this example can accurately and effectively detect eight alpha-aberrant hemoglobin gene point mutations and six beta-aberrant hemoglobin gene point mutations.

2. Positive quality control product and negative quality control product plasmid construction result

The detection result of the A tube reagent positive quality control product is CD15 heterozygous, and the detection result of the negative quality control product is wild type; the detection result of the positive quality control product of the tube B reagent is CD113 heterozygosis, and the detection result of the negative quality control product is wild type; in agreement with the expectations.

3. Results of sensitivity test

The results of the sensitivity test showed that the minimum concentration of genomic DNA that could be stably detected for each genotype in this example was 1 ng/. mu.L.

4. Results of specificity test

The specific detection result shows that the detection result of the genome DNA extracted from the whole blood sample with the clinical normal dose of sodium citrate and EDTA is consistent with the detection result of the genome DNA extracted from the whole blood sample without adding sodium citrate and EDTA. Indicating that the sodium citrate and EDTA in the clinical normal dose are not the interfering substances detected in the present example.

The results of the genomic DNA extraction and detection of blood samples from patients taking deferoxamine showed that deferoxamine is not an interfering substance for the detection of the present example.

The results of the genomic DNA assay of the hemolyzed sample show that even complete hemolysis does not interfere with the assay results of this example.

The results of genomic DNA detection of the blood samples, in which the concentrations of triglyceride and total bilirubin in the jaundice sample were 13.8mmol/L and 359.28. mu. mol/L, respectively, showed that the concentrations of triglyceride and total bilirubin in the jaundice sample were not interfering with the detection of this example, so that the case where triglyceride was less than or equal to 13.8mmol/L or total bilirubin was less than or equal to 359.28. mu. mol/L was not interfering with the detection of this example, and was not an interfering substance.

In addition, in the specificity detection of 6 samples, the first 5 samples are negative, and the result of the hepatitis B virus DNA sample is no signal, namely 6 samples have no cross reaction; the primers and probes of the present example are shown to specifically detect only 8 designed alpha-abnormal hemoglobin gene point mutations and 6 designed beta-abnormal hemoglobin gene point mutations, and are not interfered by other mutation sites.

Example two

In this example, based on the primers and probes designed in the first example, a kit for detecting abnormal hemoglobin genes was constructed in combination with the PCR melting curve technique. The method comprises the following specific steps:

[ PRODUCT NAME ]

General name: abnormal hemoglobin gene detection kit (fluorescence PCR-melting curve method)

English name: abnormal Hemoglobin Genotyping Kit (Fluorescence PCR-Multi Curve Analysis)

[ PACKAGING SPECIFICATIONS ] 24 copies/BOX, 48 copies/BOX

[ USE ]

The kit is used for detecting 8 alpha-globin gene point mutations and 6 beta-globin gene point mutations related to abnormal hemoglobin in a clinical sample, wherein the 8 alpha-globin gene point mutations comprise CD15, CD30, CD34, CD74, CD75, CD90, CD121 and CD 134. The 6 beta-globin gene point mutations include CD22-1, CD22-2, CD56, CD59, CD101 and CD 113.

[ inspection principle ]

The kit is based on fluorescence PCR amplification and melting curve analysis.

Designing specific PCR primer, amplifying to obtain certain length DNA segment containing the deletion genotype to be detected.

The detection process comprises the steps of amplifying by using asymmetric PCR (polymerase chain reaction), enriching a single-chain target sequence, and enabling a probe to be hybridized with the target sequence in the melting curve analysis process, wherein the specific implementation mode is that a corresponding probe is designed in a region needing to be detected, an upstream primer and a downstream primer are designed on the periphery of the designed probe, and a segment containing a region to be detected is amplified by using the upstream primer and the downstream primer; and (4) after the PCR amplification is finished, performing melting curve analysis.

And judging the genotype of the template according to the size of the melting point value and the shape of the melting peak appearing in different channels.

[ Main Components ]

1. The main components of the kit

The reaction solution I comprises a group of alpha globin specific primer pairs, eight alpha globin mutation site specific probes, a group of internal reference specific primer pairs, an internal reference specific probe, dNTPs and PCR reaction buffer solution; the amounts of the components are referred to in example one, table 3, "tube a reagent". The reaction solution II comprises two groups of beta globin specific primer pairs, five beta globin mutation site specific probes, dNTPs and PCR reaction buffer solution; the amounts of the components were referred to in Table 3 of example one as "tube B reagent".

2. Other major reagents required for this assay

Whole blood genomic DNA extraction reagent: a nucleic acid extraction reagent of the bioenergy biotechnology (Shenzhen) Limited (Yuejiezhen No. 20150099, Yuejiezhen No. 20200318).

[ storage conditions and term of validity ]

Storage conditions were as follows: the kit is stored at-30 to-15 ℃ to avoid repeated freeze thawing.

The validity period is as follows: 6 months.

[ APPLICATION APPARATUS ]

The kit is suitable for real-time fluorescent PCR amplification instruments with melting curve analysis functions, such as Bio-Rad CFX96, Rotor-Gene LC480 and SLAN 96P/S.

[ sample requirements ]

1. The sample source of the kit is anticoagulated whole blood, and the anticoagulant is sodium citrate or EDTA.

2. Collecting samples: and (4) extracting 1-5 mL of venous blood into a tube containing an anticoagulant, and marking sample information.

3. Blood sample preservation: the anticoagulated whole blood is placed at room temperature for no more than 24 hours, stored at 2-8 ℃ for no more than one month, stored at-30 to-15 ℃ for no more than two years, stored at-85 to-75 ℃ for a long time, and repeatedly freezing and thawing are avoided during freezing storage.

4. Blood sample transportation: when the anticoagulated whole blood is transported, an ice kettle or a foam box is required to be sealed with an ice bag, the ice bag is ensured not to be thawed, and the time limit in transit is not more than 72 hours.

[ MEASUREMENT METHOD ]

Extraction of whole blood DNA:

the nucleic acid extraction reagent (Yuejing Shenzhen 20150099, Yuejing Shenshu 20200318) of the Limited company of the bioenergy biotechnology (Shenzhen) is recommended to be used for extracting the human genome DNA.

The concentration and purity of the template DNA before PCR can be determined by a nucleic acid quantifier or an ultraviolet spectrophotometer. The kit requires that the concentration of the genomic DNA to be detected is 1-100 ng/mu L, and the purity (A260/280) is 1.6-2.2.

PCR amplification

All the components are taken out of the kit, melted at room temperature, uniformly mixed by oscillation, and centrifuged at 5000rpm for 5-10 seconds. The experimental groups of the same batch of experiments are set, the eight-tube or 96-hole PCR plate is taken out, and the experimental group and the control group are marked. The required amount of each tube of reaction liquid is equal to the number of samples to be detected, namely N +1 positive quality control +1 negative quality control +1 blank control, the reaction liquid can be directly subpackaged into 20 mu L/part for use, 5 mu L of extracted sample DNA to be detected is added into the PCR reaction liquid, and the total reaction amount is 25 mu L.

PCR amplification was performed under the following conditions: pre-denaturation at 95 ℃ for 5 min; then 50 cycles were entered: 30sec at 95 ℃, 20s at 62 ℃ and 30s at 72 ℃; after the circulation is finished, denaturation is carried out for 5min at 95 ℃; renaturation at 40 deg.C for 10 min; melting analysis at 40-90 ℃ and collecting FAM, HEX, ROX and Cy5 channel fluorescence signals.

3. Interpretation of results

[ REFERENCE VALUE ]

The Tm values of the melting peaks of each gene in 4 channels were in the following ranges:

[ interpretation of test results ]

Typical detection results of the respective mutation samples in the tube A are shown in FIGS. 2 to 5, and typical detection results of the respective mutation samples in the tube B are shown in FIGS. 6 to 9. Wherein, FIG. 2 is a melting curve of CD74 hybrid and CD75 hybrid detected by FAM fluorescence channel, W represents wild type, M represents mutant type; FIG. 3 is a melting curve of a CD34 hybrid and a CD121 hybrid detected by a HEX fluorescence channel, wherein W represents a wild type and M represents a mutant type; FIG. 4 is a melting curve of CD90 hybrid and CD134 hybrid detected by ROX fluorescence channel, wherein W represents wild type and M represents mutant type; FIG. 5 is a melting curve of CD15 hybrid and CD30 hybrid detected by CY5 fluorescence channel, wherein W represents wild type and M represents mutant; FIG. 6 shows the melting curve of CD22-1 hybrid and CD22-2 hybrid detected by FAM fluorescence channel, wherein W represents wild type and M represents mutant type; FIG. 7 shows the melting curve of the hybrid CD56 and the hybrid CD59 detected by the HEX fluorescence channel, wherein W represents the wild type and M represents the mutant type; FIG. 8 is a melting curve of CD101 heterozygote detected by ROX fluorescence channel, wherein W represents wild type, and M represents mutant type; FIG. 9 shows the melting curve of CD113 hybrid detected by CY5 fluorescence channel, wherein W represents wild type and M represents mutant type. The results of fig. 2 to 9 are in accordance with the values in the aforementioned "[ reference value ] table.

The melting peak Tm values in the tables and fig. 2 to 9 are typical reference values and result charts obtained by using a reagent a positive control, a reagent a negative control, a reagent B positive control, and a reagent B negative control as detection targets. The above numerical values can be directly used for result interpretation of the sample to be detected.

[ PRODUCT PROPERTIES INDICATOR ]

1. Assay accuracy

60 clinical positive samples and 20 clinical negative samples are used, the result shows the corresponding genotype, the research result completely accords with the sequencing result, and the positive coincidence rate and the negative coincidence rate of the product both reach 100 percent.

2. Sensitivity of analysis

The kit disclosed by the invention is used for carrying out sensitivity analysis on 8 alpha-point mutation detection sites and 6 beta-point mutation detection sites, each sample comprises 7 concentration gradients, and the lowest concentration of the genomic DNA, which can be stably detected by each genotype, is determined to be 1 ng/mu L.

3. Analysis of specificity

Through an interference screening test, the sodium citrate and the EDTA which are clinically normal in dosage are not interference substances of the product; when a sample of a patient taking past ferrioxamine is detected by the product, the detection result is not influenced, which indicates that the deferoxamine is not an interfering substance of the product; the hemolytic sample (even if completely hemolytic) does not interfere with the detection result of the kit; the concentrations of triglyceride in a lipemia sample and total bilirubin in a jaundice sample are respectively 13.8mmol/L and 359.28 mu mol/L, which reach a clinical extremely high level, but the detection of the lipemia sample and the jaundice sample is not interfered, so when the concentration of triglyceride is less than or equal to 13.8mmol/L or the concentration of total bilirubin is less than or equal to 359.28 mu mol/L, the detection result of the kit is not interfered, and the lipemia sample and the jaundice sample are not interfered substances of the product.

The product is used for detecting 6 clinical samples out of the detection range of the product, including 3G-6-PD clinical samples, 1 iron deficiency anemia clinical sample, 1 whole blood sample infected with toxoplasma gondii and 1 hepatitis B virus DNA clinical sample, wherein the first 5 samples are negative, and the hepatitis B virus DNA sample result is no signal, namely 6 samples have no cross reaction.

4. Repeatability of

The test method is characterized in that the test method is carried out on products of different batches and different persons (2 persons), the operation is carried out for 2 times a day, the operation is carried out for 2 days totally, and each reference product is tested for 3 times repeatedly. The method can repeatedly and stably detect the alpha-thalassemia genotype for multiple times under different experimental conditions, and the results show consistency.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

SEQUENCE LISTING

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<170> PatentIn version 3.3

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Claims (10)

1. A kit for detecting human abnormal hemoglobin gene mutation is characterized in that: comprises a reagent A for detecting 8 abnormal alpha-globin gene point mutations and a reagent B for detecting 6 abnormal beta-globin gene point mutations;

the reagent A comprises a group of alpha globin specific primer pairs and eight alpha globin mutation site specific probes; the upstream primer of the group of alpha globin specific primer pairs is a sequence shown by Seq ID No.1, the downstream primer is a sequence shown by Seq ID No.2, and the eight alpha globin mutation site specific probes are sequences shown by Seq ID No.3 to Seq ID No.10 in sequence;

the reagent B comprises two groups of beta globin specific primer pairs and five beta globin mutation site specific probes; in the two groups of beta globin specific primer pairs, an upstream primer of one group of beta globin specific primer pairs is a sequence shown in Seq ID No.11, and a downstream primer is a sequence shown in Seq ID No. 12; the upstream primer of the other group of beta globin specific primer pairs is a sequence shown in Seq ID No.13, and the downstream primer is a sequence shown in Seq ID No. 14; the five beta globin mutation site specific probes are sequences shown as Seq ID No.15 to Seq ID No.19 in sequence;

Seq ID No.1：5’-CGGCACTCTTCTGGTCCCCACAG-3’；

Seq ID No.2：5’-CCACTCAGACTTTATTCAAAGAC-3’；

Seq ID No.3：5’-CCGACCTTACGCCAGGCGGCC-3’；

Seq ID No.4：5’-GCCTCACCTCTCCAGGGCCTC-3’；

Seq ID No.5：5’-AAGGACAGGAACATCCTGC-3’；

Seq ID No.6：5’-GGCATGCCNNNNNNNNNCACGTGCGC-3’；

Seq ID No.7：5’-TTGGGCATNNNNNNNNNGTCCACGTGCGC-3’；

Seq ID No.8：5’-CACCCGAAGCTTGTGCGC-3’；

Seq ID No.9：5’-GGAGGCGTGCACCGCAGGG-3’；

Seq ID No.10：5’-GGAGGTCAGCACGGCGCTCACA-3’；

Seq ID No.11：5’-GGAGCAGGGAGGGCAGGAGCCAGGG-3’；

Seq ID No.12：5’-AGGGTCCCATAGACTCACCCT-3’；

Seq ID No.13：5’-GGCCCTTTTGCTAATCATGTT-3’；

Seq ID No.14：5’-AAGGCCCTTCATAATATCCCC-3’；

Seq ID No.15：5’-AACGTGGATGAAGTTGGTGGT-3’；

Seq ID No.16：5’-CTGTTATGGACAACCCNNNNNNNNNGTGAAG-3’；

Seq ID No.17：5’-CTGTTATNNNNNNNNNACCCTAAGGTGA-3’；

Seq ID No.18：5’-ACGTGGATCCTCAGAACTTCA-3’；

Seq ID No.19：5’-TGGGCCAGCTCACAGACC-3’；

wherein, the probes of the sequences shown in Seq ID No.6, Seq ID No.7, Seq ID No.16 and Seq ID No.17 are arch bridge probes, and the 'NNNNNNNNN' in the four probes represents a sequence which is not matched with a target sequence and has the length of 5-15bp, so that the four probes are in an arch bridge shape after being combined with the target; the 5 'ends of the eight alpha globin mutation site specific probes and the five beta globin mutation site specific probes are all marked with the same or different fluorescent groups, and the 3' ends are all marked with the same or different fluorescent quenching groups.

2. The kit of claim 1, wherein: the kit also comprises an internal reference specific primer pair and an internal reference specific probe for detecting the internal reference gene beta-actin, wherein the upstream primer of the internal reference specific primer pair is a sequence shown by Seq ID No.20, the downstream primer is a sequence shown by Seq ID No.21, and the internal reference specific probe is a sequence shown by Seq ID No. 22;

Seq ID No.20：5’-GCTCCATCCTGGCCTCGC-3’；

Seq ID No.21：5’-CGTCCACCGCAAATGCTTCT-3’；

Seq ID No.22：5’-CGTCAACACCCCAGCCA-3’。

3. the kit according to claim 1 or 2, characterized in that: the 5 'end of the probe with the sequence shown in Seq ID No.3 is marked with a fluorescent group CY5, and the 3' end is marked with a fluorescence quenching group BQ 3; the 5 'end of the probe with the sequence shown in Seq ID No.4 is marked with a fluorescent group CY5, and the 3' end is marked with a fluorescence quenching group BQ 3; the 5 'end of the probe with the sequence shown in Seq ID No.5 is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.6 is marked with a fluorescent group FAM, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.7 is marked with a fluorescent group FAM, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.8 is marked with a fluorescent group ROX, and the 3' end is marked with a fluorescence quenching group BQ 2; the 5 'end of the probe with the sequence shown in Seq ID No.9 is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.10 is marked with a fluorescent group ROX, and the 3' end is marked with a fluorescence quenching group BQ 2; the 5 'end of the probe with the sequence shown in Seq ID No.15 is marked with a fluorescent group FAM, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.16 is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.17 is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.18 is marked with a fluorescent group ROX, and the 3' end is marked with a fluorescence quenching group BQ 2; the 5 'end of the probe with the sequence shown in Seq ID No.19 is marked with a fluorescent group CY5, and the 3' end is marked with a fluorescence quenching group BQ 3; the 5 'end of the probe having the sequence shown in Seq ID No.22 is labeled with a fluorescent group FAM, and the 3' end is labeled with a fluorescence quenching group BQ 1.

4. Use of a kit according to any one of claims 1 to 3 for the preparation of a reagent for the detection of mutations in an aberrant hemoglobin gene.

5. A kit for detecting human abnormal hemoglobin gene mutation is characterized in that: comprises a reaction solution I and a reaction solution II;

the reaction solution I comprises a group of alpha globin specific primer pairs, eight alpha globin mutation site specific probes, a group of internal reference specific primer pairs, an internal reference specific probe, dNTPs and PCR reaction buffer solution;

the upstream primer of the group of alpha globin specific primer pairs is a sequence shown by Seq ID No.1, the downstream primer is a sequence shown by Seq ID No.2, the eight alpha globin mutation site specific probes are sequences shown by Seq ID No.3 to Seq ID No.10 in sequence, the upstream primer of the group of internal reference specific primer pairs is a sequence shown by Seq ID No.20, the downstream primer is a sequence shown by Seq ID No.21, and the internal reference specific probe is a sequence shown by Seq ID No. 22;

the reaction solution II comprises two groups of beta globin specific primer pairs, five beta globin mutation site specific probes, dNTPs and PCR reaction buffer solution;

in the two groups of beta globin specific primer pairs, an upstream primer of one group of beta globin specific primer pairs is a sequence shown in Seq ID No.11, and a downstream primer is a sequence shown in Seq ID No. 12; the upstream primer of the other group of beta globin specific primer pairs is a sequence shown in Seq ID No.13, and the downstream primer is a sequence shown in Seq ID No. 14; the five beta globin mutation site specific probes are sequences shown as Seq ID No.15 to Seq ID No.19 in sequence;

Seq ID No.1：5’-CGGCACTCTTCTGGTCCCCACAG-3’；

Seq ID No.2：5’-CCACTCAGACTTTATTCAAAGAC-3’；

Seq ID No.3：5’-CCGACCTTACGCCAGGCGGCC-3’；

Seq ID No.4：5’-GCCTCACCTCTCCAGGGCCTC-3’；

Seq ID No.5：5’-AAGGACAGGAACATCCTGC-3’；

Seq ID No.6：5’-GGCATGCCNNNNNNNNNCACGTGCGC-3’；

Seq ID No.7：5’-TTGGGCATNNNNNNNNNGTCCACGTGCGC-3’；

Seq ID No.8：5’-CACCCGAAGCTTGTGCGC-3’；

Seq ID No.9：5’-GGAGGCGTGCACCGCAGGG-3’；

Seq ID No.10：5’-GGAGGTCAGCACGGCGCTCACA-3’；

Seq ID No.11：5’-GGAGCAGGGAGGGCAGGAGCCAGGG-3’；

Seq ID No.12：5’-AGGGTCCCATAGACTCACCCT-3’；

Seq ID No.13：5’-GGCCCTTTTGCTAATCATGTT-3’；

Seq ID No.14：5’-AAGGCCCTTCATAATATCCCC-3’；

Seq ID No.15：5’-AACGTGGATGAAGTTGGTGGT-3’；

Seq ID No.16：5’-CTGTTATGGACAACCCNNNNNNNNNGTGAAG-3’；

Seq ID No.17：5’-CTGTTATNNNNNNNNNACCCTAAGGTGA-3’；

Seq ID No.18：5’-ACGTGGATCCTCAGAACTTCA-3’；

Seq ID No.19：5’-TGGGCCAGCTCACAGACC-3’；

Seq ID No.20：5’-GCTCCATCCTGGCCTCGC-3’；

Seq ID No.21：5’-CGTCCACCGCAAATGCTTCT-3’；

Seq ID No.22：5’-CGTCAACACCCCAGCCA-3’。

wherein, the probes of the sequences shown in Seq ID No.6, Seq ID No.7, Seq ID No.16 and Seq ID No.17 are arch bridge probes, and the 'NNNNNNNNN' in the four probes represents a sequence which is not matched with a target sequence and has the length of 5-15bp, so that the four probes are in an arch bridge shape after being combined with the target; the 5 'ends of the eight alpha globin mutation site specific probes, the one internal reference specific probe and the five beta globin mutation site specific probes are all marked with the same or different fluorescent groups, and the 3' ends are all marked with the same or different fluorescent quenching groups.

6. The kit of claim 5, wherein: the 5 'end of the probe with the sequence shown in Seq ID No.3 is marked with a fluorescent group CY5, and the 3' end is marked with a fluorescence quenching group BQ 3; the 5 'end of the probe with the sequence shown in Seq ID No.4 is marked with a fluorescent group CY5, and the 3' end is marked with a fluorescence quenching group BQ 3; the 5 'end of the probe with the sequence shown in Seq ID No.5 is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.6 is marked with a fluorescent group FAM, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.7 is marked with a fluorescent group FAM, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.8 is marked with a fluorescent group ROX, and the 3' end is marked with a fluorescence quenching group BQ 2; the 5 'end of the probe with the sequence shown in Seq ID No.9 is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.10 is marked with a fluorescent group ROX, and the 3' end is marked with a fluorescence quenching group BQ 2; the 5 'end of the probe with the sequence shown in Seq ID No.15 is marked with a fluorescent group FAM, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.16 is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.17 is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1; the 5 'end of the probe with the sequence shown in Seq ID No.18 is marked with a fluorescent group ROX, and the 3' end is marked with a fluorescence quenching group BQ 2; the 5 'end of the probe with the sequence shown in Seq ID No.19 is marked with a fluorescent group CY5, and the 3' end is marked with a fluorescence quenching group BQ 3; the 5 'end of the probe having the sequence shown in Seq ID No.22 is labeled with a fluorescent group FAM, and the 3' end is labeled with a fluorescence quenching group BQ 1.

7. The kit of claim 5, wherein: the kit also comprises a reaction liquid III, wherein the reaction liquid III is DNA polymerase.

8. The kit of claim 5, wherein: also comprises a positive quality control product of a reagent A and a positive quality control product of a reagent B; the positive quality control product of the reagent A is a positive plasmid containing an amplification target sequence of the group of alpha globin specific primer pairs, and the positive plasmid respectively contains a segment with known content and homozygous CD15 genotype and a segment with known content and wild genotype; the positive quality control product of the reagent B is a positive plasmid containing the amplification target sequences of the two groups of beta globin specificity primer pairs, and the positive plasmid respectively contains a segment with known content and homozygous CD113 genotype and a segment with known content and wild genotype.

9. The kit of claim 5, wherein: the kit also comprises a reagent A negative quality control product and a reagent B negative quality control product, wherein the reagent A negative quality control product is a plasmid which contains the amplification target sequence of the group of alpha globin specific primer pairs and the genotype of the amplification target sequence is wild type; the negative quality control product of the reagent B is a plasmid which contains amplification target sequences of the two groups of beta globin specific primer pairs and the genotype of which is wild type.

10. The kit according to any one of claims 5 to 9, characterized in that: a blank control is also included, the blank control being sterilized deionized water.

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