CN111961717B - Fluorescent PCR kit for simultaneously detecting deletion type and non-deletion type alpha-thalassemia genes by single tube - Google Patents
Fluorescent PCR kit for simultaneously detecting deletion type and non-deletion type alpha-thalassemia genes by single tube Download PDFInfo
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Abstract
The invention relates to the technical field of gene detection, in particular to a fluorescent PCR kit for simultaneously detecting deletion type and non-deletion type alpha-thalassemia genes by a single tube, wherein a primer and a probe in the kit are arranged in the same reaction system of the single tube, and the Tm value of the primer is higher than that of the probe. The invention has the beneficial effects that: the kit only needs to add the genome DNA of the sample to be detected into a detection reagent, performs one-time tube closing operation, detects the deletion type and non-deletion type alpha-thalassemia genes at the same time, has no intermediate link, is simple to operate and can eliminate the pollution of PCR products to experimental environments; the invention takes the current conventional fluorescent quantitative PCR instrument as an instrument platform, and can realize automatic and large-scale detection.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a fluorescent PCR kit for simultaneously detecting deletion type and non-deletion type alpha-thalassemia genes by a single tube.
Background
Thalassemia (abbreviated as "thalassemia") is one of the most common monogenic genetic diseases worldwide that has the greatest impact on human health, mainly focusing on coastal countries in the Mediterranean, southeast asia, minority africa areas and south China, with a conservative estimate of nearly 2 million people carried worldwide. The disease results from decreased synthesis of alpha and/or beta chains due to mutation of globin gene, i.e. clinically common alpha-earth deficiency and beta-earth deficiency, wherein the population carrying rate of alpha-earth deficiency is much higher than that of beta-earth deficiency. Human alpha-earthThe sea anemia gene cluster is located on chromosome 16, expresses alpha-globin, is combined with beta-globin chain, and forms functional hemoglobin tetramer by proper alpha-globin chain and beta-globin chain proportion (1:1) under normal conditions. The disease has no effective treatment means, and the prevention of the birth of the severe infant by crowd screening and prenatal diagnosis by applying the corresponding analysis technology is the accepted first choice of preventive measures at home and abroad. Therefore, the simple, practical, accurate and sensitive globin gene mutation detection method is a precondition and foundation for realizing effective prevention and control of the disease. Chinese-common gene leading to reduced alpha chain synthesis defects include absent alpha-thalassemia } -) SEA 、-- THAI 、-α 4.2 、-α 3.7 ) And non-deleted alpha-thalassemia (WS, QS, CS point mutations). In current practice, large fragment deletions are detected by the commonly used cross-split PCR technique (also known as split PCR, gap-PCR), which incorporates reverse point hybridization (PCR-RDB) to detect point mutations. The gap-PCR technology is to amplify various deletion mutations, and then to analyze products by gel electrophoresis; the PCR-RDB is to amplify the target sequence by PCR, and then to carry out mutation analysis by denaturation, hybridization, color development and other operations. In general, the gap-PCR and the PCR-RDB which are conventionally used at present are required to be detected separately, so that the cost is high, the workload is high, the operation is complicated, the detection flux is small, the automation and standardization are difficult to realize, and the problem of laboratory carrying pollution caused by the open tube operation after the PCR amplification exists, so that the current requirements of poor large-scale crowd screening and clinical routine molecular diagnosis cannot be met.
With the sequential implementation of prevention plans and measures aiming at reducing the birth rate of the infant suffering from thalassemia at present and the deep research of the molecular mechanism of thalassemia and molecular epidemiology, the high-throughput detection technology and method which are accurate, reliable, simple and practical, can realize automation and standardization, are suitable for large-scale crowd screening and conventional molecular diagnosis are technical support conditions for implementation of the items, and are more urgent needs for the molecular diagnosis of thalassemia at present. Aiming at the technical limitation of the alpha-thalassemia gene mutation detection analysis, a corresponding technical method is developed, the disposable tube closing operation is performed to prevent pollution carried by PCR products, a single reaction tube detects multiple mutation types to improve detection flux and simplify operation intensity, and the automatic detection analysis of an instrument to realize automation and standardization is the main direction of the current methodology research.
Disclosure of Invention
In order to overcome the defects in the prior art, the technical problems to be solved by the invention are as follows: provides a method for synchronously detecting alpha-thalassemia gene cluster by single reaction tube and one-time tube closing SEA 、-- THAI 、-α 4.2 、-α 3.7 Fluorescent PCR kit for deletion mutation and alpha-earth deficiency gene WS, QS and CS point mutation.
In order to solve the technical problems, the invention adopts the following technical scheme: providing a fluorescent PCR kit for simultaneously detecting deletion type and non-deletion type alpha-thalassemia genes by a single tube, wherein a primer and a probe in the kit are placed in the same reaction system by the single tube, and the primer and the probe specifically comprise:
(1) Southeast Asia deletion mutation for specifically amplifying alpha-thalassemia gene cluster SEA ) A primer pair for shortening the sequence;
thailand type deletion mutation for specifically amplifying alpha-thalassemia gene cluster THAI ) A primer pair for shortening the sequence;
left deletion mutation (-alpha) for specifically amplifying alpha-thalassemia gene cluster 4.2 ) A primer pair for shortening the sequence;
right deletion mutation (-alpha) for specifically amplifying alpha-thalassemia gene cluster 3.7 ) A primer pair for shortening the sequence;
a primer pair for specifically amplifying a normal sequence of a third exon of the alpha-thalassemia gene;
(2) Specific detection of alpha-thalassemia Gene Cluster- SEA A fluorescence labeling probe of a deletion mutation truncated sequence and a corresponding fluorescence quenching probe;
specific detection of alpha-thalassemia Gene Cluster- THAI A fluorescence labeling probe of a deletion mutation truncated sequence and a corresponding fluorescence quenching probe;
specific detection of alpha-thalassemia Gene Cluster-alpha 4.2 Fluorescence of deletion mutant truncated sequencesA label probe and a corresponding fluorescence quenching probe;
specific detection of alpha-thalassemia Gene Cluster- -alpha 3.7 A fluorescence labeling probe of a deletion mutation truncated sequence and a corresponding fluorescence quenching probe;
(3) A fluorescent marker probe for specifically detecting the sequence of the alpha-thalassemia gene point mutation WS/QS locus;
a fluorescent marker probe for specifically detecting the sequence of the CS site of the alpha-thalassemia gene point mutation;
a quenching probe corresponding to a single fluorescent probe for specifically detecting the sequence of the alpha-thalassemia gene point mutation WS/QS/CS locus;
the Tm value of said primer is higher than that of the probe;
the specific detection of alpha-thalassemia gene cluster- SEA 、-- THAI 、-α 4.2 、-α 3.7 The fluorescent marking probe of deletion mutation truncated sequence and the corresponding fluorescent quenching probe are used for determining whether the sample to be detected exists or not by modifying different fluorescent signals or different melting temperatures on the fluorescent probe SEA 、-- THAI 、-α 4.2 、-α 3.7 Deletion mutation;
the fluorescent marked probes and the corresponding fluorescent quenching probes for specifically detecting the wild type and mutant type of the alpha-thalassemia gene WS, QS and CS point mutation sequences are used for determining and distinguishing the wild type and mutant type of the WS, QS and CS point mutation sequences by modifying different fluorescent signals or different melting temperatures on the fluorescent probes.
The invention has the beneficial effects that: in the fluorescent PCR detection method for simultaneously detecting the deletion mutation and the point mutation of the gene by the single tube, the PCR amplification of the target sequence is completed at a higher annealing temperature in the initial stage of detection reaction. In this step, since the Tm value of the probe is lower than that of the amplification primer, hybridization with the template is not effected without affecting PCR amplification. The amplified PCR product can be used as a template for probe detection, and through denaturation and renaturation, each specific fluorescent probe and quenching probe are hybridized with a corresponding target sequence complementary template, and the distance between the fluorescent group of the fluorescent probe and the quenching group of the quenching probe is close to the distance without fluorescence. With the gradual rise of the programmed temperature, each fluorescent probe is firstly melted at the corresponding temperature, the fluorescent probe is dissociated from the template and far away from the quenching group to emit fluorescence, and the instrument can detect the change of the fluorescence value of a certain fluorescent channel at a certain temperature. Judging whether a detected sample has a certain deletion mutation or not according to the existence of melting peaks of each fluorescent channel; judging whether the detected sample has a certain point mutation or not according to the temperature of the melting peak of each fluorescent channel.
Compared with the prior art, the kit has the following advantages: (1) The primer probe designed by the invention has good specificity and repeatability; (2) The kit of the invention is suitable for human alpha-thalassemia gene cluster- SEA 、-- THAI 、-α 4.2 、-α 3.7 The deletion mutation, WS, QS and CS point mutation detection has good sensitivity, stability and accuracy; (3) The kit only needs to add the genome DNA of the sample to be detected into the detection reagent, and the operation of closing the tube is performed once, so that intermediate links are not needed, the operation is simple, and the pollution of the PCR product to the experimental environment can be eliminated; (4) The invention takes the current conventional fluorescent quantitative PCR instrument as an instrument platform, and can realize automatic and large-scale detection.
Drawings
FIG. 1 is a schematic diagram of the basic principle and probe position of example 1 according to the embodiment of the present invention;
FIG. 2 is a representative peak diagram of the melting analysis of example 1 of a specific embodiment of the present invention;
FIG. 3 shows an alpha-earth lean gene- & lt 1 & gt of example 1 according to an embodiment of the present invention THAI Deletion mutation melting analysis peak diagram;
FIG. 4 is a peak diagram of a point mutation melting analysis of the α -earth lean gene CS of example 1 according to an embodiment of the present invention;
FIG. 5 shows the alpha-earth-lean gene-alpha of example 1 according to an embodiment of the present invention 3.7 Deletion mutation melting analysis peak diagram;
FIG. 6 shows an example of an embodiment of the present invention1 alpha-thalassemia gene-alpha 4.2 Deletion mutation melting analysis peak diagram;
FIG. 7 shows the alpha-thalassemia genotype of example 1 according to an embodiment of the present invention SEA /α CS A point mutation melting analysis peak diagram;
FIG. 8 shows that example 1 of an embodiment of the present invention has an alpha-geolean genotype of alpha CS A point mutation melting analysis peak diagram of alpha/alpha;
FIG. 9 shows that example 1 of an embodiment of the present invention has an alpha-geolean genotype of alpha QS A point mutation melting analysis peak diagram of alpha/alpha;
FIG. 10 shows that example 1 of an embodiment of the present invention has an alpha-geolean genotype of alpha WS Point mutation melting analysis peak map of alpha/alpha.
Detailed Description
In order to describe the technical content, the constructional features, the achieved objects and effects of the present invention in detail, the following description is made in connection with the embodiments and the accompanying drawings.
The technical principle and the description of the invention are as follows: normally, the ratio of alpha and beta globin chains expressed in the human organism is appropriate (1:1), forming a biologically functional hemoglobin tetramer. When the functional gene of alpha-thalassemia is deleted, the synthesis of alpha is reduced and the beta chain is relatively excessive, alpha-thalassemia is poor. In the case of a set of human normal genomes (i.e., the nuclear genome of a cell), the α1 and α2-globin genes are in two copies each, and the deletion and/or point mutation of the α1 and/or α2-globin genes are the molecular mechanisms responsible for thalassemia, thus detecting the presence or absence of large fragment deletions and point mutations on the α -globin genes is one of the major approaches to molecular diagnosis of thalassemia.
The technical principle of the kit is shown in figure 1, and the system comprises specific detection alpha-geolean gene cluster- SEA 、-- THAI 、-α 4.2 、-α 3.7 The deletion mutant cross-breakpoint (gap-PCR) primer pair (P1-P8), and the third exon of the alpha-thalassemia gene comprise the normal range sequence primer pair (P7, P9) of WS, QS, CS point mutation sites. The Tm value of these primers is high (70-72 ℃ C.), and thus the detection is performedAt the initial stage of the reaction, the PCR amplification of the target sequence is completed at a higher annealing temperature. In this step, the Tm value of the probe is [ (]<70 ℃ below the amplification primers, will not hybridize to the template without affecting the PCR amplification. The amplified PCR product can be used as a template for detecting a subsequent probe, and the template is denatured at 95 ℃ and renatured at 40 ℃, and the fluorescent probe and the quenching probe of each mutant type and mutation site are hybridized with the corresponding target sequence complementary template, so that the fluorescent group of the fluorescent probe is close to the quenching group of the quenching probe without fluorescence.
After the first round of target sequence specific amplification, if the detected sample has some deletion mutation, the detected sample has a corresponding truncated target sequence amplification product; similarly, the third exon sequence of the alpha-earth-lean gene contained in the sample was also amplified. With the gradual rise of the programmed temperature, each fluorescent probe is melted at the corresponding temperature, the fluorescent probe is dissociated from the template and far away from the quenching group to emit fluorescence, and the instrument can detect the change of the fluorescence value of a certain fluorescent channel at a certain temperature. Judging whether the tested sample has a certain deletion mutation or not according to the existence of melting peaks and the temperature of the melting peaks of each fluorescent channel. For example, the sample to be tested has- SEA Deletion mutation, namely truncated sequence PCR product with deletion mutation, specifically detecting deletion mutation of alpha-geotrichia gene cluster southeast Asia during hybridization SEA ) The fluorescent probe and the quenching probe of (2) are hybridized with the target sequence of interest, and the corresponding labeled ROX fluorescent group is quenched. With the gradual rise of the temperature, the probe dissociates from the target sequence at 64.0+/-1.0 ℃, the ROX fluorescent group breaks away from the quenching group, and the fluorescent signal of the ROX channel obviously increases to generate a melting peak, thereby judging that the sample to be tested has- SEA Deletion mutation.
The technical scheme adopted by the invention is as follows: providing a fluorescent PCR kit for simultaneously detecting deletion type and non-deletion type alpha-thalassemia genes by a single tube, wherein a primer and a probe in the kit are placed in the same reaction system by the single tube, and the primer and the probe specifically comprise:
(1) Southeast Asia deletion process for specifically amplifying alpha-thalassemia gene clusterTransformation (-) SEA ) A primer pair for shortening the sequence;
thailand type deletion mutation for specifically amplifying alpha-thalassemia gene cluster THAI ) A primer pair for shortening the sequence;
left deletion mutation (-alpha) for specifically amplifying alpha-thalassemia gene cluster 4.2 ) A primer pair for shortening the sequence;
right deletion mutation (-alpha) for specifically amplifying alpha-thalassemia gene cluster 3.7 ) A primer pair for shortening the sequence;
a primer pair for specifically amplifying a normal sequence of a third exon of the alpha-thalassemia gene;
(2) Specific detection of alpha-thalassemia Gene Cluster- SEA A fluorescence labeling probe of a deletion mutation truncated sequence and a corresponding fluorescence quenching probe;
specific detection of alpha-thalassemia Gene Cluster- THAI A fluorescence labeling probe of a deletion mutation truncated sequence and a corresponding fluorescence quenching probe;
specific detection of alpha-thalassemia Gene Cluster-alpha 4.2 A fluorescence labeling probe of a deletion mutation truncated sequence and a corresponding fluorescence quenching probe;
specific detection of alpha-thalassemia Gene Cluster- -alpha 3.7 A fluorescence labeling probe of a deletion mutation truncated sequence and a corresponding fluorescence quenching probe;
(3) A fluorescent marker probe for specifically detecting the sequence of the alpha-thalassemia gene point mutation WS/QS locus;
a fluorescent marker probe for specifically detecting the sequence of the CS site of the alpha-thalassemia gene point mutation;
a quenching probe corresponding to a single fluorescent probe for specifically detecting the sequence of the alpha-thalassemia gene point mutation WS/QS/CS locus;
the Tm value of said primer is higher than that of the probe;
the specific detection of alpha-thalassemia gene cluster- SEA 、-- THAI 、-α 4.2 、-α 3.7 Fluorescent-labeled probes and corresponding fluorescence quenching probes lacking mutated truncated sequences by fluorescenceModifying different fluorescent signals or different melting temperatures on the probe to determine whether the sample to be detected exists SEA 、-- THAI 、-α 4.2 、-α 3.7 Deletion mutation;
the fluorescent marked probes and the corresponding fluorescent quenching probes for specifically detecting the wild type and mutant type of the alpha-thalassemia gene WS, QS and CS point mutation sequences are used for determining and distinguishing the wild type and mutant type of the WS, QS and CS point mutation sequences by modifying different fluorescent signals or different melting temperatures on the fluorescent probes.
Preferably, in the above single tube fluorescent PCR kit for simultaneously detecting deletion type and non-deletion type alpha-thalassemia gene, the Tm value of the primer is 68℃to 72℃and the Tm value of the primer is 3℃to 5℃higher than that of the probe.
Preferably, in the above-mentioned single tube fluorescent PCR kit for simultaneously detecting deletion type and non-deletion type alpha-thalassemia gene, (1) deletion mutation (-, southeast Asia type deletion mutation of alpha-thalassemia gene cluster is used for specific amplification SEA ) Primer pairs of truncated sequences, the sequences are SEQ ID No.1 and SEQ ID No.2;
thailand type deletion mutation for specifically amplifying alpha-thalassemia gene cluster THAI ) Primer pairs of truncated sequences, the sequences of which are SEQ ID No.3 and SEQ ID No.4;
left deletion mutation (-alpha) for specifically amplifying alpha-thalassemia gene cluster 4.2 ) Primer pairs of truncated sequences, the sequences of which are SEQ ID No.5 and SEQ ID No.6;
right deletion mutation (-alpha) for specifically amplifying alpha-thalassemia gene cluster 3.7 ) Primer pairs of truncated sequences, the sequences of which are SEQ ID No.7 and SEQ ID No.8;
a primer pair for specifically amplifying the normal sequence of the third exon of the alpha-thalassemia gene, wherein the sequences are SEQ ID No.7 and SEQ ID No.9;
(2) Specific detection of alpha-thalassemia Gene Cluster- SEA The sequence of the fluorescent marking probe of the deletion mutation truncated sequence and the corresponding fluorescent quenching probe is as follows: SEQ ID No.10 and SEQ ID No.11;
specific detectionAlpha-thalassemia gene cluster- THAI The sequence of the fluorescent marking probe of the deletion mutation truncated sequence and the corresponding fluorescent quenching probe is as follows: SEQ ID No.12 and SEQ ID No.13;
specific detection of alpha-thalassemia Gene Cluster-alpha 4.2 The sequence of the fluorescent marking probe of the deletion mutation truncated sequence and the corresponding fluorescent quenching probe is as follows: SEQ ID No.14 and SEQ ID No.15;
specific detection of alpha-thalassemia Gene Cluster- -alpha 3.7 The sequence of the fluorescent marking probe of the deletion mutation truncated sequence and the corresponding fluorescent quenching probe is as follows: SEQ ID No.16 and SEQ ID No.17;
(3) A fluorescent marker probe for specifically detecting the sequence of alpha-thalassemia gene point mutation WS/QS locus has the sequence: SEQ ID No.18;
a fluorescent marker probe for specifically detecting the sequence of the CS site of the alpha-thalassemia gene point mutation, which has the sequence: SEQ ID No.19;
the quenching probe corresponding to a single fluorescent probe for specifically detecting the sequence of the alpha-thalassemia gene point mutation WS/QS/CS locus has the sequence as follows: SEQ ID No.20;
preferably, in the fluorescent PCR kit for simultaneously detecting deletion type and non-deletion type alpha-thalassemia genes by using the single tube, the specific detection of alpha-thalassemia gene cluster- SEA 、-- THAI 、-α 4.2 、-α 3.7 The fluorescent probes and the quenching probes of the deletion mutation and the alpha-thalassemia gene WS, QS and CS point mutation are respectively and independently marked, and the Tm value of the melting analysis is 45-65 ℃.
Preferably, in the fluorescent PCR kit for simultaneously detecting deletion type and non-deletion type alpha-thalassemia genes by using the single tube, the specific detection of alpha-thalassemia gene cluster- SEA 、-- THAI 、-α 4.2 、-α 3.7 The Tm values of quenching probes of the deletion mutation and alpha-thalassemia gene WS, QS and CS point mutation are higher than the Tm values of corresponding fluorescent probes.
Preferably, the single tube fluorescent PCR kit for simultaneously detecting deletion type and non-deletion type alpha-thalassemia genesIn the method, the gene cluster for specifically detecting alpha-thalassemia-) SEA 、-- THAI 、-α 4.2 、-α 3.7 The fluorescent probes for deletion mutation and point mutation of the alpha-thalassemia gene WS, QS and CS and the corresponding quenching probes have the following structures:
(1) Specific detection of deletion mutation of southeast Asia type of alpha-thalassemia Gene cluster SEA ) The fluorescent probes of (2) are positioned at the 3 'end, the sense strand and the 3' end of the truncated sequence amplification product of the mutation specific primer, the Rox fluorescent groups are marked at the 3 'end, the corresponding quenching probes are positioned at the 3' downstream of the fluorescent probes, the 5 'end of the quenching probes is 2-8bp away from the 3' end of the fluorescent probes, the sense strand and the Tm value>A fluorescent probe, the 5' end of which is marked with a fluorescence quenching group;
(2) Specific detection of alpha-thalassemia Gene Cluster Thailand-type deletion mutation (-) THAI ) The fluorescent probes of (2) are positioned at the 5' end, the negative sense strand and the 5' end of the truncated sequence amplification product of the mutation specific primer, cy5 fluorescent groups are marked at the 5' end, the corresponding quenching probes are positioned at the 5' upstream of the fluorescent probes, 2-8bp from the 3' end of the fluorescent probes, the positive sense strand and the Tm value>A fluorescent probe, wherein the 3' -end is marked with a fluorescence quenching group;
(3) Specific detection of left deletion mutation (-alpha) of alpha-thalassemia gene cluster 4.2 ) The fluorescent probes of (2) are positioned at the 3' -end, the sense strand and the 5' -end of the truncated sequence amplification product of the mutation specific primer, cy5 fluorescent groups are marked at the 5' -end, and the corresponding quenching probes are positioned at the 5' -upstream of the fluorescent probes, 2-8bp from the 3' -end of the fluorescent probes, the sense strand and the Tm value>A fluorescent probe, wherein the 3' -end is marked with a fluorescence quenching group;
(4) Specific detection of right deletion mutation (-alpha) of alpha-thalassemia gene cluster 3.7 ) The fluorescent probes of (2) are positioned at the 3' -end, the sense strand and the 5' -end of the truncated sequence amplification product of the mutation specific primer, cy5 fluorescent groups are marked at the 5' -end, and the corresponding quenching probes are positioned at the 5' -upstream of the fluorescent probes, 2-8bp from the 3' -end of the fluorescent probes, the sense strand and the Tm value>A fluorescent probe, wherein the 3' -end is marked with a fluorescence quenching group;
(5) The fluorescent probe for specifically detecting non-deletion point mutation WS/QS locus is a wild type DNA sequence containing WS locus and QS locus and flanking 3-10bp range, a sense strand, and a 3' -end labeled FAM fluorescent group;
(6) The fluorescent probe for specifically detecting the non-deletion point mutation CS site is a wild type DNA sequence containing the CS site, the mutation base position is positioned in the middle 5 base ranges of the probe, the sense strand is positioned, and the 5' -end is marked with a Rox fluorescent group.
(7) The quenching probe sequence corresponding to the specific detection non-deletion point mutation WS/QS/CS site is WS/QS site fluorescent probe 3' downstream, CS site fluorescent probe 5' upstream, wild type DNA sequence 3-8bp away from the 3' end of the fluorescent probe, sense strand, tm value > fluorescent probe, and fluorescent quenching groups are marked at the 5' end and the 3' end.
FAM refers to 6-carboxyfluorescein, ROX refers to carboxy-X-rhodamine, cy5 refers to cyanine dye molecule 5, BHQ1 and BHQ2 refer to fluorescence quenching groups. If the corresponding fluorescence or quenching group is marked on the non-3 '-end of all the fluorescence probes and quenching probes, 2-5bp of base which is not matched with the target sequence is added, so that the extensibility of the non-marked 3' -end in PCR amplification reaction is prevented.
Example 1
Referring to fig. 2 to 10, a fluorescent PCR kit for simultaneously detecting deletion-type and non-deletion-type α -thalassemia genes with a single tube, comprising:
1. primer and probe
(1) A pair of specific amplified alpha-thalassemia gene cluster southeast Asia deletion mutations (-) SEA ) Primer pairs for truncated sequences are shown below:
F：5’-agcgatctgggctctgtgttctc-3’(SEQ ID NO.1)，
R：5’-agcccacgttgtgttcatggc-3’(SEQ ID NO.2)。
(2) A pair of specific amplified alpha-thalassemia gene cluster Thailand type deletion mutations (-) THAI ) Primer pairs for truncated sequences are shown below:
F：5’-cctcagcctcctccatcactcac-3’(SEQ ID NO.3)，
R：5’-gatctgcacctctgggtaggttctgtac-3’(SEQ ID NO.4)。
(3) A pair of specificity amplified alpha-ground lean gene clustersDeletion mutation (-alpha) 4.2 ) Primer pairs for truncated sequences are shown below:
F：5’-ccagtttacccatgtggtgcctc-3’(SEQ ID NO.5)，
R：5’-cccgttggatcttctcatttccc-3’(SEQ ID NO.6)。
(4) A pair of specific amplified alpha-thalassemia gene cluster right deletion mutations (-alpha) 3.7 ) Primer pairs for truncated sequences are shown below:
F：5’-cccctcgccaagtccaccc-3’(SEQ ID NO.7)，
R：5’-gcaaacctgcattgaatctgaaaagtc-3’(SEQ ID NO.8)。
(5) A pair of primers specifically amplifying the normal sequence of the third exon of the alpha-thalassemia gene is shown below:
f:5'-cccctcgccaagtccaccc-3' (SEQ ID NO. 7), which is a common primer, and which is identical in sequence to the primers SEQ ID NO.7 used in the above (4);
R：5’-agaccaggaagggccggtg-3’(SEQ ID NO.9)。
(6) Specific detection of alpha-Dilean Gene Cluster- SEA Fluorescence and quenching probes for deletion mutations:
5’-ctctgagggtgacgctgtctgctt-ROX-3’(SEQ NO.10)，
5’-BHQ2-ggcccagggaaacccaggtg-3’(SEQ NO.11)；
(7) Specific detection of alpha-thalassemia gene cluster Thailand type deletion mutation THAI ) Is a fluorescent and quenching probe of (1):
5’-Cy5-cactcctggttcatctcagcctgg-3’(SEQ NO.12)，
5’-ccttcgctgttgctgggttcaga-BHQ2-3’(SEQ NO.13)；
(8) Specific detection of alpha-thalassemia gene cluster left deletion mutation (-alpha) 4.2 ) Is a fluorescent and quenching probe of (1):
5’-Cy5-caaagatcaggaagtgctggg-3’(SEQ NO.14)，
5’-caggctgctgcctactcggacttc-BHQ2-3’(SEQ NO.15)；
(9) Specific detection of right deletion mutation (-alpha) of alpha-thalassemia gene cluster 3.7 ) Is a fluorescent and quenching probe of (1):
5’-Cy5-tgcagctggatagggta-3’(SEQ NO.16)，
5’-cagctgggacacacatggctagaac-BHQ2-3’(SEQ NO.17)；
(10) Fluorescence and quenching probes for specifically detecting alpha-thalassemia gene point mutation WS/QS/CS sites:
5’-cacgcctccctggacaagttc-FAM-3’(SEQ NO.18)；
5’-ROX-ctccaaataccgttaagctggagc-3’(SEQ NO.19)；
5’-BHQ1-cttctgtgagcaccgtgctgacct-BHQ2-3’(SEQ NO.20)。
FAM refers to 6-carboxyfluorescein, ROX refers to carboxy-X-rhodamine, cy5 refers to cyanine dye molecule 5, BHQ1 and BHQ2 refer to fluorescence quenching groups. The amplification ranges of the primers and the positions of the hybridization sites of the probes are shown in FIG. 1.
The Tm values of the primers (comprising SEQ ID NO.1 to SEQ ID NO. 9) for specifically amplifying the target sequence are all between 70 and 72 ℃.
Wherein the Tm values of the fluorescence and quenching probes (SEQ NO.10 and SEQ NO. 11) for detecting deletion mutations of the αglobin gene NG_000006.1:g.26264-45564del are 64.0℃and 65.0℃respectively;
the Tm values of the fluorescence and quenching probes (SEQ NO.12 and SEQ NO. 13) for the detection of the αglobin gene NG_000006.1:g.10664-44164del were 62.5℃and 65.0℃respectively;
the Tm values of the fluorescence and quenching probes (SEQ NO.14 and SEQ NO. 15) for the detection of the αglobin gene NG_000006.1:g.30908-35164del are 58.5℃and 64.5℃respectively;
the Tm values of the fluorescence and quenching probes (SEQ NO.16 and SEQ NO. 17) for the detection of the αglobin gene NG_000006.1:g.34164-37967del were 55.0deg.C and 63.5deg.C, respectively;
the Tm values of the fluorescence and quenching probes (SEQ No.18, SEQ No.19 and SEQ No. 20) for the point mutation site HBA2:c.369C > G, HBA2:c.377T > C and HBA2:c.427T > C were 63.5 ℃, 59.5 ℃ and 65.0 ℃, respectively.
The Tm values of the primers are higher than the Tm values of the fluorescent and quenching probes, and the Tm values of the quenching probes are higher than the Tm values of the fluorescent probes.
2. The other components are as follows:
TaKaRa LA Taq enzyme and its associated LA Taq Buffer, dNTPS were purchased from Takara, and betaine was purchased from Sigma, USA.
2. The application method of the kit comprises the following steps:
(1) The reaction system is shown in Table 1;
TABLE 1
(2) The reaction procedure is:
pre-denaturation at 95℃for 7min;95℃45sec+64℃1min+72℃2min,50 cycles; extending at 72 ℃ for 5min; denaturation at 95℃for 5min; renaturation at 40 ℃ for 30min; melting analysis is carried out at 45-70 ℃ and FAM, ROX, cy channel fluorescence signals are collected.
The instrument used was a macrostone SLAN 96P fluorescent quantitative PCR instrument, purchased from Shanghai macrostone medical science and technology Co.
(3) Sample treatment:
gDNA samples were diluted to 50-100 ng/. Mu.l with sterilized double distilled water for use. Wherein the gDNA specimen can be obtained by the following method: peripheral whole blood samples were extracted, EDTA anticoagulated, and gDNA samples were obtained by extraction using a Tian Gen column type peripheral blood genomic DNA column type extraction reagent (Beijing Tian Gen Biotechnology Co.).
(4) Sample detection:
adding the gDNA sample to be detected into the reaction system, performing amplification detection on a fluorescent quantitative PCR instrument according to the reaction program, and recording a melting analysis map and a corresponding melting temperature value.
(5) Data analysis and result judgment:
the instrument matching software automatically displays a melting analysis peak diagram according to the change of the melting analysis fluorescent signal, and the temperature corresponding to the peak tip (the lowest point of the inverted peak) of the peak diagram is the melting temperature;
as shown in fig. 3: cy5 channel has a melting peak at 62.5.+ -. 1.0deg.C, and it can be determined that the sample has-) THAI Deletion mutation.
As shown in fig. 4: the ROX channel has a melting peak at 50.5.+ -. 1.0 ℃ and this sample can be judged to have a CS point mutation.
As shown in fig. 5: cy5 channel has a melting peak at 55.0.+ -. 1.0 ℃ and it can be determined that the sample has- α 3.7 Deletion mutation.
As shown in fig. 6: cy5 channel has a melting peak at 58.5.+ -. 1.0deg.C, and it can be determined that the sample has- α 4.2 Deletion mutation.
As shown in fig. 7: the ROX channel has two melting peaks at 64.0.+ -. 1.0 ℃ and 50.5.+ -. 1.0 ℃, which can determine that the sample is-) SEA Deletion mutation and CS Point mutation, thereby determining the genotype of the sample as- SEA /α CS α。
As shown in fig. 8: the ROX channel has two melting peaks at 50.5+ -1.0deg.C and 59.5+ -1.0deg.C, and can determine that the sample has wild type and CS point mutation, thereby determining that the genotype of the sample is alpha CS α/αα。
As shown in fig. 9: FAM channel has two melting peaks at 60.0+ -1.0deg.C and 63.5+ -1.0deg.C, which can determine that the sample has wild type and QS point mutation, and can determine that the genotype of the sample is alpha QS α/αα。
As shown in fig. 10: FAM channel has two melting peaks at 53.0+ -1.0deg.C and 63.5+ -1.0deg.C, which can determine that the sample has wild type and WS point mutation, and can determine that the genotype of the sample is alpha WS α/αα。
The melting temperatures of 7 mutation types detected in the invention are designed to be 45-68 ℃, and the fluorescence channels and melting temperatures corresponding to the 7 mutation types are shown in Table 2. Table 2 shows alpha-thalassemia gene- SEA 、-- THAI 、-α 4.2 、-α 3.7 Deletion and WS, QS, CS point mutations correspond to fluorescence channels and melting temperature tables. The test results can be obtained by obtaining the melting temperature of the test sample according to the above-described procedure, and then analyzing the mutant genotype thereof in combination with Table 2.
TABLE 2
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.
SEQUENCE LISTING
<110> sub-energy biotechnology (Shenzhen) Limited at university of south medical science
<120> fluorescent PCR kit for simultaneously detecting deletion type and non-deletion type alpha-thalassemia gene by single tube
<160> 20
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213> artificial sequence
<400> 1
agcgatctgg gctctgtgtt ctc 23
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<212> DNA
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<400> 2
agcccacgtt gtgttcatgg c 21
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<400> 3
cctcagcctc ctccatcact cac 23
<210> 4
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<400> 4
gatctgcacc tctgggtagg ttctgtac 28
<210> 5
<211> 23
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<400> 5
ccagtttacc catgtggtgc ctc 23
<210> 6
<211> 23
<212> DNA
<213> artificial sequence
<400> 6
cccgttggat cttctcattt ccc 23
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<400> 7
cccctcgcca agtccaccc 19
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<211> 27
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<213> artificial sequence
<400> 8
gcaaacctgc attgaatctg aaaagtc 27
<210> 9
<211> 19
<212> DNA
<213> artificial sequence
<400> 9
agaccaggaa gggccggtg 19
<210> 10
<211> 24
<212> DNA
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<400> 10
ctctgagggt gacgctgtct gctt 24
<210> 11
<211> 20
<212> DNA
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<400> 11
ggcccaggga aacccaggtg 20
<210> 12
<211> 24
<212> DNA
<213> artificial sequence
<400> 12
cactcctggt tcatctcagc ctgg 24
<210> 13
<211> 23
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<400> 13
ccttcgctgt tgctgggttc aga 23
<210> 14
<211> 21
<212> DNA
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<400> 14
caaagatcag gaagtgctgg g 21
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<211> 24
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caggctgctg cctactcgga cttc 24
<210> 16
<211> 17
<212> DNA
<213> artificial sequence
<400> 16
tgcagctgga tagggta 17
<210> 17
<211> 25
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<213> artificial sequence
<400> 17
cagctgggac acacatggct agaac 25
<210> 18
<211> 21
<212> DNA
<213> artificial sequence
<400> 18
cacgcctccc tggacaagtt c 21
<210> 19
<211> 24
<212> DNA
<213> artificial sequence
<400> 19
ctccaaatac cgttaagctg gagc 24
<210> 20
<211> 24
<212> DNA
<213> artificial sequence
<400> 20
cttctgtgag caccgtgctg acct 24
Claims (1)
1. A fluorescent PCR kit for simultaneously detecting deletion type and non-deletion type alpha-thalassemia genes by a single tube is characterized in that a primer and a probe in the kit are placed in the same reaction system by the single tube, and the primer and the probe specifically comprise:
(1) Deletion mutation of southeast Asia type for specifically amplifying alpha-thalassemia gene cluster SEA Primer pairs of truncated sequences, the sequences are SEQ ID No.1 and SEQ ID No.2;
thailand type deletion mutation for specifically amplifying alpha-thalassemia gene cluster THAI Primer pairs of truncated sequences, the sequences of which are SEQ ID No.3 and SEQ ID No.4;
left deletion mutation-alpha for specific amplification of alpha-thalassemia gene cluster 4.2 Primer pairs of truncated sequences, the sequences of which are SEQ ID No.5 and SEQ ID No.6;
right deletion mutation-alpha for specific amplification of alpha-thalassemia gene cluster 3.7 Primer pairs of truncated sequences, the sequences of which are SEQ ID No.7 and SEQ ID No.8;
a primer pair for specifically amplifying the normal sequence of the third exon of the alpha-thalassemia gene, wherein the sequences are SEQ ID No.7 and SEQ ID No.9;
(2) Specific detection of deletion mutation of southeast Asia type of alpha-thalassemia gene cluster-) SEA The sequence of the fluorescent marked probe and the corresponding fluorescent quenching probe with truncated sequences is: SEQ ID No.10 and SEQ ID No.11;
specific detection of alpha-thalassemia Gene Cluster Thailand type deletion mutation-) THAI The sequence of the fluorescent marked probe and the corresponding fluorescent quenching probe with truncated sequences is: SEQ ID No.12 and SEQ ID No.13;
specific detection of alpha-thalassemia Gene Cluster left deletion mutation-alpha 4.2 The sequence of the fluorescent marked probe and the corresponding fluorescent quenching probe with truncated sequences is: SEQ ID No.14 and SEQ ID No.15;
specific detection of right deletion mutation-alpha of alpha-thalassemia gene cluster 3.7 The sequence of the fluorescent marked probe and the corresponding fluorescent quenching probe with truncated sequences is: SEQ ID No.16 and SEQ ID No.17;
(3) A fluorescent marker probe for specifically detecting the sequence of alpha-thalassemia gene point mutation WS/QS locus has the sequence: SEQ ID No.18;
a fluorescent marker probe for specifically detecting the sequence of the CS site of the alpha-thalassemia gene point mutation, which has the sequence: SEQ ID No.19;
the quenching probe corresponding to the fluorescent labeling probe for specifically detecting the sequence of the alpha-thalassemia gene point mutation WS/QS/CS locus has the sequence as follows: SEQ ID No.20.
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| CN103911455A (en) * | 2014-04-16 | 2014-07-09 | 龙驹 | Kit for quickly detecting common deletion-form alpha-thalassemia and application method thereof |
| CN106868196B (en) * | 2017-04-20 | 2021-05-18 | 亚能生物技术（深圳）有限公司 | Probe, primer and kit for detecting thalassemia gene mutation |
| CN109112199A (en) * | 2017-06-23 | 2019-01-01 | 陈治中 | It is a kind of for detecting the genetic chip, amplifing reagent and kit of α-thalassemia |
| CN110257505B (en) * | 2019-07-02 | 2020-06-05 | 广西壮族自治区人民医院 | Non-deletion α thalassemia point mutation rapid detection kit and detection method |
| CN110577990B (en) * | 2019-09-12 | 2021-03-16 | 南方医科大学 | Kit for detecting thalassemia gene mutation |
| CN111471763B (en) * | 2020-06-17 | 2023-06-02 | 厦门安普利生物工程有限公司 | Primer, probe and reaction buffer solution combination and kit for multiplex real-time fluorescence PCR detection of alpha-thalassemia |
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