CN110423807B

CN110423807B - Primer combination and kit for detecting deletion type alpha-thalassemia

Info

Publication number: CN110423807B
Application number: CN201910763808.8A
Authority: CN
Inventors: 罗锋; 韩勋领; 孙松松; 张军; 谢海迎
Original assignee: Chongqing Puluotong Gene Medicine Research Institute Co ltd
Current assignee: Chongqing Puluotong Gene Medicine Research Institute Co ltd
Priority date: 2019-08-19
Filing date: 2019-08-19
Publication date: 2022-02-15
Anticipated expiration: 2039-08-19
Also published as: CN110423807A

Abstract

The invention relates to an in-vitro molecular diagnosis technology, in particular to a primer combination and a kit for detecting deletion type alpha-thalassemia. The primer combination can stably detect-alpha^3.7And- (alpha)^20.5The two deletion types overcome the problems of unstable amplification system due to high GC content of the alpha-globin gene region; meanwhile, the annealing temperatures of the primers are in the same temperature range, so that the phenomena of nonspecific amplification and unequal efficiency caused by different annealing temperatures are avoided, in addition, the compatibility of the primers is good, no dimer is generated, the kit overcomes the influence among the primers, the multiple single-tube thalassemia of 7 genotypes can be detected, the requirements of clinical rapid and convenient detection of thalassemia are met, conditions are created for more comprehensive thalassemia screening, and scientific basis is provided for prenatal examination and thalassemia diagnosis of pregnant fetuses.

Description

Primer combination and kit for detecting deletion type alpha-thalassemia

Technical Field

The invention relates to an in-vitro molecular diagnosis technology, in particular to a primer combination and a kit for detecting deletion type alpha-thalassemia.

Background

Thalassemia (Thalassemia, abbreviated as Thalassemia) is a genetic hemolytic disease widely prevalent in the world, and is a group of disabling lethal genetic hemolytic proteinopathies seriously threatening human health caused by the loss or reduction of globin chain synthesis due to globin gene deletion or defect, and imbalance of alpha/beta chain ratio for forming hemoglobin, and is also one of the largest single-gene genetic diseases in the world. At least 3.5 million people all over the world carry the gene, and the gene is mainly distributed in coastal countries of the Mediterranean sea and countries of southeast Asia. The disease is common in southern areas of China, is one of the genetic diseases with the highest incidence and the greatest influence in southern provinces of Yangtze river in China, particularly in Guangxi, Guangdong and Hainan, and has a tendency of spreading to the north.

Thalassemia is classified into alpha-thalassemia and beta-thalassemia according to whether the deletion defect occurs in the alpha chain or the beta chain of the globin gene. The alpha globin gene is positioned at 16p13.3, normal human has 2 alpha globin genes on each chromosome 16, if 1 alpha gene on the chromosome is deleted or defective, the synthesis of alpha chain is only partially inhibited, so the alpha globin gene is called alpha⁺Barren wheat; if 2 alpha-genes on the chromosome are all deleted or defective, the gene is called alpha⁰And (4) barren soil. Only 1 alpha-gene is abnormal, i.e. alpha⁺In the state of thalassemia and heterozygosis, no obvious clinical symptoms generally exist, and the phenotype is static; when 2 a-genes are abnormal, i.e. alpha⁺Thalassemia homozygote or alpha⁰In the heterozygote state, a considerable amount of alpha-chain synthesis still exists, the symptom is slight, and the phenotype is a standard type; when 3 alpha-genes are abnormal, i.e. alpha⁰And alpha⁺In the heterozygote state of thalassemia, the patient can synthesize only a small amount of alpha-chains, and the symptom is moderate hemolytic anemia and the phenotype is hemoglobin H disease (Hb H disease); when 4 alpha-genes are abnormal, i.e. alpha⁰In the state of thalassemia homozygote, the alpha-chain cannot be generated. The hemoglobin of the fetus normally has a major component of HbF, and due to the lack of alpha-chains, the excess gamma-chains polymerize to form tetramers, Hb Bart's. Hb Bart's have high oxygen affinity, cannot solve the physiological function of intrauterine oxygen supply of a fetus, and then causes fetal edema, so that the fetus is fatigued in the womb or in the pregnancy ending within the latter half hour of delivery.

According to the affected cause of alpha-globin gene mutation, the gene is divided into deletion type and non-deletion type, and more than 95 percent of alpha deficiency is clarified to be caused by the deletion type. At least 36 of the α thalassemia deficiency types identified so far, 8 of them occur in china. The most common of Chinese is the right deletion (-alpha)^3.7) Left side deletion (-alpha)^4.2) And southeast Asia deletion (-^SEA) Relatively rare or rare ones^THAI、-(α)^20.5、--^MED、--^HWAnd-^FILIs absent. According to different combinations of common deletion types in individuals, the four types of the symptoms of the resting type, the standard type, the haemoglobin H disease and the Hb Bart's fetal edema syndrome can be expressed. The existing deletion type thalassemia detection method and products in the market mostly adopt the technology of Gap-PCR combined electrophoresis method or gene chip and the like to detect the common three deletion type alpha thalassemia (-one)^SEA、-α^3.7And-alpha^4.2) While ignoring the importance of other rare or rare deletions causing severe anemia leading to disease. Such as Thailand defect type-^THAI) There are data showing that-^THAIAbout 0.63%. The Thailand type alpha-thalassemia gene deletion fragment is inferior to southeast Asia gene deletion (-one)^SEA) The fragments are also long-^THAISelf or-^SEABy itself or with-alpha^3.7Or-alpha^4.2Mutual co-inheritance can cause lethal Hb Bart's fetuses or HbH disease that severely affects quality of life. Homozygote (-one) of the southeast subtype^SEA/--^SEA) And Thailand type-^THAI/--^THAI) Or southeast Asia complex Thailand type (-one)^SEA/--^THAI) Since all 4 alphaglobin genes are deleted or defective, no alpha chain is generated at all, and the alpha-thalassemia is the most serious one, namely, the severe alphathalassemia. Therefore, the screening and diagnosis of the rare alpha-thalassemia should be noticed in the areas with high incidence of the alpha-thalassemia, and it is important to prevent the birth of the severe thalassemia caused by missed diagnosis. As the global population mobility increases, different ethnicities and crowds become married with each other, and the diffusion rate of human genes among different crowds increases. Popular-sand-taste in mediterranean region^MEDAlso spreads rapidly into China, which brings a serious test for the prevention of thalassemia in China.

At present, no ideal treatment method for alpha-thalassemia exists, and selective elimination of severe alpha-thalassemia fetuses through genetic screening and prenatal diagnosis is a primary way for controlling the disease and an effective prevention measure. Therefore, a simple, rapid and accurate genotyping technical means is urgently needed for the detection of deletion type alpha-thalassemia. The existing alpha-thalassemia screening methods comprise blood routine parameter detection and analysis, erythrocyte osmotic fragility test, hemoglobin electrophoresis test and the like, have low specificity and are easy to generate false negative. For the molecular diagnosis of the alpha-thalassemia, the Southern Blot hybridization technology is a gold standard, has high accuracy, but is complex to operate, large in sample consumption, time-consuming, labor-consuming and small in detection flux, and is not suitable for routine detection; other TaqMan probe technologies, Denaturing High Performance Liquid Chromatography (DHPLC), direct DNA sequencing, DNA microarray technologies, and the like. However, these methods are complicated in detection process, long in detection time, expensive in reagent or instrument, and special in labeled primers, and thus are not suitable for wide clinical popularization and application.

At present, most of the deficient thalassemia detection methods and products in the market adopt Gap-PCR combined electrophoresis method or gene chip technology for detection, and the related information of domestic main alpha-thalassemia detection products is shown in Table 1:

TABLE 1 domestic main alpha-thalassemia detection products

As can be seen from the above, most of the kits for diagnosing the deletion type alpha-thalassemia in China at the present stage are developed based on the multiple Gap-PCR technology and can only realize 3 common alpha-thalassemia deletion genes (-one-^SEA、-α^3.7And-alpha^4.2) There are also few products for realizing 4 deletion genes (-one) based on PCR reverse dot hybridization technique^SEA、-α^3.7、-α^4.2And-^THAI) Detection of (3). However, PCR-reverse dot hybridization is to design corresponding amplification primers and probes according to mutation or deletion sites of each genotype, immobilize the probes on a nylon membrane by using biotin labeled primers and amino labeled probes and using a DNA chip (nylon membrane) as a substrate, hybridize the PCR products amplified by specific primers with the probes immobilized on the DNA chip, and interpret the signal color developing cassette to diagnose the anemia. I.e., complicated detection process, long detection time, expensive reagent or instrument, and special labeling guideAnd the like, thus being not suitable for wide clinical popularization and application. Although the Gap-PCR technology is widely used for diagnosing deletion type alpha-thalassemia, the CG content of the area where the alpha globin gene is located is high, so that an amplification system is unstable, and the condition of missing detection is easy to occur, particularly the condition of alpha^3.7And- (alpha)^20.5Detection of heterozygous deletion type. Except for 3 common alpha-thalassemia deleted genes (-one)^SEA、-α^3.7And-alpha^4.2) At present, no detection method capable of simultaneously, simply and rapidly detecting-^SEA、-α^3.7、-α^4.2、--^THAI、-(α)^20.5、--^MEDAnd-^FIL7 deletion type kits cause the thalassemia to be detected very inconveniently, easily cause omission and generate great harm.

Disclosure of Invention

In order to solve the problems of high GC content and unstable amplification system in the alpha-globin gene region, the method for stably detecting alpha^3.7And- (alpha)^20.5A deletion type primer set.

A primer combination for detecting deletion type alpha-thalassemia comprises the following primers:

α2-3.7-F：5’-TTCCTTCCTCACCCCACATCCC-3’(SEQ ID NO.1)；

20.5-F：5’-GCCCAACATCCGGAGTACATG-3’(SEQ ID NO.2)；

3.7-20.5-R1：5’-CTCAAAGCACTCTAGGGTCCAGCG-3’(SEQ ID NO.3)；

3.7-20.5-R2：5’-AAAGCACTCTAGGGTCCAGCG-3’(SEQ ID NO.4)；

3.7-20.5-R3：5’-CTCAAAGCACTCTAGGGTCCA-3’(SEQ ID NO.5)。

the primer group can stably detect-alpha^3.7And- (alpha)^20.5Thalassemia of the deficiency type.

Preferably, the primer combination further comprises-alpha^4.2A primer group:

4.2-F：5’-TCCCGGTTTACCCATGTGGTGCCTC-3’(SEQ ID NO.6)；

4.2-R：5’-CCCGTTGGATCTTCTCATTTCCC-3’(SEQ ID NO.7)。

preferably, the primer combination isAlso includes-^SEAA primer group:

SEA-F：5’-CGATCTGGGCTCTGTGTTCTCAGT-3’(SEQ ID NO.8)；

SEA-R：5’-AGCCCACGTTG7TGTTCATGGC-3’(SEQ ID NO.9)。

preferably, the primer combination further comprises-^THAIA primer group:

THAI-F：5’-GACCATTCCT8CAGCGTGGGTG-3’(SEQ ID NO.10)；

THAI-R：5’-CAAGTGGGCTGAGCCCTTGAG-3’(SEQ ID NO.11)。

preferably, the primer combination further comprises-^MEDA primer group:

MED-F：5’-TACCCTTTGCAAGCACACGTAC-3’(SEQ ID NO.12)；

MED-R：5’-TCAATCTCCGACAGCTCCGAC-3’(SEQ ID NO.13)。

preferably, the primer combination further comprises-^FILA primer group:

FIL-F：5’-TTTAAATGGGCAAAACAGGCCAGG-3’(SEQ ID NO.14)；

FIL-R：5’-ATAACCTTTATCTGCCACATGTAGC-3’(SEQ ID NO.15).

preferably, the primer combination further comprises the following primer groups for evaluating the quality of the DNA template, wherein the upstream primer of the primer group is the same as the sequence of alpha 2-3.7-F, and the downstream primer of the primer group is alpha 2-R: 5'-TTATTCAAAGACCAGGAAGGGCC-3' (SEQ ID NO. 16).

The application of the primer combination in detecting the deletion type alpha-thalassemia.

A kit for detecting deletion type alpha-thalassemia comprises a PCR reaction solution, a standard molecular weight marker and a loading buffer solution (loading buffer), wherein the PCR reaction solution comprises the primer combination, dNTP, DNA polymerase, the buffer solution and deionized water.

Preferably, the DNA polymerase is TaKaRa-Multiplex PCR Assay Kit.

Preferably, the Buffer is a2 × Multiplex PCR Buffer. The buffer solution is a reaction solution for the above-mentioned preferable DNA polymerase, and can improve the annealing specificity of the primer, and not only can perform Multiplex PCR, but also can reduce the amount of the Extra band and Smear when the Extra band and Smear are present in large amounts and when the Single PCR reaction in which the target amplified fragment is difficult to obtain is performed, and can specifically amplify the target fragment in a short time.

Preferably, the PCR reaction solution further comprises a PCR enhancer, wherein the PCR enhancer is BSA (bovine serum albumin).

The PCR reaction solution also comprises a PCR stabilizer, wherein the PCR stabilizer is DMSO.

Preferably, in the primer combination, the concentration of each primer is 0.06-0.10. mu. mol/L.

More preferably, in the primer combination, the concentration of the primer alpha 2-3.7-F is 0.08. mu. mol/L, the concentration of the primer 20.5-F is 0.08. mu. mol/L, the concentration of the primer 3.7-20.5-R1 is 0.06. mu. mol/L, the concentration of the primer 3.7-20.5-R2 is 0.06. mu. mol/L, the concentration of the primer 3.7-20.5-R3 is 0.06. mu. mol/L, the concentration of the primer 4.2-F is 0.08. mu. mol/L, the concentration of the primer 4.2-R is 0.08. mu. mol/L, the concentration of the primer SEA-F is 0.08. mu. mol/L, the concentration of the primer THAI-R is 0.08. mu. mol/L, the concentration of the primer MED-F is 0.08 mu mol/L, the concentration of the primer MED-R is 0.08 mu mol/L, the concentration of the primer FIL-F is 0.08 mu mol/L, and the concentration of the primer FIL-R is 0.08 mu mol/L.

Preferably, the PCR reaction solution has a total volume of 23 μ L, and includes: 2 × Multiplex PCR Buffer 12.5 μ L; 4U/. mu.L Multiplex PCR Enzyme 0.125. mu.L; 1.25 μ L of 20mg/mL BSA; DMSO 1.2 μ L; 10umol/L primer alpha 2-3.7F 0.2 mu L; 10 mu mol/L primer 20.5-F0.2 mu L; 10umol/L primer 3.7-20.5-R10.15 μ L; 10umol/L primer 3.7-20.5-R20.15 μ L; 3.7-20.5-R30.15 mu L of 10 mu mol/L primer; 10 mu mol/L primer 4.2-F0.2 mu L; 10 mu mol/L primer 4.2-R0.2 mu L; 10 mu mol/L primer SEA-F0.2 mu L; 10 mu mol/L primer SEA-R0.2 mu L; 10 mu mol/L primer THAI-F0.2 mu L; 10 mu mol/L primer THAI-R0.2 mu L; 0.2 mu L of 10 mu mol/L primer MED-F; 10umol/L primer MED-R0.2 mu L; 0.2 mu L of 10 mu mol/L primer FIL-F; 0.2 mu L of 10 mu mol/L primer FIL-R; the balance being DD H₂O。

The invention also provides a using method of the kit, which comprises the following steps:

(1) collecting samples: extracting an anticoagulation whole blood DNA template of a sample to be detected by using the whole blood DNA extraction kit, wherein the concentration of the DNA template is 10-30 ng/mu L;

(2) and (3) PCR amplification: taking out a tube of PCR reaction solution, instantly separating for 5 seconds, marking, then adding 2uL of the extracted DNA template, and obtaining an amplification product according to a PCR amplification program; meanwhile, another tube of reaction solution is taken in each experiment, 2 microliter of pure water is added as a template, and negative control is performed;

(4) and (3) electrophoresis detection: adding a sample loading buffer solution into the amplification product, uniformly mixing, then spotting into 0.8-1.5% agarose gel containing 0.01% nucleic acid dye, and carrying out electrophoresis to obtain a detection result;

(5) and (4) judging a result: when the detection result has only one band of 1791bp, the sample is normal; when two bands appear in the detection result, one band is a 1791bp band, the other band is a non-1791 bp band, and the sample is in a heterozygous deletion type; when only one non 1791bp band exists in the detection result, the sample is homozygous deletion; when two bands appear in the detection result, and both bands are non 1791bp bands, the sample is in a double heterozygous deletion type.

Preferably, the nucleic acid dye is selected from one of EB, GelGreen, SYBR Green I, GoldView, GelRed and GelGreen.

Preferably, the non 1791bp band is a deletion band of each gene, and the size of each deletion band is shown in table 2:

TABLE 2 size of each deletion type band

Deletion type	-α^3.7	αα -α	^4.2 --	^SEA --	^THAI	-(α)^20.5 --	^MED --	^FIL
									Size (bp)	2005-2012	1791	1632	1349	1153	1007-1010	807	546

Note: alpha (1791bp) is an internal control band, namely when the detection result has only one band of 1791bp, the sample is a normal type; when two bands appear in the detection result, one band is a 1791bp band, the other band is a non-1791 bp band, and the sample is in a heterozygous deletion type; when only one non 1791bp band exists in the detection result, the sample is homozygous deletion; when two bands appear in the detection result, and both bands are non 1791bp bands, the sample is in a double heterozygous deletion type.

Preferably, the PCR amplification procedure is:

the final addition of 12 ℃ reaction for 12s in the amplification procedure is added by default in most instruments, and the specific function is to prevent the degradation of products under the condition that the instruments are not taken out in time after the amplification is finished.

The invention has the beneficial effects that:

(1) the primer combination of the invention can stably detect-alpha^3.7And- (alpha)^20.5The two deletion types overcome the problems of unstable amplification system due to high GC content of the alpha-globin gene region; meanwhile, the annealing temperature of each primer is in the same temperature range, so that the phenomena of non-specific amplification and unequal efficiency caused by different annealing temperatures are avoided, and in addition, each primer has good compatibility and no dimer is generated.

(2) The kit overcomes the influence among primers, can detect the thalassemia of 7 genotypes in a single tube in a multiple way, meets the requirements of clinical rapid and convenient detection of the thalassemia, creates conditions for more comprehensive thalassemia screening, and provides scientific basis for prenatal and prenatal examination and thalassemia diagnosis of pregnant fetuses.

(3) The kit is an enzyme and buffer system with good sensitivity and specificity, the reaction system of the kit can detect the DNA template as low as 10ng, has low requirement on the purity of the DNA, is easy to amplify, has a single amplification band, and generates no non-specific band which is easy to interpret.

(4) The PCR reaction solution of the kit comprises the PCR enhancer and the concentration of each component in the PCR reaction solution is set, so that templates with different GC contents in each area achieve similar amplification efficiency, and the determination of a detection result is facilitated.

Description of the drawings:

FIG. 1 is a diagram showing the amplification of-. alpha.in example 1 of the present invention^3.7And- (alpha)^20.5Designing a primer combination;

FIG. 2 shows the primer set-alpha of different primer combinations in example 1 of the present invention^3.7And- (alpha)^20.5The amplification result; wherein 1 is alpha and 2 is alpha ^3.73 is- (. alpha.)^20.5M is DNA marker;

FIG. 3 is a graph showing a comparison of the effects of different BSA concentrations on the assay results in example 2 of the present invention; wherein A is the result of amplification without BSA; b is the result of amplification with a BSA concentration of 600ng/uL in the PCR reaction solution; c is the result of amplification with BSA concentration of 800 ng/. mu.L in the PCR reaction solution; d is the BSA concentration in the PCR reaction solution of 1000 ng/. mu.L; wherein 1 is-alpha^3.7(ii) a2 is α α; 3 is-alpha^4.2(ii) a 4 is-^SEA(ii) a 5 is-^THAI(ii) a 6 is- (. alpha.)^20.5(ii) a 7 is-^MED(ii) a 8 is-^FIL(ii) a M is DNAmarker;

FIG. 4 is a graph showing a comparison of the effects of different DMSO concentrations on the assay results in example 2 of the present invention; wherein, A is the result of amplification without DMSO; b is the amplification result of DMSO with the volume concentration of 4% in the PCR reaction solution; c is the amplification result of DMSO with the volume concentration of 4.8% in the PCR reaction solution; d is the amplification result of DMSO at a volume concentration of 6% in the PCR reaction solution, wherein 1 is-alpha^3.7(ii) a2 is α α; 3 is-alpha^4.2(ii) a 4 is-^SEA(ii) a 5 is-^THAI(ii) a 6 is- (. alpha.)^20.5(ii) a 7 is-^MED(ii) a 8 is-^FIL(ii) a M is DNA marker;

FIG. 5 is a graph showing the comparison of the effects of different combinations of DMSO and BSA concentrations in the reaction solution in example 2 of the present invention on the assay results; a is the result of 1000ng/uL BSA and 4% DMSO amplification; b is the result of 1000ng/uL BSA and 4.8% DMSO amplification; c: amplification results with 1000ng/uL BSA and 6% DMSO; wherein 1 is-alpha^3.7(ii) a2 is α α; 3 is-alpha^4.2(ii) a 4 is-^SEA(ii) a 5 is-^THAI(ii) a 6 is- (. alpha.)^20.5(ii) a 7 is-^MED(ii) a 8 is-^FIL(ii) a M is DNA marker;

FIG. 6 shows the results of the detection of 7 α -thalassemia deficiency type samples after system optimization in example 2 of the present invention; detecting the template: 1 is-alpha^3.7(ii) a2 is α α; 3 is-alpha^4.2(ii) a 4 is-^SEA(ii) a 5 is-^THAI(ii) a 6 is- (. alpha.)^20.5(ii) a 7 is-^MED(ii) a 8 is-^FIL(ii) a M is DNA marker;

FIG. 7 shows the results of amplification of 10ng and 20ng DNA templates after system optimization in example 2 of the present invention, where α α: normal; alpha/-alpha^3.7：-α^3.7Heterozygous deletion type; alpha/-alpha^4.2:-α^4.2Heterozygous deletion type; alpha/-^THAI：--^THAIHeterozygous deletion type; alpha/- (alpha)^20.5:-(α)^20.5Heterozygous deletion type; alpha/-^MED:--^MEDHeterozygous deletion type; alpha/-^FIL:--^FILHeterozygous deletion type; marker is molecular weight Marker.

Detailed Description

To further illustrate the technical solution of the present invention, the following detailed description is made with reference to the accompanying drawings.

Example 1 Using the present invention for the Stable detection of-. alpha.^3.7And- (alpha)^20.5

1. Detection of-alpha^3.7And- (alpha)^20.5Primer combination

The research finds that the alpha is^3.7And- (alpha)^20.5The GC content in the region is high, and a complex secondary structure is easily formed to prevent the effective amplification of the product. The amplification result of the region based on the conventional primer design is not ideal, the requirement on the purity of the template is high, the amplification result is sometimes, the omission is easily caused, and great risk is brought to clinical diagnosis.

The invention aims to realize simultaneous detection of-alpha by single-tube multiplex PCR amplification^3.7And- (alpha)^20.5Two genotypes are adopted, particularly the problem that a secondary structure is easily formed in a region with high GC content to prevent effective amplification of a specific product is avoided, a large amount of experimental research is carried out, then an amplification primer and a reaction system are optimally designed based on the enrichment effect of the amplification product, and conventional primers alpha 2-3.7-F and 20.5-F are designed at one end with low GC content, and the sequences of the primers are 5'-TTCCTTCCTCACCCCACATCCC-3' (SEQ ID No.1) and 5'-GCCCAACATCCGGAGTACATG-3' (SEQ ID No. 2); three consecutive overlapping primers 3.7-20.5-R1, 3.7-20.5-R2 and 3.7-20.5-R3 with sequences of 5' -CTCAAAGCACTCTAGGGTCCAGCG-3, respectively, are designed at the end with higher GC content to enhance the amplification effect (see FIG. 1)' (SEQ ID NO.3), 5'-AAAGCACTCTAGGGTCCAGCG-3' (SEQ ID NO.4) and 5'-CTCAAAGCACTCTAGGGTCCA-3' (SEQ ID NO.5) (see Table 3).

TABLE 3 amplification of-. alpha.^3.7And- (alpha)^20.5Primer combination nucleotide sequence of (1)

Primer name	Sequence numbering	Sequence of
			α2-3.7-F	SEQ ID NO.1	5’-TTCCTTCCTCACCCCACATCCC-3’
3.7-20.5-R1	SEQ ID NO.3	5’-CTCAAAGCACTCTAGGGTCCAGCG-3’
			3.7-20.5-R2	SEQ ID NO.4	5’-AAAGCACTCTAGGGTCCAGCG-3’
3.7-20.5-R3	SEQ ID NO.5	5’-CTCAAAGCACTCTAGGGTCCA-3’
			20.5-F	SEQ ID NO.2	5’-GCCCAACATCCGGAGTACATG-3’
α2-R	SEQ ID NO.16	5’-TTATTCAAAGACCAGGAAGGGCC-3’

2. Amplification of alpha^3.7And- (alpha)^20.5Reaction system

According to amplification-alpha^3.7And- (alpha)^20.5The primer combinations are different, the optimal PCR reaction solution formula system is determined by optimizing each component in the reaction system and a large number of comparative experiments, and the optimal PCR reaction solution formula system is shown in a table 4:

TABLE 4 reaction System reagent composition and content

Note: the DNA template loading was 2. mu.L (10ng) and the total reaction volume was 25. mu.L.

3. Amplification of alpha^3.7And- (alpha)^20.5Reaction procedure

The invention determines the optimal amplification-alpha by optimizing each component in the reaction system and a large number of comparative experiments^3.7And- (alpha)^20.5The reaction procedure is shown in Table 5.

The apparatus used for the PCR reaction: the specific model of the Bio-Rad thermal cycler T100 or the Langji PCR amplimer A200 has no requirement. Preferably, the PCR amplification procedure is shown in Table 5, and the agarose gel electrophoresis analysis is performed after all cycles have been completed.

Sample source: all samples are derived from anticoagulated peripheral blood samples with determined genotypes, and the samples are collected from anticoagulated peripheral blood or umbilical cord blood and other samples.

Sample detection: and (3) carrying out amplification detection on a sample (whole blood) to be detected with a known genotype detected and determined by a conventional method on a PCR instrument according to the reaction system and the reaction program, and carrying out agarose gel electrophoresis analysis after all cycles are finished.

TABLE 5 amplification-alpha^3.7And- (alpha)^20.5Reaction procedure

4. Electrophoretic detection of amplification products

mu.L of 10 × loading Buffer 2.5. mu.L of the amplification product was added to the 25. mu.L of the amplification product, mixed well, and electrophoresed through 1.2% agarose gel (containing nucleic acid dye) at a voltage of 5v/cm for 50 minutes.

5. Data analysis and result determination

And (3) analyzing and interpreting the result according to the agarose gel electrophoresis of the PCR product:

1) and (3) normal: the detection sample has only 1 band, 1790 bp.

2) Heterozygous deletion: the detection result shows 2 bands, wherein 1 band is 1790bp, and the other band is a deletion-type band.

3) Homozygous deletion: the detection result has 1 deletion type band and no 1790 bp.

4) Double heterozygous deletion: the detection result has 3 bands, namely 1790bp band and two deletion type bands.

The kit of the invention passes through p-alpha^3.7And- (alpha)^20.5Detection of both mutation types showed good amplification and discrimination of the two mutations. By designing conventional primers alpha 2-3.7-F and 20.5-F at the end with low GC content and three consecutive overlapping primers 3.7-20.5-R1, 3.7-20.5-R2 and 3.7-20.5-R3 at the end with high GC content, alpha-alpha can be obviously detected^3.7And- (alpha)^20.5Two types of mutations (see FIG. 2). Therefore, when the kit is used for detecting the thalassemia genes, the result is accurate; and the wild type sample can only detect a normal band, has higher specificity and has visual result.

Example 2 detection of 7 kinds of deletion-type thalassemias by the primer combination and the kit of the present invention

In order to realize higher amplification efficiency of the primers, a batch of different types of PCR stabilizers and enhancers are selected for screening optimization, so as to increase the yield of the required PCR products or reduce non-specific products. The classes of PCR enhancers can be broadly classified according to their mechanism of action: (1) co-solvents for amplification of GC-rich and complex secondary structure templates; (2) a stabilizer for protecting the activity of DNA polymerase; (3) auxiliary enhancers for optimizing primer and template binding.

Common stabilizers and enhancers include 1-5% DMSO (dimethyl sulfoxide), 1-5% glycerol, 1.25-10% (v/v) formamide, 0.5-2 mM betaine, 0.01-0.1% (w/v) BSA (bovine serum albumin), 0.1-1M trehalose, etc. DMSO and BSA with different concentrations are mainly selected as a stabilizer and a reinforcing agent for verification, and the preferable DMSO concentrations are as follows: 4% -6%; preferred BSA concentrations are: 600ng/uL-1000 ng/uL. The more preferable scheme is as follows: the DMSO concentration was: 4.8%, BSA concentration: 1000 ng/uL.

1. Effect of different concentrations of BSA on amplification results

The invention optimizes the concentration of the reinforcing agent BSA in the reaction system through a large number of comparative experiments, which is shown in Table 6.

TABLE 6 reaction systems with different BSA concentrations

The procedure is described in example 1, and the PCR amplification reaction conditions are described in Table 5 of example 1, where the DNA template types are in turn-. alpha.^3.7，αα，-α^4.2，-^SEA，--^THAI，-(α)^20.5，--^MED，--^FIL。

And (3) electrophoretic detection of an amplification product: as a result, as shown in FIG. 3, the preferred embodiment of the present invention is B, C, D, and the BSA concentration in the PCR reaction solution is 600-1000 ng/. mu.L.

2. Effect of different concentrations of DMSO on amplification results

The concentration of the stabilizer DMSO in the reaction system is optimized through a large number of comparative experiments, and the table 7 shows.

TABLE 7 reaction systems with different DMSO action concentrations

And (3) electrophoretic detection of an amplification product: the results are shown in FIG. 4, and the preferred embodiment of the present invention is B, C, D, and the volume percentage of DMSO in the PCR reaction solution is 4-6%.

Effect of the Combined Effect of BSA and DMSO on the amplification results

The invention optimizes the concentration of the combined effect of BSA and DMSO in the reaction system through a large number of comparative experiments, which is shown in Table 8.

TABLE 8 BSA and DMSO concentration reaction System

And (3) electrophoretic detection of an amplification product: the results are shown in FIG. 5, which is a preferred embodiment B of the invention, with a preferred DMSO concentration of 4.8% and a BSA concentration of 1000 ng/uL.

4. Determining the concentration of the primer and the concentration of other components in the reaction system:

a large number of experiments prove that the final concentration range of the primers in the reaction system is 0.1-1 mu mol/L, the final concentration of MgCl2 is 2mM, the final concentration of dNTP in a mixed solution of four kinds of deoxyadenosine triphosphate (dATP, dGTP, dCTP and dTTP) with equal concentration is 100nM-500nM, the final concentration of DMSO is 4.8%, the final concentration of BSA is 1000ng/uL, the final concentration of DNA polymerase is 1-4U/reaction, and the optimal PCR reaction system formula is finally determined by using an orthogonal test method and through different concentration experiment comparison, wherein the optimal PCR reaction system formula is shown in a table 9.

TABLE 9 optimal reaction System

And (3) electrophoretic detection of an amplification product: the results are shown in FIG. 6, where the preferred DMSO concentration of the present invention is 4.8% and the BSA concentration is 1000 ng/uL.

5. Influence of template amount on amplification result of detection system

The template amount in the reaction system has great influence on the amplification result, and the preferable template amount range is 10-300 ng. The kit is an enzyme and buffer system with good sensitivity and specificity, can detect a DNA template as low as 10ng, has low requirement on the purity of DNA, is easy to amplify, has a single amplification band, does not generate a non-specific band, and is easy to interpret, as shown in figure 7.

SEQUENCE LISTING

<110> Chongqing Puluotong Gene medical research institute Co., Ltd

<120> primer combination and kit for detecting deletion type alpha-thalassemia

<130> 2018.12.10

<160> 16

<170> PatentIn version 3.5

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ttccttcctc accccacatc cc 22

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ctcaaagcac tctagggtcc agcg 24

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tttaaatggg caaaacaggc cagg 24

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ttattcaaag accaggaagg gcc 23

Claims

1. A primer combination for detecting deletion type alpha-thalassemia comprises the following primers:

α2-3.7-F：5’-TTCCTTCCTCACCCCACATCCC-3’；

20.5-F：5’-GCCCAACATCCGGAGTACATG-3’；

3.7-20.5-R1：5’-CTCAAAGCACTCTAGGGTCCAGCG-3’；

3.7-20.5-R2：5’-AAAGCACTCTAGGGTCCAGCG-3’；

3.7-20.5-R3：5’-CTCAAAGCACTCTAGGGTCCA-3’。

2. the primer combination of claim 1, wherein the primer combination further comprises one or more of the following primer sets:

- α 4.2 primer set:

4.2-F：5’-TCCCGGTTTACCCATGTGGTGCCTC-3’；

4.2-R：5’-CCCGTTGGATCTTCTCATTTCCC-3’；

-SEA primer set:

SEA-F：5’-CGATCTGGGCTCTGTGTTCTCAGT-3’；

SEA-R：5’-AGCCCACGTTGTGTTCATGGC-3’；

-THAI primer set:

THAI-F：5’-GACCATTCCTCAGCGTGGGTG-3’；

THAI-R：5’-CAAGTGGGCTGAGCCCTTGAG-3’；

-MED primer set:

MED-F：5’-TACCCTTTGCAAGCACACGTAC-3’；

MED-R：5’-TCAATCTCCGACAGCTCCGAC-3’；

-FIL primer set:

FIL-F：5’-TTTAAATGGGCAAAACAGGCCAGG-3’；

FIL-R：5’-ATAACCTTTATCTGCCACATGTAGC-3’。

3. the primer combination of claim 2, wherein the primer combination further comprises a primer set for evaluating the quality of the DNA template, wherein the upstream primer has the same sequence as α 2-3.7-F, and the downstream primer has a sequence of α 2-R: 5'-TTATTCAAAGACCAGGAAGGGCC-3' are provided.

4. A kit for detecting deletion type α -thalassemia, comprising a PCR reaction solution, a standard molecular weight marker and a loading buffer solution, wherein the PCR reaction solution comprises the primer combination of any one of claims 1 to 3, dntps, DNA polymerase, BSA, DMSO, a buffer solution and deionized water.

5. The kit of claim 4, wherein the Buffer is a2 x Multiplex PCR Buffer.

6. The kit according to claim 4, wherein the concentration of each primer in the primer combination is 0.06-0.10. mu. mol/L.

7. The kit according to claim 4, wherein when the primer set is the primer set according to claim 2 or 3, the concentration of the primer α 2-3.7-F is 0.08. mu. mol/L, the concentration of the primer 20.5-F is 0.08. mu. mol/L, the concentration of the primer 3.7-20.5-R1 is 0.06. mu. mol/L, the concentration of the primer 3.7-20.5-R2 is 0.06. mu. mol/L, the concentration of the primer 3.7-20.5-R3 is 0.06. mu. mol/L, the concentration of the primer 4.2-F is 0.08. mu. mol/L, the concentration of the primer 4.2-R is 0.08. mu. mol/L, the concentration of the primer SEA-F is 0.08. mu. mol/L, and the concentration of the primer SEA-R is 0.08. mu. mol/L, the concentration of the primer THAI-F is 0.08 mu mol/L, the concentration of the primer THAI-R is 0.08 mu mol/L, the concentration of the primer MED-F is 0.08 mu mol/L, the concentration of the primer MED-R is 0.08 mu mol/L, the concentration of the primer FIL-F is 0.08 mu mol/L, and the concentration of the primer FIL-R is 0.08 mu mol/L.