CN111471763B

CN111471763B - Primer, probe and reaction buffer solution combination and kit for multiplex real-time fluorescence PCR detection of alpha-thalassemia

Info

Publication number: CN111471763B
Application number: CN202010552793.3A
Authority: CN
Inventors: 魏劭; 林培蕊; 雷彩霞; 魏超
Original assignee: Nanjing Anpu Precision Medical Test Co ltd; XIAMEN AMPLLY BIOTECHNOLOGY CO Ltd
Current assignee: Nanjing Anpu Precision Medical Test Co ltd; XIAMEN AMPLLY BIOTECHNOLOGY CO Ltd
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2023-06-02
Anticipated expiration: 2040-06-17
Also published as: CN111471763A

Abstract

The invention provides a primer, a probe and a reaction buffer solution combination and a kit for multiplex real-time fluorescence PCR detection of alpha-thalassemia, belonging to the technical field of molecular biology detection; the primer, probe and reaction buffer combination comprises 8 groups of primer and probe combinations and 4 reaction buffers; the 4 reaction buffers comprise a B-4 reaction buffer, a B-2 reaction buffer, a J-1 reaction buffer and an H-1 reaction buffer. The combination of the primer, the probe and the reaction buffer solution of the invention ^SEA 、‑‑ ^THAI 、‑α ^3.7 、‑α ^4.2 Amplification of Long fragment deletions and alpha ^CS α/αα、α ^QS α/αα、α ^WS The method has the characteristics of strong specificity, high sensitivity, short time consumption and the like, and simultaneously has the characteristic of distinguishing different mutant types through different fluorescent channels in the same reaction tube.

Description

Primer, probe and reaction buffer solution combination and kit for multiplex real-time fluorescence PCR detection of alpha-thalassemia

Technical Field

The invention relates to the technical field of molecular biology detection, in particular to a primer, a probe, a reaction buffer solution combination and a kit for multiplex real-time fluorescence PCR detection of alpha-thalassemia.

Background

Alpha-thalassemia is caused by a defect in the alpha-globin gene at the end of human chromosome 16 and can be divided into two major categories, namely, deletion type and non-deletion type, wherein the incidence of deletion type is far higher than that of non-deletion type. Alpha-thalassemia belongs to autosomal monogenic genetic disease, highIs mainly used for the people in China in the south, especially in the south, southwest and other areas, and the people in China mainly have right side deficiency (-alpha) ^3.7 ) Left absence (-alpha) ^4.2 ) And southeast Asia type (-) ^SEA ) Three deletions, alpha ^CS α/αα、α ^QS α/αα、α ^WS Alpha/alpha three non-missing types and sporadic case-Thailand missing types-) ^THAI ) And waiting for alpha-thalassemia genotypes. -alpha ^3.7 The main deletion of the type is alpha 1 gene, and the deletion fragment is 3.7Kb long; -alpha ^4.2 The type is deleted alpha 2 gene, the length of the deleted fragment is 4.2Kb; and-) ^SEA The two functional genes, namely alpha 1 and alpha 2, are all deleted, and the deletion fragment is as long as 20Kb. - - ^THAI Deletion fragment ratio-) ^SEA The deleted fragments are also long, and comprise alpha 1 and alpha 2 genes, so that moderate and severe alpha thalassemia can be formed clinically. Alpha ^CS α/αα、α ^QS α/αα、α ^WS The three point mutations of alpha/alpha are all located on the alpha 2 globin gene.

Clinically, patients with α -thalassemia are classified as type 4: i.e. resting, light, standard, i.e. HbH disease (hemoglobinopathies), and heavy (Bart's fetuses). Resting, also known as thalassemia 2 or alpha + -thalassemia, is essentially asymptomatic, has no obvious hematological changes, and is a carrier lacking an alpha-gene. Light weight, also known as alpha-thalassemia 1 or alpha 0-thalassemia, has mild anemia symptoms, manifesting as mild anemia, due to the deletion of 2 alpha genes. The intermediate type (HbH) is the most common type of alpha-thalassemia clinically, has obvious and serious anemia symptoms, and has the symptoms of anemia from 1 year old or so, and serious hepatosplenomegaly and jaundice, emaciation with yellowish complexion and bone changes and life support by blood transfusion. HbH patients have a total or equivalent deletion of three alpha genes. Heavy ground is one of the most serious, homozygous state of alpha 0, where 4 alpha globin genes are deleted or defective, so that no alpha chain is produced at all, and the fetus is born as a dead fetus. At present, no effective treatment method exists for the disease, and only genetic consultation and prenatal gene diagnosis are used for preventing the birth of infants suffering from thalassemia so as to achieve the purpose of prenatal.

The most common methods currently on the market for the detection of the alpha thalassemia gene are the gap PCR (gap-PCR) technique and the reverse dot hybridization (RDB) technique.

The Gap-PCR technique is to design primers complementary to sequences on both sides of a deleted sequence, and the deletion allows the distance between the pair of primers which are far apart in normal DNA sequences to be close due to the end-to-end ligation, and allows a fragment of a specific length to be amplified. The genotype of the sample is detected according to the size of the electrophoresis fragment by agarose gel electrophoresis, which is an effective method for detecting the deficiency type thalassemia, but the method can only detect the deficiency type in alpha thalassemia.

The RDB technology is to hybridize the PCR product with the specific probe fixed on the membrane strip based on PCR amplification, and observe the result by exciting fluorescence or a series of color reaction, the method has high sensitivity, but the method can only detect non-deletion thalassemia.

There is currently a lack of a device for achieving ^SEA 、-- ^THAI 、-α ^3.7 、-α ^4.2 Amplification of Long fragment deletions and alpha ^CS α/αα、α ^QS α/αα、α ^WS Alpha/alpha three non-deletion type synchronous typing detection method.

Disclosure of Invention

The invention aims to provide a primer, a probe and a reaction buffer solution combination and a kit for multiplex real-time fluorescence PCR detection of alpha-thalassemia, and the primer, the probe and the kit can realize that ^SEA 、-- ^THAI 、-α ^3.7 、-α ^4.2 Amplification of Long fragment deletions and alpha ^CS α/αα、α ^QS α/αα、α ^WS Alpha/alpha three non-deletion type synchronous typing detection.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a primer, probe and reaction buffer solution combination for multiplex real-time fluorescence PCR detection of alpha-thalassemia, which comprises 8 groups of primer and probe combinations and 4 reaction buffers;

the 8 groups of primer and probe combinations comprise 1 group of primer and probe combinations to 8 groups of primer and probe combinations; the 1 st group primer and the probe combination to the 8 th group primer and the probe combination are respectively and independently packaged;

the 4 reaction buffers comprise a B-4 reaction buffer, a B-2 reaction buffer, a J-1 reaction buffer and an H-1 reaction buffer; the B-4 reaction buffer solution, the B-2 reaction buffer solution, the J-1 reaction buffer solution and the H-1 reaction buffer solution are respectively and independently packaged;

the group 1 primer and probe combination comprises: an SEA upstream primer, an SEA downstream primer, an SEA probe, an internal standard 01 upstream primer, an internal standard 01 downstream primer and an internal standard 01 probe; the nucleotide sequences of the SEA upstream primer, the SEA downstream primer and the SEA probe are respectively shown as SEQ ID NO. 1-SEQ ID NO. 3; the nucleotide sequences of the internal standard 01 upstream primer, the internal standard 01 downstream primer and the internal standard 01 probe are respectively shown in SEQ ID NO. 28-SEQ ID NO. 30;

The group 2 primer and probe combinations include: THAI upstream primer, THAI downstream primer, THAI probe, internal standard 01 upstream primer, internal standard 01 downstream primer, and internal standard 01 probe; the nucleotide sequences of the THAI upstream primer, the THAI downstream primer and the THAI probe are respectively shown in SEQ ID NO. 4-SEQ ID NO. 6; the nucleotide sequences of the internal standard 01 upstream primer, the internal standard 01 downstream primer and the internal standard 01 probe are respectively shown in SEQ ID NO. 28-SEQ ID NO. 30;

the 3 rd primer and probe combination includes: 4.2A upstream primer, 4.2A downstream primer, 4.2A probe, internal standard 03 upstream primer, internal standard 03 downstream primer and internal standard 03 probe; the nucleotide sequences of the 4.2A upstream primer, the 4.2A downstream primer and the 4.2A probe are respectively shown as SEQ ID NO. 7-SEQ ID NO. 9; the nucleotide sequences of the internal standard 03 upstream primer, the internal standard 03 downstream primer and the internal standard 03 probe are respectively shown in SEQ ID NO. 34-SEQ ID NO. 36;

the 4 th set of primer and probe combinations include: 4.2B upstream primer, 4.2B downstream primer, 4.2B probe, internal standard 02 upstream primer, internal standard 02 downstream primer and internal standard 02 probe; the nucleotide sequences of the 4.2B upstream primer, the 4.2B downstream primer and the 4.2B probe are respectively shown as SEQ ID NO. 10-SEQ ID NO. 12; the nucleotide sequences of the internal standard 02 upstream primer, the internal standard 02 downstream primer and the internal standard 02 probe are respectively shown in SEQ ID NO. 31-SEQ ID NO. 33;

The 5 th set of primer and probe combinations include: 3.7 upstream primer, 3.7 downstream primer, 3.7 probe, internal standard 02 upstream primer, internal standard 02 downstream primer and internal standard 02 probe; the nucleotide sequences of the 3.7 upstream primer, the 3.7 downstream primer and the 3.7 probe are respectively shown as SEQ ID NO. 13-SEQ ID NO. 15; the nucleotide sequences of the internal standard 02 upstream primer, the internal standard 02 downstream primer and the internal standard 02 probe are respectively shown in SEQ ID NO. 31-SEQ ID NO. 33;

the 6 th set of primer and probe combinations include: WS upstream primer, WS downstream primer, first WS probe, second WS probe, internal standard 01 upstream primer, internal standard 01 downstream primer and internal standard 01 probe; the nucleotide sequences of the WS upstream primer, the WS downstream primer, the first WS probe and the second WS probe are respectively shown as SEQ ID NO. 16-SEQ ID NO. 19; the nucleotide sequences of the internal standard 01 upstream primer, the internal standard 01 downstream primer and the internal standard 01 probe are respectively shown in SEQ ID NO. 28-SEQ ID NO. 30;

the 7 th set of primer and probe combinations include: a QS upstream primer, a QS downstream primer, a first QS probe, a second QS probe, an internal standard 01 upstream primer, an internal standard 01 downstream primer and an internal standard 01 probe; the nucleotide sequences of the QS upstream primer, the QS downstream primer, the first QS probe and the second QS probe are respectively shown as SEQ ID NO. 20-SEQ ID NO. 23; the nucleotide sequences of the internal standard 01 upstream primer, the internal standard 01 downstream primer and the internal standard 01 probe are respectively shown in SEQ ID NO. 28-SEQ ID NO. 30;

The 8 th set of primer and probe combinations include: CS upstream primer, CS downstream primer, first CS probe, second CS probe, internal standard 01 upstream primer, internal standard 01 downstream primer and internal standard 01 probe; the nucleotide sequences of the CS upstream primer, the CS downstream primer, the first CS probe and the second CS probe are respectively shown as SEQ ID NO. 24-SEQ ID NO. 27; the nucleotide sequences of the internal standard 01 upstream primer, the internal standard 01 downstream primer and the internal standard 01 probe are respectively shown in SEQ ID NO. 28-SEQ ID NO. 30;

the 5 'ends of the SEA probe, the THAI probe, the 4.2A probe, the 4.2B probe, the 3.7 probe, the second WS probe, the second QS probe and the second CS probe are respectively marked with a fluorescence reporter group FAM, and the 3' ends are respectively marked with a quenching gene BHQ1;

the 5 'ends of the first WS probe, the first QS probe and the first CS probe are respectively marked with a fluorescent reporter group ROX, and the 3' ends of the first WS probe, the first QS probe and the first CS probe are respectively marked with a quenching gene BHQ2;

the 5 'ends of the internal standard 01 probe, the internal standard 02 probe and the internal standard 03 probe are respectively marked with a fluorescent reporter group HEX, and the 3' ends are respectively marked with a quenching gene BHQ1;

the B-4 reaction buffer comprises the following components in concentration: tris hydrochloride 3.8mM, tris 32.2mM, potassium chloride 150mM, magnesium chloride 6.25mM, triton 0.25 mM, ammonium sulfate 12.5mM, deoxyribonucleoside triphosphate 0.75mM and deoxyuridine triphosphate 1.5mM;

The B-2 reaction buffer comprises the following components in concentration: tris hydrochloride 3.8mM, tris 32.2mM, potassium chloride 150mM, magnesium chloride 6.25mM, triton 0.25 mM, ammonium sulfate 12.5mM, tetramethylammonium chloride 20mM, deoxyribonucleoside triphosphate 0.75mM and deoxyuridine triphosphate 1.5mM;

the J-1 reaction buffer comprises the following components in concentration: tris hydrochloride 3.8mM, tris 32.2mM, potassium chloride 125mM, magnesium chloride 6.25mM, ammonium sulfate 25mM, dimethyl sulfoxide 12.50% (by volume), betaine 2.5M, 7-deaza-dGTP 0.075mM, deoxyribonucleoside triphosphates 1mM and deoxyuridine triphosphate 2mM;

the H-1 reaction buffer comprises the following components in concentration: 50mM of 4-hydroxyethyl piperazine ethane sulfonic acid, 25mM of ammonium sulfate, 125mM of potassium chloride, 7.5mM of magnesium chloride, 2.50% by volume of formamide, 0.4mM of deoxyribonucleoside triphosphate and 0.8mM of deoxyuridine triphosphate. The invention also provides a kit comprising the primer, the probe and the reaction buffer solution combination according to the scheme.

Preferably, the kit further comprises an enzyme mixture, a dilution buffer, a TE buffer and paraffin oil.

Preferably, the enzyme mixed solution comprises the following components: taq DNA polymerase 4.0U/. Mu.L, uracil glycosylase 0.004U/. Mu.L and enzyme stock A (50 mM Tris-HCl, pH8.0, 100mM NaCl,0.1mM EDTA.2Na, 1% TritonX-100 (vol.), 50% glycerol (vol.)); the dilution buffer comprises the following components: 1mM of tris (hydroxymethyl) aminomethane hydrochloride and 9mM of tris (hydroxymethyl) aminomethane; the TE buffer comprises the following components: tris hydrochloride 5.6mM, tris 4.4mM and disodium ethylenediamine tetraacetate 1mM.

Preferably, the kit further comprises a negative control and a positive control; the negative control comprises normal saline; the positive control comprises: pGEM-T-Easy-SEA plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-THAI plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-4.2A plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-4.2B plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-3.7 plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-WS plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-QS plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-CS plasmid 1.0X10 ⁴ copies/. Mu.L, wild-type human genomic DNA.

Preferably, the stock concentration of each primer or each probe in each set of primer and probe combinations is 50. Mu.M.

The invention has the beneficial effects that: the invention provides a primer, probe and reaction buffer solution combination for multiplex real-time fluorescence PCR detection of alpha-thalassemia, which comprises 8 groups of primer and probe combinations and 4 reaction buffers; the 4 reaction buffers comprise a B-4 reaction buffer, a B-2 reaction buffer, a J-1 reaction buffer and an H-1 reaction buffer.

The primer, the probe and the reaction buffer solution can synchronously judge the deletion type and non-deletion type homozygous heterozygous mutation of the alpha-thalassemia gene; for the missing part, finding the position of the broken end, designing a pair of primers and probes on two sides of the broken end of the missing segment, and carrying out target segment amplification by the missing part to ensure that the distance between the pair of primers and probes which are far apart in the normal DNA sequence is close due to the connection of the broken end, so as to emit light; for the non-deletion type, designing a pair of primers at two sides of a mutation site of the non-deletion type alpha thalassemia and respectively designing a pair of wild type and mutant probes at an SNP site; the homozygosity and heterozygosity of the point mutation can be directly judged according to different fluorescent signals; the homozygous deletion of the deleted fragment was then combined with whether the mutant ROX channel exhibited fluorescent properties, thereby judging whether the heterozygous deletion or the homozygous deletion was present.

The invention increases the-alpha by adding the reinforcing agents such as DMSO, betaine or TMAC and the like into the reaction buffer solution ^4.2 And-alpha ^3.7 The amplification efficiency of the G+C template with equal height is improved, and the 7-deaza-dGTP is added, so that the specificity and high fidelity of the amplified product can be improved.

In the invention, mutation conditions are indicated by FAM, wild type is indicated by ROX, and internal standard is indicated by HEX; thereby realizing synchronous amplification of one sample and right-side deletion type (-alpha) ^3.7 ) Left absence (-alpha) ^4.2 ) Southeast Asia type (-) ^SEA ) Thailand deletion type ^THAI ) Judgment of the cases of the homozygous heterozygous mutations of seven types of alpha CS alpha/alpha, alpha QS alpha/alpha and alpha WS alpha/alpha.

The primer, probe and reaction buffer solution combination provided by the invention has the characteristics of strong specificity, high sensitivity, short time consumption and the like, and simultaneously has the characteristic of distinguishing different mutant types through different fluorescent channels in the same reaction tube.

The primer, the probe and the reaction buffer solution can realize the synchronous detection of the alpha-thalassemia gene deletion type and the non-deletion type fluorescent PCR without adopting two methodologies for detection, and the operation is simpler. Compared with the traditional deletion type gap-PCR method, the method has the advantages of short time consumption, good specificity, high sensitivity, simple operation, safety and high degree of automation. Compared with the RDB technical method, the method is simple to operate, short in time consumption, high in automation degree and less prone to pollution. Compared with the gap-PCR method and the RDB method, the method does not need to open the cover in the whole process, and reduces the serious consequences such as false positive and false negative of experimental results caused by laboratory pollution.

Drawings

FIG. 1 is a national negative reference-FAM pathway (0001, 0002, 0010);

FIG. 2 is a national negative reference-HEX pathway (0001, 0002, 0010);

FIG. 3 is a national negative reference-ROX pathway (0001, 0002, 0010);

FIG. 4 shows the FAM channel (0003, 0004, 0009) as a reference for other mutations outside the detection range of the kit;

FIG. 5 shows HEX channels (0003, 0004, 0009) which are other mutation type references outside the detection range of the kit;

FIG. 6 shows the other mutant reference-ROX channels (0003, 0004, 0009) outside the detection range of the kit;

FIG. 7 is a national positive reference (YSH 2015-0005);

FIG. 8 is a national positive reference (YSH 2015-0006);

FIG. 9 is a national positive reference (YSH 2015-0007);

FIG. 10 is a national positive reference (YSH 2015-0008);

FIG. 11 is a national positive reference (YSH 2015-0013);

FIG. 12 is a national positive reference (YSH 2015-0019);

FIG. 13 is a national positive reference (YSH 2015-0021);

FIG. 14 is a national positive reference (YSH 2015-0022);

FIG. 15 is a national positive reference (YSH 2015-0029);

FIG. 16 is a national positive reference (YSH 2015-0030);

FIG. 17 is a national positive reference (YSH 2015-0032).

Detailed Description

the H-1 reaction buffer comprises the following components in concentration: 50mM of 4-hydroxyethyl piperazine ethane sulfonic acid, 25mM of ammonium sulfate, 125mM of potassium chloride, 7.5mM of magnesium chloride, 2.50% (volume ratio) of formamide, 0.4mM of deoxyribonucleoside triphosphate and 0.8mM of deoxyuridine triphosphate.

In the present invention, the nucleotide sequences of the 8 sets of primer and probe combinations are shown in Table 8.

TABLE 8 nucleotide sequences of the 8-set primer and probe combinations of the invention

In the present invention, the method consists ofAt-alpha ^4.2 The missing broken end point is undefined, through clinical specimen verification, a pair of primer probes are independently designed, the missing detection phenomenon can occur, in order to avoid the missing detection phenomenon, the position of the other broken end is searched, a pair of primers and probes are respectively designed at two sides of the broken end of the missing segment by combining the positions of the two broken end sites, and the missing makes the distance between the pair of primers and probes which are far away from each other in the normal DNA sequence close due to the connection of the broken end, and the target segment is amplified, so that the light is emitted; wherein-alpha ^4.2B Fragment (-alpha) ^4.2 Comprises-alpha ^4.2A And-alpha ^4.2B ) The high G+C content results in low PCR amplification efficiency, and the amplification efficiency is improved by adding TMAC enhancer into the reaction buffer.

In the present invention, due to-alpha ^3.7 The deletion end point is undefined, and the position fragment is longer, so that the need of long fragment amplification is avoided when a primer probe is designed for detection, the G+C content is very high, the conditions of low amplification efficiency, poor specificity and the like easily occur in the PCR amplification process, the amplification efficiency is improved by adding enhancers such as betaine, DMSO and the like into a reaction buffer, and the amplification specificity is improved by adding a 7-deaza-dGTP enhancer.

The invention designs a primer and a probe according to target genes as gene sequences containing various mutations, and selects sequences of conserved regions near the mutations as internal reference genes according to the characteristics of human genome; the application of the reference gene is as follows: and controlling false negative, and proving the reliability of the detection result.

In the invention, the 1 st primer and probe combination to the 3 rd primer and probe combination are preferably combined with a B-4 reaction buffer; the 4 th primer and the probe combination are preferably combined with a B-2 reaction buffer; the 5 th primer and probe combination is preferably used in combination with a J-1 reaction buffer; the 6 th primer and probe combination to the 8 th primer and probe combination are preferably used in combination with an H-1 reaction buffer solution respectively.

The invention also provides a kit comprising the primer, the probe and the reaction buffer solution combination according to the scheme.

In the invention, the kit also comprises an enzyme mixed solution,Dilution buffer, TE buffer and paraffin oil; the enzyme mixed solution comprises the following components: taq DNA polymerase 4.0U/. Mu.L, uracil glycosylase 0.004U/. Mu.L and enzyme stock A (50 mM Tris-HCl, pH8.0, 100mM NaCl,0.1mM EDTA.2Na, 1% Triton X-100 (vol.), 50% glycerol (vol.); the dilution buffer comprises the following components: 1mM of tris (hydroxymethyl) aminomethane hydrochloride and 9mM of tris (hydroxymethyl) aminomethane; the TE buffer comprises the following components: tris hydrochloride 5.6mM, tris 4.4mM and disodium ethylenediamine tetraacetate 1mM. The kit also comprises a negative control and a positive control; the negative control comprises normal saline; the positive control comprises: pGEM-T-Easy-SEA plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-THAI plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-4.2A plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-4.2B plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-3.7 plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-WS plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-QS plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-CS plasmid 1.0X10 ⁴ copies/. Mu.L, wild-type human genomic DNA. The stock concentration for each primer or each probe in each set of primer and probe combinations was 50. Mu.M.

The method for using the kit preferably comprises the following steps:

1) Extracting genome DNA of a sample to be detected;

2) Taking the genome DNA of the sample to be detected as a template, and carrying out multiplex real-time fluorescence PCR amplification reaction by utilizing the combination of the primer, the probe and the reaction buffer solution according to the scheme; in the multiplex real-time fluorescence PCR amplification reaction process, a full-automatic medical PCR analysis system (Xiamen An Puli biological engineering Co., ltd.) is used for collecting fluorescence signals, and fluorescein is set as FAM, ROX and HEX; the fluorescence signal collection is set at 58 ℃ for 30s;

taking 3-12 cycles of fluorescent signals as a baseline, analyzing a baseline threshold value, and adjusting a threshold line to be just above the highest point of a normal negative control amplification curve (an irregular noise line) based on the Ct value=0.0;

3) Analyzing the detection result; the FAM channel, the ROX channel and the HEX channel of the negative control reaction well should have no amplification curve, the FAM channel, the ROX channel and the HEX channel of the positive control reaction well should have amplification curves and Ct value should be less than 38, otherwise, the experiment is regarded as invalid;

The HEX channel of the sample to be tested should have an amplification curve, and the Ct value should be less than 38; if the HEX channel has no amplification curve, the quality of the extracted genome does not meet the experimental requirement, and the genome needs to be extracted again;

if the FAM channel of the reaction hole of the sample to be detected has no amplification curve, judging that the detection result is that the alpha-thalassemia mutation is absent;

if the FAM channel of the reaction well 1 to 5 of the sample to be detected has an amplification curve and the Ct value is less than or equal to 38, the homozygous deletion and heterozygous deletion are judged according to the ROX channel of the reaction well 6 to 8. If the ROX channel has an amplification curve and the Ct value is less than or equal to 38, judging that heterozygous deletion exists; if the ROX channel has no amplification curve, judging that the ROX channel is homozygous missing;

if the FAM channel of the reaction hole of the sample to be detected is provided with an amplification curve and the Ct value is less than or equal to 38, the homozygous and heterozygous judgment is carried out according to the ROX channel of the reaction hole of the sample to be detected. If the corresponding ROX channel has an amplification curve and the Ct value is less than or equal to 38, judging that the ROX channel is heterozygous mutation corresponding to mutation; if the corresponding ROX channel has no amplification curve, judging that the ROX channel is homozygously mutated corresponding to the mutation;

if two or more amplification curves exist in the FAM channel of the sample to be detected, judging that the sample to be detected has compound mutation.

Firstly extracting genome DNA of a sample to be detected; the sample preferably comprises a sample of a-thalassemia mutant genes; the method for extracting the genomic DNA of the sample to be detected is not particularly limited, and conventional methods in the art can be adopted.

After obtaining the genome DNA of the sample to be detected, the invention uses the genome DNA of the sample to be detected as a template, and uses the combination of the primer, the probe and the reaction buffer solution to carry out multiplex real-time fluorescence PCR amplification reaction; in the multiple real-time fluorescence PCR amplification reaction process, fluorescence signals are collected by using a full-automatic medical PCR analysis system (Xiamen An Puli biological engineering Co., ltd.) instrument, and fluorescein is set as FAM, ROX and HEX; the fluorescence signal collection is set at 58 ℃ for 30s; taking 3-12 cycles of fluorescent signals as a baseline, analyzing a baseline threshold value, and adjusting a threshold line to be just above the highest point of a normal negative control amplification curve (an irregular noise line) based on the Ct value=0.0; the amplified system is preferably 50. Mu.L; the procedure for amplification is preferably: 38 ℃ for 5min;95 ℃ for 10min;95 ℃, 15s,58 ℃, 50s (30 s post-reading fluorescence), 72 ℃,45 s,45 cycles; 38 ℃ for 30s.

After the detection result is obtained, the invention analyzes the detection result; the FAM channel, the ROX channel and the HEX channel of the negative control reaction well should have no amplification curve, the FAM channel, the ROX channel and the HEX channel of the positive control reaction well should have amplification curves and Ct value should be less than 38, otherwise, the experiment is regarded as invalid;

The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

1. Sample of

Thalassemia nucleic acid detecting national reference (lot number: 360014-201701), mutation types see table 2 below:

TABLE 2 types of mutations corresponding to the samples

(II) Experimental procedure

1. Reagent preparation (reagent preparation area)

(1) And taking out the reaction buffer solution, the amplification solution, the A enzyme mixed solution, the dilution buffer solution, the paraffin oil, the TE buffer solution and the like in the kit, and standing at room temperature to fully dissolve the reaction buffer solution, the amplification solution, the A enzyme mixed solution, the dilution buffer solution, the paraffin oil, the TE buffer solution and the like for standby.

(2) Preparing an amplification solution: and adding 240 mu L of TE buffer into the amplification tube, fully and uniformly mixing, centrifuging for 5 seconds at 3000-5000 g, and transferring to a specimen processing area.

(3) Enzyme configuration: according to the number of the samples, preparing 0.5 mu L of enzyme mixed solution and 9.5 mu L of dilution buffer solution, fully and uniformly mixing, centrifuging for 5 seconds at 3000-5000 g, and transferring to a sample treatment area.

2. Sample adding (sample treatment area)

Adding 10 mu L of 8 kinds of amplification solutions into a PCR reaction tube respectively, adding 20 mu L of 4 kinds of reaction buffer solutions into the corresponding PCR reaction tube respectively (SEA, THAI and detection holes of 4.2A are added with B-4 reaction buffer solution, detection holes of 4.2B are added with B-2 reaction buffer solution, detection holes of 3.7 are added with J-1 reaction buffer solution, detection holes of WS, QS and CS are added with H-1 reaction buffer solution), adding 10 mu L of enzyme mixed solution prepared in the step (3) into the PCR reaction tube, sequentially adding 10 mu L of reference DNA and reference substance into the corresponding PCR reaction tube respectively, sequentially adding 25 mu L of paraffin oil, shaking and mixing uniformly, slightly centrifuging, and placing into a fluorescent PCR amplification instrument. (the reaction plate layout is shown in the table)

TABLE 3 reaction plate layout

Sequence number

Name of the name

1

2

…

9

10

11

12

1

SEA

Sample 1

Sample 2

…

Sample 9

Sample 10

Negative control

Positive control

2

THAI

Sample 1

Sample 2

…

Sample 9

Sample 10

Negative control

Positive control

3

4.2A

Sample 1

Sample 2

…

Sample 9

Sample 10

Negative control

Positive control

4

4.2B

Sample 1

Sample 2

…

Sample 9

Sample 10

Negative control

Positive control

5

3.7

Sample 1

Sample 2

…

Sample 9

Sample 10

Negative control

Positive control

6

WS

Sample 1

Sample 2

…

Sample 9

Sample 10

Negative control

Positive control

7

QS

Sample 1

Sample 2

…

Sample 9

Sample 10

Negative control

Positive control

8

CS

Sample 1

Sample 2

…

Sample 9

Sample 10

Negative control

Positive control

3. On-machine detection (amplification detection zone)

(1) Circulation condition setting

38 ℃ for 5 minutes and 95 ℃ for 10 minutes; the following cycle is entered: 95℃for 15 seconds, 58℃for 50 seconds (30 seconds after reading fluorescence), 72℃for 45 seconds, 45 cycles; 38 ℃ for 30 seconds.

(2) Instrument detection channel selection

Fluorescein was set as FAM, ROX and HEX, and internal standard control fluorescence signal was HEX; the mutant fluorescence signal of the deletion mutation is FAM; the mutant fluorescence signal of the point mutation is FAM, the wild fluorescence signal is ROX, the collection of the fluorescence signal is set at 58 ℃ for 30 seconds, and the specific setting method is referred to the instruction manual of the instrument.

4. Setting of baseline and threshold values

When the instrument is used for analysis, 3-12 cycles of fluorescent signals are taken as a baseline, the instrument automatically analyzes a baseline threshold value, if the noise of a few sample amplification curves is too large, the threshold line is manually adjusted to be just above the highest point of a normal negative control amplification curve (an irregular noise line), and the Ct value=0.0 is used as the standard. The threshold cycle number is the number of cycles that the fluorescent signal undergoes when reaching a set threshold.

(III) interpretation of test results

1. The FAM, ROX and HEX channels of the negative control wells should have no amplification curves, the FAM, ROX and HEX channels of the positive control wells should have amplification curves and Ct values less than 38, otherwise the experiment is considered ineffective.

2. The HEX channel of the sample to be tested should have an amplification curve and a Ct value of less than 38. If the HEX channel has no amplification curve, the quality of the extracted genome does not meet the experimental requirements, and the genome needs to be extracted again.

3. If the FAM channel of the reaction hole of the sample to be detected has no amplification curve, judging that the detection result is that the alpha-thalassemia mutation is absent.

4. If the FAM channel of the reaction well 1 to 5 of the sample to be detected has an amplification curve and the Ct value is less than or equal to 38, the homozygous deletion and heterozygous deletion are judged according to the ROX channel of the reaction well 6 to 8. If the ROX channel has an amplification curve and the Ct value is less than or equal to 38, judging that heterozygous deletion exists; if the ROX channel has no amplification curve, the ROX channel is judged to be homozygous deletion.

5. If the FAM channel of the reaction hole of the sample to be detected is provided with an amplification curve and the Ct value is less than or equal to 38, the homozygous and heterozygous judgment is carried out according to the ROX channel of the reaction hole of the sample to be detected. If the corresponding ROX channel has an amplification curve and the Ct value is less than or equal to 38, judging that the ROX channel is heterozygous mutation corresponding to mutation; and if the corresponding ROX channel has no amplification curve, judging that the corresponding mutation is homozygous mutation.

6. If two or more amplification curves exist in the FAM channel of the sample to be detected, judging that the sample to be detected has compound mutation.

TABLE 4 determination of results for FAM channel, ROX channel, and HEX channel

(detection of deletion mutation of alpha-thalassemia by the reaction solution 1-5 of the kit, and detection of Point mutation of alpha-thalassemia by the reaction solution 6-8.)

Examples of common mutations of alpha-thalassemia: (+ represents amplification curve, -represents no amplification curve)

TABLE 5 mutation cases corresponding to different mutation types

(IV) detection results

1. Experimental data analysis, results are shown in fig. 1 to 17 (fonts in the figures are indicative characters of software, and have no influence on the result judgment).

1) The national negative reference was tested, and as a result no mutation was detected.

2) Other types of mutation outside the scope of the kit were detected as a reference, and no mutation was detected as a result.

3) And detecting the national positive reference of the alpha-thalassemia gene mutation, wherein the result is the corresponding genotype.

As can be seen from fig. 1 to 3, the kit of the invention detects the amplification-free curve of FAM channel of national negative reference products YSH2015-001, YSH2015-002, YSH 2015-010; the HEX channel has an amplification curve, and the Ct value is smaller than 38; the ROX channel has an amplification curve, and the Ct value should be less than 38, and the result is judged as negative.

As can be seen from fig. 4 to 6, the FAM channel has no amplification curve in other mutation type reference products YSH2015-003, YSH2015-004 and YSH2015-009 outside the detection range of the kit detection kit of the present invention; the HEX channel has an amplification curve, and the Ct value is smaller than 38; the ROX channel has an amplification curve, and the Ct value should be less than 38, and the result is judged as negative.

As can be seen from FIGS. 7 to 17, the detection results of the kit of the present invention, namely, the detection results of the national positive reference YSH2015-0005, YSH2015-0006, YSH2015-0007, YSH2015-0008, YSH2015-0013, YSH2015-0019, YSH2015-0021, YSH2015-0022, YSH2015-0029, YSH2015-0030 and YSH2015-0032, are the corresponding genotypes.

In summary, the kit of the invention can detect the corresponding genotype by detecting the national reference.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> Xiamen An Puli bioengineering Co., ltd

Nanjing Anpu precision medical test Co.,Ltd.

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Claims

1. The kit for the multiplex real-time fluorescent PCR detection of the alpha-thalassemia is formed by combining primers, probes and reaction buffers for the multiplex real-time fluorescent PCR detection of the alpha-thalassemia, wherein the combination comprises 8 groups of primer and probe combinations and 4 reaction buffers; the 4 reaction buffers comprise a B-4 reaction buffer, a B-2 reaction buffer, a J-1 reaction buffer and an H-1 reaction buffer; the 8 groups of primer and probe combinations comprise 1 group of primer and probe combinations to 8 groups of primer and probe combinations; the 1 st group primer and the probe combination to the 8 th group primer and the probe combination are respectively and independently packaged; the B-4 reaction buffer solution, the B-2 reaction buffer solution, the J-1 reaction buffer solution and the H-1 reaction buffer solution are respectively and independently packaged;

the J-1 reaction buffer comprises the following components in concentration: tris hydrochloride 3.8mM, tris 32.2mM, potassium chloride 125mM, magnesium chloride 6.25mM, ammonium sulfate 25mM, dimethyl sulfoxide at 12.50% by volume, betaine 2.5M, 7-deaza-dGTP0.075mM, deoxyribonucleoside triphosphate 1mM and deoxyuridine triphosphate 2mM;

The H-1 reaction buffer comprises the following components in concentration: 50mM of 4-hydroxyethyl piperazine ethane sulfonic acid, 25mM of ammonium sulfate, 125mM of potassium chloride, 7.5mM of magnesium chloride, 2.50% formamide by volume, 0.4mM of deoxyribonucleoside triphosphate and 0.8mM of deoxyuridine triphosphate;

the 1 st primer and probe combination-3 rd primer and probe combination are respectively combined with a B-4 reaction buffer solution;

the 4 th primer and probe combination is combined with a B-2 reaction buffer solution;

the 5 th primer and probe combination is combined with J-1 reaction buffer;

the 6 th primer and the probe combination-8 th primer and the probe combination are respectively combined with an H-1 reaction buffer solution;

the kit also comprises an enzyme mixed solution, a dilution buffer solution, a TE buffer solution and paraffin oil;

in each set of primer and probe combinations of the kit, the concentration of the use stock of each primer or each probe is 50. Mu.M.

2. The kit of claim 1, wherein the enzyme cocktail comprises the following components: 4.0U/. Mu.L of TaqDNA polymerase, 0.004U/. Mu.L of uracil glycosylase and an enzyme stock solution A consisting of 50mM Tris-HCl pH8.0, 100mM NaCl, 0.1mM EDTA.2Na, 1% Triton X-100 by volume and 50% glycerol by volume; the dilution buffer comprises the following components: 1mM of tris (hydroxymethyl) aminomethane hydrochloride and 9mM of tris (hydroxymethyl) aminomethane; the TE buffer comprises the following components: tris hydrochloride 5.6mM, tris 4.4mM and disodium ethylenediamine tetraacetate 1mM.

3. The kit of claim 1, further comprising a negative control and a positive control; the negative control comprises normal saline; the positive control comprises: pGEM-T-Easy-SEA plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-THAI plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-4.2A plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-4.2B plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-3.7 plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-WS plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-QS plasmid 1.0X10 ⁴ The copies/. Mu. L, pGEM-T-Easy-CS plasmid 1.0X10 ⁴ copies/. Mu.L, wild-type human genomic DNA.