CN116144750A

CN116144750A - Primer probe set, digital PCR kit and application

Info

Publication number: CN116144750A
Application number: CN202211403875.7A
Authority: CN
Inventors: 朱琦; 周燕; 王馨月
Original assignee: Hefei Xingzhi Biotechnology Co ltd
Current assignee: Hefei Xingzhi Biotechnology Co ltd
Priority date: 2022-11-10
Filing date: 2022-11-10
Publication date: 2023-05-23

Abstract

The application relates to a primer probe set, a digital PCR kit and application, wherein the primer probe set comprises: a primer probe group for detecting the pathogenic micro variation of the SMN1 gene, which comprises upstream primers SMN1-1F to SMN1-7F, downstream primers SMN1-1R to SMN1-7R and probes MGB1-1 to MGB1-7; primer probes for detecting SMN1 exon7, including an upstream primer SMN1-exon7F, a downstream primer SMN1-exon7R and a probe MGB1-exon7; primer probes for detecting SMN2 exon7 include an upstream primer SMN2-exon7F, a downstream primer SMN2-exon7R, and a probe MGB2-exon7. According to the technical scheme, the copy number variation conditions of the SMN1 and the SMN2 can be detected simultaneously, and detection of small pathogenic variation of the SMN1 gene is included, so that the omission ratio is reduced.

Description

Primer probe set, digital PCR kit and application

Technical Field

The application relates to a primer probe group, a digital PCR kit and application, and belongs to the technical field of gene detection.

Background

Spinal muscular atrophy (Spinal muscular atrophy, SMA) is a common autosomal recessive inherited neurological disorder. Clinical features are degeneration and degeneration of spinal cord anterior horn cells, leading to symmetric muscle weakness and muscle atrophy. The children suffering from childhood spinal muscular atrophy are inherited in autosomal recessive, the incidence rate of children is 1/5000-1/10000, the carrying rate of children in the crowd is 1/35-1/50, SMA is a first genetic disease which causes death of the infants, and the children are arranged at the 2 nd position of the lethal autosomal genetic disease.

SMA is caused by mutations in the SMN1 gene and the SMN2 gene in vivo, the SMN1 and SMN2 genes being highly homologous, with only a 5 nucleotide difference between the two, SMN1 being the major functional gene and also the SMA causative gene. The normal SMN1 gene encodes 100% of motor neuron survival proteins (SMN proteins), which are essential for neurological and motor function. The mutated SMN1 gene renders normal SMN protein incapable of being produced in vivo, resulting in skeletal muscle atrophy, further affecting motor, swallowing, and respiratory capacity in SMA patients. The SMN2 gene can also code SMN protein, and multiple copies can be formed on the same chromosome, but because of the alternative splicing of SMN2 pre-mRNA caused by c.840C > T variation, 90% of the generated transcription product is unstable delta 7 truncated protein and is extremely easy to degrade, so that the normal physiological function of the complete SMN protein cannot be exerted, and the SMN2 is the supplement of the SMN1 function and belongs to a modified gene.

Studies have shown that 95% of patients are due to homozygous deletions of the SMN1 biallelic gene, i.e., the [0+0] genotype; 5% of patients are caused by a complex heterozygous mutation of SMN1, i.e. one allele is deleted and the other allele undergoes a slight pathogenic variation, being of the [0+1d ] genotype. SMN1 deletions are mostly exon7 combined with exon 8 co-deletions, and a small portion is only exon7 deletions, with SMN1 deletions usually referring to exon7 deletions, since exon 8 is in the non-coding region. Parents with normal phenotypes typically carry one or two normal SMN1 genes. A person carrying only one normal SMN1 gene is called a carrier. In addition, by clinical typing of SMA populations: type 0: SMN2 has a copy number of 1, an unusual type that occurs in utero, is not actively treated, dies often within 1 year of age, and is one of the most serious types; type I: SMN2 has a copy number of 1-2, the most common type, and is onset less than 6 months, usually die within 2 years of age from aspiration pneumonia, which is severe. Type II: SMN2 is onset at a copy number of 3, 6-18 months, and requires ventilator maintenance for respiration as the disease progresses, often surviving to 4 years old. Type III: the copy number of SMN2 is 3-4, the disease is developed when the copy number is more than 18 months, the child patient can walk independently and independently, the survival is basically not affected, and the infant patient is light and can always survive to the adult stage. Type IV: the copy number of SMN2 is 4-8, the disease is more than 30 years old, the patient can walk independently and independently, and the patient has no digestive system and respiratory system symptoms and is adult. It can be seen that the copy number of the SMN2 gene has a certain correlation with the severity of SMA. However, based on the large number of SMA patient populations, for a particular patient, the severity of the disease cannot be estimated based entirely on the number of copies of SMN2, some patients have a high number of copies of SMN2 but a very severe condition, and others have a mild condition but a low number of copies. In summary, since SMA disease is currently not amenable to scientifically certified treatment to cure or alleviate a patient's condition, it places a considerable burden on the home and society, and there is a great need to reduce the incidence of genetic disease by proper prenatal screening.

The current common methods for detecting SMA diseases in clinic mainly comprise multiple ligation dependent probe amplification (MLPA), fluorescent quantitative PCR (q-PCR), high-throughput measurement and other technologies. q-PCR is the most economical and least time-consuming method, costs about $1, and requires about 3 hours from a cost and time-consuming standpoint. MLPA costs high, about $ 10, requiring about 24 hours to complete the experiment. Generally, q-PCR is relatively simple and convenient to operate and low in cost, and is suitable for crowd screening, but the specificity is slightly inferior to that of the MLPA method, and the SMN2 copy number needs to be detected by additionally designing a probe. Likewise, neither could detect minor variations in the SMN1 gene and the [2+0] genotype.

Disclosure of Invention

The application aims to provide a primer probe set, a digital PCR kit and application, which can detect the copy number variation of SMN1 and SMN2 at the same time, wherein the detection of the pathogenic micro variation of the SMN1 gene is included, so as to reduce the omission ratio.

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

in a first aspect, there is provided a primer probe set comprising:

primer probe group for detecting the pathogenic micro variation of the SMN1 gene, comprising upstream primers SMN1-1F to SMN1-7F shown in SEQ ID NO.1 to SEQ ID NO.7, downstream primers SMN1-1R to SMN1-7R shown in SEQ ID NO.8 to SEQ ID NO.14 and probes MGB1-1 to MGB1-7 shown in SEQ ID NO.15 to SEQ ID NO. 21;

the primer probe for detecting the SMN1 exon7 comprises an upstream primer SMN1-exon7F shown as SEQ ID NO.22, a downstream primer SMN1-exon7R shown as SEQ ID NO.23 and a probe MGB1-exon7 shown as SEQ ID NO. 24;

the primer probe for detecting the SMN2 exon7 comprises an upstream primer SMN2-exon7F shown as SEQ ID NO.25, a downstream primer SMN2-exon7R shown as SEQ ID NO.26 and a probe MGB2-exon7 shown as SEQ ID NO. 27.

In some possible embodiments, the probes MGB1-1 through MGB1-7, MGB1-exon7, and MGB2-exon7 are labeled with different fluorescent lights.

In some possible embodiments, the probes MGB1-1 through MGB1-7 are labeled with a 6-FAM fluorescent dye; and/or, the MGB1-exon7 is marked with HEX fluorescent dye; and/or, the MGB2-exon7 is marked with ROX fluorescent dye.

In some possible embodiments, the detection region of the primer probe set for detecting a small pathogenic variation of the SMN1 gene comprises g.70220952dup, g.70240540T > a, g.70240546c > T, g.70247796g > T, g.7023831g > a, g.70238374-70238375del, and g.70247763t > G.

In some possible embodiments, further comprising an upstream primer, a downstream primer and a probe for detecting the reference gene.

In some possible embodiments, the sequences of the upstream primer, the downstream primer and the probe for detecting the reference gene are shown in SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30, respectively.

In a second aspect, there is provided the use of a primer probe set according to the first aspect for the preparation of a product for screening spinal muscular atrophy.

In a third aspect, there is provided a digital PCR kit comprising the primer probe set of the first aspect, and a PCR reaction reagent.

In some possible embodiments, the digital PCR kit is suitable for use in microdroplet digital PCR or chip digital PCR.

In some possible embodiments, the PCR reaction reagents include a buffer solution and an enzyme.

By means of the embodiments of the present application,

the application designs primer probe groups which have specificity and do not have primer dimers and hairpin structures aiming at small pathogenicity variation of the SMN1 gene, SMN1 exon7 and SMN2 exon7, and single-tube detection is carried out on the SMN1 and the SMN2 through a digital PCR technology, and disease screening and typing are carried out simultaneously. In addition, the problem that the conventional detection method cannot simply and effectively detect the small pathogenic variation of the SMN1 gene is solved, and the detection method is different from all SMA detection products on the market, so that the detection of the small pathogenic variation of the SMN1 gene is increased at extremely low cost, and the SMA omission rate is reduced. Meanwhile, the digital PCR technology is suitable for detecting various types of samples, and can accurately quantify low-concentration samples.

The primer probe set and the digital PCR kit can carry out large-scale sample screening detection on spinal muscular atrophy before delivery, and are low in cost, simple to operate, short in time consumption and clear and reliable in result.

The foregoing description is only an overview of the present invention, and is intended to provide a better understanding of the present invention, as it is embodied in the following description, with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

Fig. 1 to fig. 7 are diagrams of the results of SMN1 pathogenic micro variation primer probe verification provided in the embodiments of the present application.

Detailed Description

The following description of the embodiments of the present invention will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown. 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.

In the description of the present application, it is noted that the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion unless otherwise expressly specified and defined. For example, a process, method, system, article, or apparatus that comprises a list of steps or elements is not limited to only those listed steps or elements but may include other steps or elements not listed or inherent to such process, method, article, or apparatus. The specific meaning of the terms in this application will be understood by those of ordinary skill in the art in a specific context. Furthermore, in the description of the present application, unless otherwise indicated, "a plurality" means two or more. "and/or", describes an association relationship of an association object, and indicates that there may be three relationships, for example, a and/or B, and may indicate: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.

The present application provides, in a first aspect, a primer-probe set, as shown in table 1, comprising:

the primer probe for detecting the SMN2 exon7 comprises an upstream primer SMN2-exon7F shown as SEQ ID NO.25, a downstream primer SMN2-exon7R shown as SEQ ID NO.26 and a probe MGB2-exon7 shown as SEQ ID NO. 27;

the method comprises the steps of,

the upstream primer, the downstream primer and the probe are used for detecting an internal reference gene, wherein the internal reference primer is a housekeeping gene with relatively stable expression level, and the housekeeping gene is used as an internal control gene, such as conserved genes of beta-actin, glyceraldehyde phosphate dehydrogenase, ribosomal ribonucleic acid and the like. The sequences of the reference gene, the upstream primer, the downstream primer and the probe adopted by the application are respectively shown as SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO. 30.

Indeed, in other embodiments, other conserved genes may be used as reference genes, and those skilled in the art will choose them according to their actual needs.

Table 1 selected genes and related primer probes

Primer dimer and hairpin structure do not exist between the designed sequences, the upstream primer, the downstream primer and the probe sequence in between, so that the requirements of specificity and sensitivity are ensured; meanwhile, the annealing temperature of the designed primer pair needs to be 58-62 ℃, and the size of the generated amplicon is about 60-80 bp.

In this example, MGB probes were designed for the amplified region, all point mutation probes for small pathogenic variations of the SMN1 gene were labeled with the same fluorescent dye (6-FAM) at the 5' end, SMN1-exon7 target probes were labeled with HEX fluorescent dye at the 5' end, and SMN2-exon7 target probes were labeled with ROX fluorescent dye at the 5' end. In addition, the reference gene was labeled at the 5' end with Cy5 fluorescent dye by designing a primer and a probe to be matched in the same manner as described above.

Fig. 1 to 7 show the results of SMN1 pathogenic micro variation primer probe verification in the examples of the present application.

As shown in fig. 1 to 7, experiments prove that the primer probe designed for the SMN1 pathogenicity tiny variation can basically distinguish wild type from mutation variation.

Based on the primer probe set, the embodiment adopts digital PCR to detect the related DNA sample. Specific: to the sample detection reaction tube, 0.5. Mu.l of a primer probe mixed solution, 10. Mu.l of a 4XdPCR Probe MasterMix (QIAGEN) reaction premix and (2-100 ng) of a DNA sample were added, and sterile water was added to make up to 40. Mu.l of a reaction system. The reaction mixture was applied to dPCR nanowell plate wells and amplified by the QIAcity One 5Plex system (QIAGEN). The amplification conditions are as follows: the reaction was repeated 36 times at 95℃for 2min,95℃for 30s,58℃for 30s, and 72℃for 30 s. After amplification is finished, the instrument automatically performs data analysis, detects fluorescent signals in the reaction chamber, calculates copy number by using poisson distribution, and calculates a formula of copy number concentration: c= -ln (1-P)/V, where (P is the positive droplet proportion and V is the droplet volume)

In this example, the following method was used to analyze the results: and according to the fluorescence signals of the dPCR, the copy numbers CTn (n=1, 2, 3) of the detection channels of each target point are respectively obtained, and the copy number CR of the reference gene is obtained. Based on the ratio of the copy number of each target detection channel to the copy number of the reference gene (TGRn=CTn/CR, n=1, 2, 3), and carrying out SMA risk assessment by combining with SMN1 amplification channel copy number z value data standardized statistical analysis, wherein the SMA typing assessment is mainly based on an SMN2 amplification channel TGR, and the SMN1 gene pathogenicity minor variation amplification channel TGR is an auxiliary parameter for the assessment.

Wherein, the liquid crystal display device comprises a liquid crystal display device,

(1) the formula of TGR is as follows:

since the reference gene usually has 2copies in 1 diploid genome, the copy number of the target gene can be deduced from TGR. In theory, TGR is 0, 0.5, 1, 1.5, 2, respectively, corresponding to the copy number of the target gene being 0, 1,2,3, 4copies, respectively. Through data statistical analysis, when TGR is more than or equal to 0 and less than 0.24, the target gene copy number is 0copy; when TGR is more than 0.45 and less than 0.72, heterozygously deleting the target gene, wherein the copy number of the target gene is 1copy; when TGR is more than 0.78 and less than 1.23, the copy number of the target gene is normal, and the copy number is 2copies; when TGR is more than 1.45 and less than 1.7, the copy number is repeated, and the copy number is 3copies; when TGR is more than 1.9 and less than 2.16, the copy number is repeated, and the copy number is 4copies; when TGR >2.8, the copy number is repeated, the copy number is >4copies.

(2) Z= (R2-X)/sigma, wherein the R value is the TGR value of the internal reference gene corresponding to the detection channel target point, the specific definition R2 represents the TGR value of the detection channel of the amplification copy number of the sample SMN1 to be detected, X is the average value of the R values of the detection channels of the healthy sample group SMN1, and sigma is the standard deviation of the R values of the detection channels of the healthy sample group SMN 1. Specifically, a healthy sample group is firstly tested, and the average value X (X= 1.089322969) of the ratio of the amplified copy number of the detection channel of the healthy sample group SMN1 to the copy number of the reference gene and the standard deviation sigma (sigma= 0.059070907) of the ratio are determined.

SMA risk assessment was as follows: if the TGR of the SMN1 channel is more than or equal to 0 and less than 0.24, the SMA is in high risk of disease; if the SMN1 channel, the TGR is more than 0.45 and less than 0.72, and the z value is more than-3.6, the SMA carries high risk; if the SMN1 channel is 0.78 < TGR < 1.23, and the absolute value of 0 < z < 3, then SMA is at low risk. If the z value or TGR is out of the defined range, the detection is needed in the gray area, and if the detection result is still in the gray area, the gene sequencing is recommended to be rechecked. In particular, SMA is at high risk if the SMN1 pathogenicity minor variation amplification channel TGR >0.2 and the SMN1 amplification channel z value is < -3.6.

SMA typing was evaluated as follows: the copy number of SMN2 is 1-0; the copy number of SMN2 is 1-2-I type; the copy number of SMN2 is 3-II; the copy number of SMN2 is 3-4-III; the copy number of SMN2 is 4-8-V type. And (3) injection: SMN2 copy number varies from person to person, and in general, the lower the SMN2 copy number, the more severe the disease.

Based on the evaluation mode and the kit, the application adopts a digital PCR technology to detect SMA screening volunteer samples, wherein the samples comprise 1 patient, 3 carriers and 2 healthy people.

In this example, the same volunteer took a whole blood sample and a throat swab sample for testing. Whole blood sample extraction kit adopts blood/cell/tissue genomic DNA extraction kit (DP 304, tiangen product), and throat swab sample extraction kit adopts oral swab genomic DNA extraction kit (DP 322, tiangen product).

The specific detection results are shown in table 2:

TABLE 2

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After comparison, the evaluation result after the digital PCR detection of the application accords with the clinical detection of volunteers.

To sum up: the application designs primer probe groups which have specificity and do not have primer dimers and hairpin structures aiming at small pathogenicity variation of the SMN1 gene, SMN1 exon7 and SMN2 exon7, and single-tube detection is carried out on the SMN1 and the SMN2 through a digital PCR technology, and disease screening and typing are carried out simultaneously. In addition, the problem that the conventional detection method cannot simply and effectively detect the small pathogenic variation of the SMN1 gene is solved, and the detection method is different from all SMA detection products on the market, so that the detection of the small pathogenic variation of the SMN1 gene is increased at extremely low cost, and the SMA omission rate is reduced. Meanwhile, the digital PCR technology is suitable for detecting various types of samples, and can accurately quantify low-concentration samples.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A primer probe set, comprising:

2. The primer probe set of claim 1, wherein the probes MGB1-1 to MGB1-7, MGB1-exon7 and MGB2-exon7 are labeled with different fluorescent lights.

3. The primer probe set of claim 2, wherein the probes MGB1-1 to MGB1-7 are labeled with a 6-FAM fluorescent dye; and/or, the MGB1-exon7 is marked with HEX fluorescent dye; and/or, the MGB2-exon7 is marked with ROX fluorescent dye.

4. The primer probe set of claim 1, wherein the detection region of the primer probe set for detecting small pathogenic variations in SMN1 gene comprises g.70220952dup, g.70240540T > a, g.70240546c > T, g.70247796g > T, g.70238311G > a, g.70238374-70238375del, and g.70247763t > G.

5. The primer probe set of claim 1, further comprising an upstream primer, a downstream primer and a probe for detecting a reference gene.

6. The primer probe set according to claim 5, wherein the sequences of the upstream primer, the downstream primer and the probe for detecting the reference gene are shown as SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30, respectively.

7. Use of a primer-probe set according to any one of claims 1 to 6 for the preparation of a product for screening spinal muscular atrophy.

8. A digital PCR kit comprising the primer probe set of any one of claims 1 to 6, and a PCR reaction reagent.

9. The digital PCR kit of claim 8, wherein the digital PCR kit is suitable for use in microdroplet digital PCR or chip digital PCR.

10. The digital PCR kit of claim 8, wherein the PCR reaction reagents include a buffer solution and an enzyme.