阅读说明：本技术 引物组、包含该引物组的试剂盒以及该引物组的用途 (Primer group, kit containing primer group and application of primer group ) 是由 何逖 覃嘉嘉 黄冉冉 于 2020-12-07 设计创作，主要内容包括：本发明公开了用于检测SMA等相关突变位点的引物组、包含该引物组的试剂盒以及该引物组的用途。(The invention discloses a primer group for detecting related mutation sites such as SMA (shape memory alloy), a kit containing the primer group and application of the primer group.)

1. The primer group for detecting related mutation sites in SMA comprises an amplification primer and an extension primer, wherein the sequence of the amplification primer is shown as SEQ ID Nos. 7-12, and the sequence of the extension primer is shown as SEQ ID Nos. 60-62.

2. A kit for detecting a relevant mutation site in SMA, comprising the primer set of claim 1.

3. The kit of claim 2, further comprising PCR amplification reaction reagents, alkaline phosphatase treatment reagents, and single base extension reaction reagents.

4. The primer set of claim 1 or the kit of claim 2, wherein the information of the mutation site is SMA 1E 7: NM _000344.4: c.840c > T, SMA 2E 7: NM _017411.4: c.840t > C, SMA 1E 8: NM _000344.4: c.239 ═ SMA 2E 8: NM _017411.4: c.239 ═ m.

5. Use of the primer set of claim 1 in the preparation of a kit for screening SMA.

6. The use of claim 5, wherein the screening SMA comprises the steps of:

s1, extracting DNA from a sample;

s2, performing multiplex PCR amplification reaction by using the amplification primer in the claim 1 and the DNA in the step S1;

s3, carrying out dephosphorylation reaction on the amplification product obtained in the step S2;

s4, carrying out extension reaction on the product obtained in the step S3 by using the extension primer in claim 2;

s5, purifying the product obtained in the step S4;

s6, measuring the molecular weight of the extended primer in the purified product obtained in the step S5 by using a MassArray technology, and detecting the base type of the related mutation site by using the difference of the molecular weight between different bases;

s7, judging whether the examinee has SMA or not based on the base type detected in the step S6.

7. Use according to claim 6, wherein the sample is from a human, preferably from a body fluid, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, lung lavage fluid, cerebrospinal fluid, stool and a tissue sample, more preferably fresh or frozen anticoagulation.

Technical Field

The invention relates to the technical field of gene detection, in particular to a primer group, a kit containing the primer group and application of the primer group.

Background

SMA is a typical disease that afflicts humans severely and is characterized by: (1) is a disease listed in the first rare disease name list of the country; (2) the early diagnosis and early screening have great significance, and once death occurs, the disability rate is high; (3) the pathogenic rate of SMA is 0.6-2 ten thousandth; (4) the pathogenic mechanism and site are clear.

In the prior art, the hot point mutation of the disease can be detected by gene detection technology to diagnose whether the disease is ill or not. In general, gene detection techniques that may be specifically used include MLPA, RT-PCR, and NGS. The advantages and disadvantages of these gene detection techniques are shown in the following table:

as can be seen from the above table, MLPA, RT-PCR and NGS all have the disadvantages of complicated process and high cost.

Meanwhile, the MassArray technology is a method for distinguishing detection sites by single base extension and utilizing the difference of molecular weight among different bases, can directly and accurately detect the type of the base of the detected site, and has strong specificity; fluorescent probes are not used, so that the fluorescent interference of similar sites is avoided, and the detection result is accurate. The technology can realize a reaction to complete an experiment, does not use a fluorescent probe and has low experiment cost.

Therefore, it is necessary to provide a method for gene detection of SMA by using the MassArray technology, so as to achieve the following advantages: the method can detect the mutation of a plurality of gene loci in one reaction, eliminates the interference among reaction holes, has simple operation, low screening cost and low requirement on samples, can well detect dry blood spots and whole blood samples, and is suitable for common screening.

Disclosure of Invention

In order to solve the technical problems, the first aspect of the invention provides a primer set for detecting related mutation sites in SMA, wherein the primer set comprises an amplification primer and an extension primer, the sequence of the amplification primer is shown in SEQ ID Nos. 7-12, and the sequence of the extension primer is shown in SEQ ID Nos. 60-62.

In a second aspect, the present invention provides a kit for detecting a mutation site of interest in SMA, comprising a primer set according to the first aspect.

In one embodiment, the kit according to the second aspect of the present invention further comprises a PCR amplification reaction reagent, an alkaline phosphatase treatment reagent and a single base extension reaction reagent.

In one embodiment, the primer set according to the first aspect of the invention or the kit according to the second aspect of the invention, wherein the information of the mutation site is SMA 1E 7: NM _000344.4: c.840c > T, SMA 2E 7: NM _017411.4: c.840t > C, SMA 1E 8: NM _000344.4: c.239 ═ SMA 2E 8: NM _017411.4: c.239 ═ 239.

A third aspect of the invention provides the use of a primer set according to the first aspect of the invention in the preparation of a kit for screening SMA.

In one embodiment, in the use according to the third aspect of the invention, the screening SMA comprises the steps of:

s1, extracting DNA from a sample;

s2, performing multiplex PCR amplification reaction by using the amplification primer of the first aspect of the invention and the DNA in the step S1;

s3, carrying out dephosphorylation reaction on the amplification product obtained in the step S2;

s4, carrying out extension reaction on the product obtained in the step S3 by using the extension primer in the second aspect of the invention;

s5, purifying the product obtained in the step S4;

s6, measuring the molecular weight of the extended primer in the purified product obtained in the step S5 by using a MassArray technology, and detecting the base type of the related mutation site by using the difference of the molecular weight between different bases;

s7, judging whether the examinee has SMA or not based on the base type detected in the step S6.

The invention achieves the following beneficial technical effects:

(1) the MassArray technology is used for detecting SMA, the interpretation algorithm is high in accuracy, high in flux and low in screening cost; the requirement on the sample is low, and both the dried blood spot and the whole blood sample can be well detected, so that the method is suitable for common screening;

(2) the MassArray technology detects that detection sites are distinguished by single base extension and the difference of molecular weight among different bases, so that the type of the base of the detected site can be directly and accurately detected, and the specificity is strong; fluorescent probes are not used, so that the fluorescent interference of similar sites is avoided, and the detection result is accurate. The experiment is completed in one reaction, a fluorescent probe is not used, and the experiment cost is low.

Drawings

FIG. 1 is a mass spectrum of SMA detection sites.

FIG. 2 is the mass spectrum of reference gene RPP 40.

Detailed Description

The disclosure may be understood more readily by reference to the following detailed description of preferred embodiments of the application and the examples included therein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In case of conflict, the present specification, including definitions, will control.

As used herein, the term "prepared from …" is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion.

The conjunction "consisting of …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4," "1 to 3," "1-2 and 4-5," "1-3 and 5," and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

As used herein, the term "SMA" (spinal muscular atrophy) is an autosomal recessive inherited progressive motor neuron disease, characterized primarily by progressive degeneration of the anterior horn cells of the spinal cord and the motor nuclei of the brainstem. Clinically, it is mainly manifested as progressive, symmetrical muscle weakness and atrophy, with the proximal extremity heavier than the distal one and the lower extremity heavier than the upper one. The patient occupies about 1/10000 in the newborn, and the carrier of the pathogenic gene occupies about 1/50. According to the onset age and clinical manifestations, the disease can be classified into type 4, namely spinal muscular atrophy type I (SMA 1, also called Werdnig-Hoffmann disease), 6-18 months onset muscular atrophy type II (SMA 2), childhood or juvenile onset spinal muscular atrophy type III (SMA 3, also called Kugelher-Welander disease) and adult onset spinal muscular atrophy type IV (SMA 4). The SMA causative genes of the 4 subtypes are identical and are motor neuron survival gene 1 (SMN 1) [ OMIM600354 ]. The SMN1 gene is located on chromosome 5, has a total length of about 20kb and contains 9 exons. It is highly homologous to its immediate neighbors, SMN2 and SMN1, differing by only 5 nucleic acids. SMN2 is a regulatory gene whose copy number is inversely proportional to the severity of the disease state of SMA.

First primer set, kit comprising first primer set and application of first primer set

The first aspect of the invention provides a primer group for detecting related mutation sites in SMA, wherein the primer group comprises an amplification primer and an extension primer, the sequence of the amplification primer is shown in SEQ ID Nos. 7-12, and the sequence of the extension primer is shown in SEQ ID Nos. 60-62.

In a second aspect, the present invention provides a kit for detecting a mutation site of interest in SMA, comprising a primer set according to the first aspect.

In one embodiment, the kit according to the second aspect of the present invention further comprises a PCR amplification reaction reagent, an alkaline phosphatase treatment reagent and a single base extension reaction reagent.

In one embodiment, the primer set according to the first aspect of the invention or the kit according to the second aspect of the invention, wherein the information of the mutation site is SMA 1E 7: NM _000344.4: c.840c > T, SMA 2E 7: NM _017411.4: c.840t > C, SMA 1E 8: NM _000344.4: c.239 ═ SMA 2E 8: NM _017411.4: c.239 ═ 239.

A third aspect of the invention provides the use of a primer set according to the first aspect of the invention in the preparation of a kit for screening SMA.

In one embodiment, in the use according to the third aspect of the invention, the screening SMA comprises the steps of:

s1, extracting DNA from a sample;

s2, performing multiplex PCR amplification reaction by using the amplification primer of the first aspect of the invention and the DNA in the step S1;

s3, carrying out dephosphorylation reaction on the amplification product obtained in the step S2;

s4, carrying out extension reaction on the product obtained in the step S3 by using the extension primer in the second aspect of the invention;

s5, purifying the product obtained in the step S4;

s6, measuring the molecular weight of the extended primer in the purified product obtained in the step S5 by a Massa array technology, and detecting the base type of the related mutation site by using the difference of the molecular weight between different bases;

s7, judging whether the examinee has SMA or not based on the base type detected in the step S6.

In one embodiment, in the use according to the third aspect of the invention, the sample is from a human, preferably from a body fluid, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, lung lavage, cerebrospinal fluid, stool and tissue sample, more preferably fresh or frozen anticoagulation.

In one embodiment, in the use according to the third aspect of the invention, the dephosphorylation reaction is effected using alkaline phosphatase, preferably SAP.

In one embodiment, in the use according to the third aspect of the invention, the purification is effected using a resin.

In one embodiment, in the use according to the third aspect of the invention, the detection of the base type of the relevant mutation site using the difference in molecular weight between the different bases is achieved by using the software Typers 4.0.

PREFERRED EMBODIMENTS

The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, is intended to be illustrative, and not restrictive, and it is intended that all such modifications and equivalents be included within the scope of the present invention.

1. Preparation of reaction mixed liquid

1.1 PCR primer mixture preparation

The following table, PCR Primer MIX configuration table:

and preparing the amplification primers into mixed solution PCR Primer MIX, wherein the final concentration of the PCR amplification primers in the mixed solution is 0.5-1 mu M, and preferably, the PCR amplification reaction is carried out in the same reaction hole. (ii) a

The amplification primer information is shown in the following table:

1.2 preparation of PCR reaction mixture

And (3) PCR reaction mixed solution system:

1.3 preparation of SAP reaction mixture

SAP reaction mixture system:

1.4 preparation of extension primer mixture

Table below, iPLEX Primer MIX configuration table:

detecting disease	Detection of genes	SNP ID	Extension primer	Concentration of extension primer in the mixture
					Internal reference	RPP40	RPP40	RPP40_E	5～15μM
SMA	SMN1、SMN2	SMN_E7	SMN1-2_E7_E	5～15μM
					SMA	SMN1、SMN2	SMN_E8	SMN1-2_E8_E	5～15μM

Preparing extension primers into a mixed solution iPLEX Primer MIX, wherein the final concentration of the extension primers in the mixed solution is 5-15 mu M, and preferably, the extension reaction is carried out in the same reaction hole;

the extension primer information is as follows:

1.5 preparation of extension reaction mixture

Extension reaction mixed liquid system

2. Genomic DNA acquisition

Extracting genome DNA from fresh or frozen anticoagulated blood specimen by using a Ranunculus paramagnetic particle method blood genome DNA extraction kit,

and (3) extracting the genome DNA of the blood spot card specimen by using a Ranunculus armeniaca blood spot genome DNA extraction kit.

gDNA was used as a negative control, a normal human sample. Human Genomic DNA purchased from Promega under accession number G1471.

3. Multiple amplification step

3.1PCR amplification step

And (3) uniformly mixing the PCR reaction mixed liquid prepared by 1.2, subpackaging the mixture into reaction wells corresponding to 384-well plates, subpackaging 4 mu l of each well, and then adding 1 mu l of the sample to be detected. Negative control gDNA and blank control water were included for each test. Adhering a film, slightly centrifuging, placing on a gene amplification instrument, and carrying out amplification according to the following PCR program:

3.2 SAP purification step

Mu.l of SAP reaction mixture (1.3 configuration) was added to each well of PCR amplification reaction product after 3.1PCR, and the mixture was attached to a pad, centrifuged slightly, placed on a gene amplification apparatus, and purified according to the following SAP procedure:

37℃	40min
		85℃	5min
8℃	Hold

3.3 elongation reaction step

Mu.l of the extension reaction mixture (1.5 configuration) was added to 3.2 SAP-terminated SAP purified reaction products per well, attached to a membrane, centrifuged slightly, placed on a gene amplification apparatus, and extended according to the following extension reaction procedure:

4. desalting treatment and mass spectrometer spectroscopy

After the extension reaction procedure was completed, the reaction mixture was centrifuged instantaneously. To each well, 16. mu.L of sterilized double distilled water (6 mg of Clean Resin (Resin, Clean Resin from Agena) was added. Mix by inversion for 15min, centrifuge with 3200x g for 5 min. And (5) sample application and spectrum printing of the sample. The mass spectrometer was spectrally resolved using the Massarray method, and the specific detection sites are shown in the following table:

5. data analysis and interpretation of test results

Original files exported by instrument matching software Typers 4.0, xml files, path View → plate data disc; the results in the Call column are the genotype results of the SNP detection sites

The results of mass spectrometry after amplification of the above extended primers are shown in the following table:

comparison of test results

7 samples: 1 case of yin ginseng, 5 cases of normal people and 1 case of patients with mutation

The results of comparison between the Massarray method test and the gold standard method test are shown in the following table:

from the test results in the table, compared with the gold-labeled method, the Massarray method of the invention obtains at least the same or even better test results, and obtains obvious beneficial technical effects.

The foregoing examples are merely illustrative and serve to explain some of the features of the present disclosure. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the application. As used in the claims, the term "comprising" and its grammatical variants are also logically inclusive of different and varying phrases, such as, but not limited to, "consisting essentially of" or "consisting of. Where desired, numerical ranges are provided and sub-ranges therebetween are included. Variations in these ranges are also self-explanatory to those skilled in the art and should not be considered to be dedicated to the public, but rather should be construed to be covered by the appended claims where possible. And that advances in science and technology will result in possible equivalents or sub-substitutes not currently contemplated for reasons of inaccuracy in language representation, and such changes should also be construed where possible to be covered by the appended claims.