Digital PCR kit for detecting spinal muscular atrophy and application thereof

文档序号：872168 发布日期：2021-03-19 浏览：2次中文

阅读说明：本技术 一种检测脊髓性肌萎缩症数字pcr试剂盒及其应用 (Digital PCR kit for detecting spinal muscular atrophy and application thereof ) 是由陈芊如王芳郭娜郭永祝令香杨文军于 2020-12-21 设计创作，主要内容包括：本发明提供一种检测脊髓性肌萎缩症数字PCR试剂盒,所述数字PCR试剂盒是在一管数字PCR反应中同时检测待测样中SMN1外显子7、SMN1外显子8和SMN2外显子7以及内参基因的数字PCR试剂盒,在至少四个荧光通道中同时检测SMN1外显子7、SMN1外显子8和SMN2外显子7和内参基因。本发明试剂盒基于数字PCR技术,可以一管完成SMN1外显子7、8和SMN2的拷贝数定量检测,可应用于SMA临床确诊、携带者筛查和疾病分型。(The invention provides a digital PCR kit for detecting spinal muscular atrophy, which is a digital PCR kit for simultaneously detecting SMN1 exon 7, SMN1 exon 8, SMN2 exon 7 and an internal reference gene in a sample to be detected in one tube of digital PCR reaction, and simultaneously detecting SMN1 exon 7, SMN1 exon 8, SMN2 exon 7 and the internal reference gene in at least four fluorescence channels. The kit can complete copy number quantitative detection of the SMN1 exons 7 and 8 and the SMN2 in one tube based on a digital PCR technology, and can be applied to clinical diagnosis of SMA, carrier screening and disease typing.)

1. The digital PCR kit for detecting the spinal muscular atrophy is characterized by being a digital PCR kit for simultaneously detecting SMN1 exon 7, SMN1 exon 8, SMN2 exon 7 and an internal reference gene in a sample to be detected in one tube of digital PCR reaction; the absolute copy number of the SMN1 exon 7, the SMN1 exon 8 and the SMN2 exon 7 in the sample to be detected is determined by simultaneously detecting the SMN1 exon 7, the SMN1 exon 8, the SMN2 exon 7 and the reference gene in at least four fluorescence channels, calculating the ratio of the SMN1 exon 7, the SMN1 exon 8 and the SMN2 exon 7 to the reference gene in the sample to be detected, and determining the absolute copy number of the SMN1 exon 7, the SMN1 exon 8 and the SMN2 exon 7 in the sample to be detected.

2. The PCR kit of claim 1, wherein the kit comprises an upstream primer SEQ ID NO: AGCTATTTTTTTTAACTTCCTTTATTTTC, and the downstream primer SEQ ID NO: GAGCACCTTCCTTCTTTTTG parts by weight; and detecting SMN1 exon 7 probe SEQ ID NO: TTGTCTGAAACCC and detection SMN2 exon 7 probe SEQ ID NO: 10: TTTTGTCTAAAACC are provided.

3. The PCR kit of claim 1, wherein the kit comprises an upstream primer SEQ ID NO: GACTCTATTTTGAAAAACCAT, and the downstream primer SEQ ID NO: ATTTTCTCAACTGCCTCAC, 4: ATTTTCTCAACTGCCTCAC; and detecting SMN1 exon 8 probe SEQ ID NO: CCCACC + C + CAGTCT, wherein '+' is a locked nucleic acid modification.

4. The PCR kit of claim 1, wherein the kit comprises primers and probes for amplifying and detecting at least two reference genes.

5. The PCR kit of claim 4, wherein the kit comprises an upstream primer SEQ ID NO: GTTTGGGTTCAGGTCTTT, and the downstream primer SEQ ID NO: TACAATGAAAACATTCAGAAGTC, and detecting the probe of reference gene RPS27A SEQ ID NO: TTGTCTACCACTTGCAAAGCTG parts by weight; and an upstream primer SEQ ID NO: GAAGAAACCTCGGCCAT, and the downstream primer SEQ ID NO: CCTCACACTTGGCTTTC and the probe for detecting the reference gene RPP30 SEQ ID NO: 13: AGATGAAGATTGTCTTCCAGCTTCCA are provided.

6. The PCR kit according to claim 2 or 3, wherein the fluorescent group labeled at the 5 'end of the detection probe for detecting exon 7 of SMN1, exon 8 of SMN1 and exon 7 of SMN2 and the internal reference gene is FAM, FITC, Cy3, VIC, HEX, ROX, TET, NED, Cy5 or Cy5.5, and the label at the 3' end is MGB, BHQ-1, BHQ-2 or BHQ-3; each probe is labeled with a different fluorescence.

7. Use of a kit according to any one of claims 1 to 6 for the detection of spinal muscular atrophy.

8. A method for simultaneously determining absolute copy numbers of SMN1 exon 7, SMN1 exon 8 and SMN2 exon 7 in a sample to be detected in a digital PCR reaction by using digital PCR is characterized by comprising the steps of simultaneously detecting the SMN1 exon 7, the SMN1 exon 8 and the SMN2 exon 7 and an internal reference gene in a sample to be detected in one tube of digital PCR reaction, and simultaneously detecting the SMN1 exon 7, the SMN1 exon 8 and the SMN2 exon 7 and the internal reference gene in at least four fluorescence channels; and determining the absolute copy numbers of the SMN1 exon 7, the SMN1 exon 8 and the SMN2 exon 7 in the sample to be detected by calculating the ratio of the SMN1 exon 7, the SMN1 exon 8 and the SMN2 exon 7 to the reference gene in the sample to be detected.

9. The method according to claim 8, wherein when the reference gene is a 2-copy gene, the absolute copy number of the test gene is determined by the following numerical PCR ratio of the test gene to the reference gene

Ratio of Absolute number of copies <0.1 0 0.3-0.7 1 0.8-1.2 2 1.3-1.7 3 1.8-2.2 4 2.3-2.7 5 2.8-3.2 6 Others Can not determine

。

Technical Field

The invention relates to the technical field of biology, in particular to a digital PCR kit for detecting spinal muscular atrophy and application thereof.

Background

Autosomal recessive disease Spinal Muscular Atrophy (SMA) is a severe neuromuscular disease characterized by degeneration of alpha motor neurons in the Spinal cord, leading to progressive weakness and paralysis of the proximal muscles. SMA is the second most common lethal autosomal recessive disease after cystic fibrosis, with a prevalence of 1/6000-1/10000 and a carrier rate of 1/40-1/60.

SMA is caused by a mutation or deletion in the SMN1 gene. The SMN1 gene of normal people is 2 copies of the gene, 95 percent of SMA patients have homozygous deletion (0 copies) of SMN1 exon 7, most of the SMN1 genes have simultaneous deletion of exons 7 and 8, only a few of the SMN patients have deletion of exon 7 only, and the SMN patients have reports that the SMN patients have deletion of exon 8 only. In the remaining 5% of patients, the SMN1 gene contained other mutations, with two more common mutations in the chinese population, c.22dupa for exon 1 and c.683t > a for exon 5, respectively.

Since SMA is an autosomal recessive inheritance, a carrier is only 1 copy of the normal SMN1 gene, and although the disease of SMA is not shown, if parents and parents are the carriers, the probability that children are SMA patients, SMA pathogenic gene carriers and healthy every time they are pregnant is 25%, 50% and 25% respectively. In addition, 2 copies of normal SMN1 were located on one allele in some people, and SMN1 exon 7 was homozygously deleted in the other allele, which was also a carrier. Once the SMA is attacked, no economic and effective treatment means is available, and psychological and economic heavy burden is brought to families, so that the birth rate of SMA children in people is reduced by prenatal screening at the present stage, and the screening strategy is in accordance with the hygiene and economics.

The amount of SMN2 is critical in determining the severity of the SMA phenotype. SMA can be divided into four clinical types according to the age and clinical course of disease. Type I SMA is characterized by severe general muscle weakness and low muscle tone at birth or within 3 months of birth, death due to respiratory failure usually occurs within two years after birth, and about 80% of patients with type I SMA possess 1 or 2 copies of the SMN2 gene. Type II children can sit but cannot stand or walk independently and generally survive more than 4 years, with about 82% of type II SMA patients having 3 copies of the SMN2 gene. Type III SMA is in the infancy or adolescence stage, and the patient can learn to walk independently and has long survival time, and about 96% of type III SMA patients have 3 or 4 copies of SMN2 gene. In cases where the first symptoms occur between 20 and 30 years of age, SMA type IV, about 100% of patients with SMA type IV possess 4 or 6 copies of the SMN2 gene. SMN1 and SMN2 genes differ by only 5 bases, with a C > T change in exon 7 of SMN2 resulting in disruption of the splice regulator, thereby leaving most transcripts of SMN2 devoid of exon 7. SMN mRNA lacking exon 7 encodes amino acids that do not oligomerize efficiently and degrade rapidly, so SMN2 does not produce as much full-length SMN protein as SMN1, and SMN2 plays a dose-compensating role in SMA patients.

The current clinically common SMA detection methods include Restriction Fragment Length Polymorphism (RFLP), Multiplex Ligation Probe Amplification (MLPA) and fluorescent quantitative PCR (qPCR). RFLP firstly utilizes PCR primers to amplify SMN1 and SMN2 exons, then uses restriction enzyme to digest SMN2 amplification products, and finally displays the result through gel electrophoresis. RFLP is simple and convenient to operate and has low requirements on equipment, but the RFLP can only detect the homozygous deletion of the SMN1 exon 7. MLPA is a gold standard for detection of SMA that first hybridizes to a target sequence using a specific probe comprising two oligonucleotides that can be ligated only if the two oligonucleotides hybridize successfully, followed by multiplex PCR amplification to produce products of different lengths. Products of different sizes were then analyzed on a capillary sequencer, and the change in peak height reflected the deletion of the gene. The qPCR utilizes a fluorescent probe to target a target sequence, and carries out real-time detection on multiple PCR reactions, and a standard curve method or a comparative threshold method is adopted to obtain gene copy number information. The standard curve method firstly constructs a standard curve based on DNA with known copy number, and deduces the information of gene copy number by taking the curve as a reference standard; the comparative thresholding method compares the Ct value of the sample with the Ct value of the calibration factor and the result must be corrected with the internal control gene (housekeeping gene). qPCR is simple and rapid to operate, but the quantitative determination needs to be carried out by depending on a standard curve, and the existing kit cannot simultaneously detect the SMN1 exons 7 and 8 and the SMN2 gene.

At present, no technical scheme for realizing one-tube detection of the SMN1 exons 7 and 8 and the SMN2 gene based on digital PCR is available.

Disclosure of Invention

In order to solve the above problems, the present invention provides a digital PCR kit for detecting spinal muscular atrophy, wherein the digital PCR kit is a digital PCR kit for simultaneously detecting SMN1 exon 7, SMN1 exon 8, SMN2 exon 7 and an internal reference gene in a sample to be detected in one-tube digital PCR reaction; the absolute copy number of the SMN1 exon 7, the SMN1 exon 8 and the SMN2 exon 7 in the sample to be detected is determined by simultaneously detecting the SMN1 exon 7, the SMN1 exon 8, the SMN2 exon 7 and the reference gene in at least four fluorescence channels, calculating the ratio of the SMN1 exon 7, the SMN1 exon 8 and the SMN2 exon 7 to the reference gene in the sample to be detected, and determining the absolute copy number of the SMN1 exon 7, the SMN1 exon 8 and the SMN2 exon 7 in the sample to be detected.

In one embodiment, the kit comprises an upstream primer SEQ ID NO: AGCTATTTTTTTTAACTTCCTTTATTTTC, and the downstream primer SEQ ID NO: GAGCACCTTCCTTCTTTTTG parts by weight; and detecting SMN1 exon 7 probe SEQ ID NO: TTGTCTGAAACCC and detection SMN2 exon 7 probe SEQ ID NO: 10: TTTTGTCTAAAACC are provided.

In one embodiment, the kit comprises an upstream primer SEQ ID NO: GACTCTATTTTGAAAAACCAT, and the downstream primer SEQ ID NO: ATTTTCTCAACTGCCTCAC, 4: ATTTTCTCAACTGCCTCAC; and detecting SMN1 exon 8 probe SEQ ID NO: CCCACC + C + CAGTCT, wherein '+' is a locked nucleic acid modification.

In one embodiment, the kit comprises primers and probes for amplifying and detecting at least two reference genes.

In one embodiment, the kit comprises an upstream primer SEQ ID NO: GTTTGGGTTCAGGTCTTT, and the downstream primer SEQ ID NO: TACAATGAAAACATTCAGAAGTC, and detecting the probe of reference gene RPS27A SEQ ID NO: TTGTCTACCACTTGCAAAGCTG parts by weight; and an upstream primer SEQ ID NO: GAAGAAACCTCGGCCAT, and the downstream primer SEQ ID NO: CCTCACACTTGGCTTTC and the probe for detecting the reference gene RPP30 SEQ ID NO: 13: AGATGAAGATTGTCTTCCAGCTTCCA are provided.

In one embodiment, the 5 '-end labeled fluorophore of the detection probe for detecting exon 7 of SMN1, exon 8 of SMN1, and exon 7 of SMN2 and the reference gene is FAM, FITC, Cy3, VIC, HEX, ROX, TET, NED, Cy5, or Cy5.5, and the 3' -end label is MGB, BHQ-1, BHQ-2, or BHQ-3; each probe is labeled with a different fluorescence.

In one embodiment, the invention provides the use of the above kit for detecting spinal muscular atrophy.

In one embodiment, the invention provides a method for simultaneously determining the absolute copy number of SMN1 exon 7, SMN1 exon 8 and SMN2 exon 7 in a sample to be tested in one digital PCR reaction by using digital PCR, the method comprising simultaneously detecting SMN1 exon 7, SMN1 exon 8 and SMN2 exon 7 and an internal reference gene in a sample to be tested in one tube of digital PCR reaction, and simultaneously detecting SMN1 exon 7, SMN1 exon 8 and SMN2 exon 7 and an internal reference gene in at least four fluorescence channels; and determining the absolute copy numbers of the SMN1 exon 7, the SMN1 exon 8 and the SMN2 exon 7 in the sample to be detected by calculating the ratio of the SMN1 exon 7, the SMN1 exon 8 and the SMN2 exon 7 to the reference gene in the sample to be detected.

In one embodiment, when the reference gene is a 2-copy gene, the absolute copy number of the test gene is determined by the following digital PCR ratio of the test gene to the reference gene

Ratio of	Absolute number of copies
		<0.1	0
0.3-0.7	1
		0.8-1.2	2
1.3-1.7	3
		1.8-2.2	4
2.3-2.7	5
		2.8-3.2	6
Others	Can not determine

。

The invention provides a spinal muscular atrophy detection kit, which is based on a digital PCR technology, can complete copy number quantitative detection of SMN1 exons 7 and 8 and SMN2 in one tube, and can be applied to clinical diagnosis of SMA, carrier screening and disease typing.

Compared with the existing single internal reference gene method, the kit of the invention uses two internal reference genes to determine the absolute copy number of the gene to be detected, and the two internal reference genes can make up the defect that the experimental result can not be determined or is wrong possibly caused by the single internal reference, so that the result is more accurate and has higher reliability, and the accuracy and the reliability of the method are improved.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a one-dimensional graph of digital PCR fluorescence detection of two sets of primer probes, namely, SMN1 exon 7 and SMN2 exon 7, wherein the X axis is the number of a droplet, the Y axis is the intensity of a fluorescence signal, and marked 1 and 2 in the graph correspond to a positive droplet signal and a negative droplet signal respectively;

FIG. 2 is a one-dimensional graph of digital PCR fluorescence detection of SMN1 exon 8 three sets of primer probes, wherein the X-axis is the number of droplets, the Y-axis is the intensity of fluorescence signals, and the 1 and 2 marked in the graph correspond to a positive droplet signal and a negative droplet signal respectively;

FIG. 3 is a two-dimensional graph of the results of normal human SMA detection, in which the FAM probe is SMN1 exon 7, the VIC probe is SMN2 exon 7, the Cy5 probe is SMN1 exon 8, the ROX probe is reference gene RPS27A, and the Cy5.5 probe is reference gene RPP 30; the figure shows the corresponding targets of 12 clusters, wherein 1 represents a negative droplet not containing SMN1 exon 7 and SMN2 exon 7, 2 represents a SMN1 exon 7 positive droplet, 3 represents a SMN2 exon 7 positive droplet, 4 represents a positive droplet containing both SMN1 and SMN2 exon 7, 5 represents a negative droplet not containing SMN1 exon 8 and RPS27A, 6 represents a SMN1 exon 8 positive droplet, 7 represents a RPS27A positive droplet, 8 represents a positive droplet containing both SMN1 exon 8 and RPS27A, 9 represents a negative droplet not containing SMN1 exon 7 and RPP30, 10 represents a SMN1 exon 7 positive droplet, 11 represents a RPP30 positive droplet, 12 contains both SMN1 exon 7 and RPP 30;

FIG. 4 is a two-dimensional graph of SMA detection results of SMA carriers (heterozygous deletion), wherein the FAM probe is SMN1 exon 7, the VIC probe is SMN2 exon 7, the Cy5 probe is SMN1 exon 8, the ROX probe is reference gene RPS27A, and the Cy5.5 probe is reference gene RPP 30; the figure shows the corresponding targets of 12 clusters, wherein 1 represents a negative droplet not containing SMN1 exon 7 and SMN2 exon 7, 2 represents a SMN1 exon 7 positive droplet, 3 represents a SMN2 exon 7 positive droplet, 4 represents a positive droplet containing both SMN1 and SMN2 exon 7, 5 represents a negative droplet not containing SMN1 exon 8 and RPS27A, 6 represents a SMN1 exon 8 positive droplet, 7 represents a RPS27A positive droplet, 8 represents a positive droplet containing both SMN1 exon 8 and RPS27A, 9 represents a negative droplet not containing SMN1 exon 7 and RPP30, 10 represents a SMN1 exon 7 positive droplet, 11 represents a RPP30 positive droplet, 12 contains both SMN1 exon 7 and RPP 30;

FIG. 5 is a two-dimensional graph of SMA detection results of SMA patients (homozygous deletion), wherein the FAM probe is SMN1 exon 7, the VIC probe is SMN2 exon 7, the Cy5 probe is SMN1 exon 8, the ROX probe is reference gene RPS27A, and the Cy5.5 probe is reference gene RPP 30; the figure shows the targets corresponding to 6 clusters, where 1 represents a negative droplet not containing SMN1 exon 7 and SMN2 exon 7, 2 represents a SMN2 exon 7 positive droplet, 3 represents a negative droplet not containing SMN1 exon 8 and RPS27A, 4 represents a RPS27A positive droplet, 5 represents a negative droplet not containing SMN1 exon 8 and RPS27A, and 6 represents a RPP30 positive droplet.

Detailed Description

In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described with reference to the following examples, and it is obvious that the described examples are only a part of the examples of the present application, and not all examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application. The invention is further described with reference to the following figures and examples.

EXAMPLE one primer Probe Screen for exon 7, 8 SMN1 and exon 7 SMN2

First, SMN1 exon 7 and SMN2 exon 7 primer probe screening

Primer probe design for exon 7 of SMN1 and exon 7 of SMN2

Sequences of the SMN1 and SMN2 genes were downloaded from NCBI for sequence analysis. Since the SMN1 exon 7 and the SMN2 exon 7 are different by only one base, probes specifically targeting SMN1 and SMN2 exon 7 are designed aiming at the different bases of the two genes respectively, and the two genes are amplified by using the same pair of primers. The following table is the candidate primer probe sequences:

2. plasmid reference synthesis, digestion and quantification

To verify the amplification effect of the primer probe, Jinwei Zhi corporation synthesized a plasmid reference containing the target sequence, a plasmid with SMN1 exon 7 and SMN2 exon 7, respectively, with an insertion sequence of 300 bp. The plasmid reference product is subjected to enzyme digestion by using ScaI-HF restriction endonuclease, the linearized plasmid reference product after enzyme digestion is quantified by using a Qubit 3.0(Thermo Scientific) fluorescence detector, and the linearized plasmid reference product is diluted to 10000 copies/mu L by using a nucleic acid diluent and is stored at-20 ℃ in a subpackaging manner until being used.

Preparation of PCR reaction System

PCR reaction systems (total volume 30. mu.L) were prepared according to the following table.

Composition (I)	Volume (μ L)	Final concentration (nM)
			2x ddPCR Mix	15	-
Upstream primer (20. mu.M)	0.6	400
			Downstream primer (20. mu.M)	0.6	400
Probe (20 μ M)	0.3	200
			Stencil (10000 copy/uL)	1	-
Water (W)	12.5	-

4. Digital PCR workflow

(1) Preparing micro-droplets: a droplet generation chip (Beijing, Inc.) and a sample preparation instrument (Xinyi, manufacturing technology, Inc.) were used, a 30. mu.L PCR reaction system was added to a sample well of the droplet generation chip, 180. mu.L droplet generation oil was added to an oil well, the chip and an 8-up tube were placed in the preparation instrument, and a rubber pad was covered to prepare a micro-droplet.

(2) And (3) PCR amplification: the 8-linked discharge tube containing the micro-droplets is placed on a PCR instrument for amplification, and the amplification program is set as shown in the following table:

(3) micro-droplet detection: after the PCR was completed, the 8-row tube and the droplet detection chip (Xinyi manufacturing technology (Beijing) Co., Ltd.) were placed in a jig, 430. mu.L and 500. mu.L of detection oil were added to the oil holes, respectively, and the chip was covered with a rubber pad and placed in a chip analyzer (Xinyi manufacturing technology (Beijing) Co., Ltd.) for droplet detection. The chip analyzer comprises 5 fluorescence channels (FAM/VIC/ROX/Cy5/Cy5.5), 5 lasers with the wavelengths of 473 nm, 532 nm, 580 nm, 640 nm and 710nm are used as excitation light sources to excite fluorescent dyes in micro-droplets, and finally fluorescence excited in the micro-droplets is collected through a fluorescence detection light path.

(4) Signal processing and data analysis: the method comprises the steps that each of tens of thousands of micro-droplets prepared by a droplet generation chip and a sample preparation instrument is an independent PCR reactor, most of the micro-droplets do not contain a target gene to be detected or contain at least one target gene to be detected, after PCR amplification, FAM/VIC/ROX/Cy5/Cy5.5 fluorescence signal detection is carried out on each micro-droplet by a chip analyzer, the peak height of the micro-droplet signal is recorded, and the droplet containing the target gene to be detected can detect a corresponding fluorescence signal. And (3) digitizing the fluorescence intensity in the micro-droplets through a fluorescence classification threshold, judging the micro-droplets with stronger fluorescence as '1' (positive), judging the micro-droplets with weaker fluorescence as '0' (negative), counting the number of '1' and '0', and correcting through a Poisson distribution model to calculate the total copy number of the FAM/VIC/ROX/Cy5/Cy5.5 labeled target genes put into the template.

5. Screening results

5.1 quantitative accuracy and stability assessment

In the experiment, each group of primer probes is repeatedly tested for 3 times, the quantitative result of the copy number is counted, and the average value, the standard deviation and the variation coefficient are calculated, so that the quantitative accuracy and the stability of each group of primer probes are evaluated. Results as shown in the table below, the quantitative results of the first set of primer probes for exon 7 of SMN1 are closer to the theoretical copy number, and the coefficient of variation is comparable to the second set; the quantitative results of the first set of primer probes for exon 7 of SMN2 were closer to the theoretical copy number, and the coefficient of variation was comparable for the second set.

5.2 fluorescent Signal intensity and concentration assessment

FIG. 1 is a digital PCR fluorescence detection one-dimensional graph of two sets of primer probes, namely SMN1 exon 7 and SMN2 exon 7, wherein the X axis is the number of liquid drops, and the Y axis is the fluorescence signal intensity. As shown in FIG. 1 and the following table, the intensity of the positive fluorescence signal of the first set of primer probes is higher, and the difference between the positive signal and the negative signal of the first set of primer probes is larger, i.e., the discrimination is better, and it can be seen from FIG. 1 that the aggregation degree of the first set is better.

Combining the above results, a first set of primer probes was selected for exon 7 of SMN1 and exon 7 of SMN 2.

Second, SMN1 exon 8 primer probe screening

1. Primer Probe sequence design

Sequences of the SMN1 and SMN2 genes were downloaded from NCBI for sequence analysis. Since SMN1 exon 8 and SMN2 exon 8 differ by only one base, probes specifically targeting SMN1 exon 8 were designed for the different bases of these two genes. The following table is the candidate primer probe sequences:

'+' is Locked Nucleic Acid (LNA) modification

2. Plasmid reference synthesis, digestion and quantification

To verify the amplification effect of the primer probe, a plasmid reference containing exon 8 of SMN1 was synthesized by Kingchi corporation with an insertion sequence of 300 bp. The plasmid reference product is subjected to enzyme digestion by using ScaI-HF restriction endonuclease, the linearized plasmid reference product after enzyme digestion is quantified by using a Qubit 3.0(Thermo Scientific) fluorescence detector, and the linearized plasmid reference product is diluted to 10000 copies/mu L by using a nucleic acid diluent and is stored at-20 ℃ in a subpackaging manner until being used.

Preparation of PCR reaction System

PCR reaction systems (total volume 30. mu.L) were prepared according to the following table.

4. Digital PCR workflow

5. Screening results

5.1 quantitative accuracy and stability assessment

In the experiment, each group of primer probes is repeatedly tested for 3 times, the quantitative result of the copy number is counted, and the average value, the standard deviation and the variation coefficient are calculated, so that the quantitative accuracy and the stability of each group of primer probes are evaluated. Results the results are shown in the table below, where the quantification of the third set of primer probes for exon 8 of SMN1 is closer to the theoretical copy number, with a coefficient of variation comparable to that of the first set.

5.2 fluorescent Signal intensity and concentration assessment

FIG. 2 is a digital PCR fluorescence detection one-dimensional graph of SMN1 exon 8 three primer probes, wherein the X axis is the number of droplets and the Y axis is the fluorescence signal intensity. As shown in FIG. 2 and the following table, the intensity of the positive fluorescence signal of the third group of primer probes is higher, the difference between the positive and negative signals of the primer probes is larger, i.e., the discrimination is better, and the first group is the worst, and the second group is the worst. It can also be seen from fig. 2 that the third set of signals is more focused.

Combining the above results, a third set of primer probes for exon 8 of SMN1 was selected.

EXAMPLE 2 detection of human blood samples for SMA

Blood sample collection and DNA extraction

Blood samples of normal, SMA carrier (SMN1 heterozygous deletion) and SMA patient (SMN1 homozygous deletion) were collected, DNA in the blood samples was extracted using a DNA extraction kit, the amount and purity of DNA was determined using a Nanodrop 2000 spectrophotometer and a Qubit 3.0(Thermo Scientific) fluorescence detector, and the DNA template was diluted to 10 ng/. mu.l. All DNA samples were stored at-80 ℃ until use.

Second, primers and probes

In this experiment, 5 different gene sequences were amplified, namely SMN1 exon 7, SMN1 exon 8, SMN2 exon 7, reference gene RPS27A and reference gene RPP 30. The 5' end of the probe was labeled with 5 different fluorophores, each with independent excitation and emission wavelengths to avoid interference with each other, using FAM, VIC, ROX, Cy5 and Cy5.5 fluorophores. The 3' end of the probe is labeled by a quenching group, and MGB, BHQ-2 and BHQ-3 are used for labeling in the experiment. The specific primers and probes used are shown in the following table:

'+' is Locked Nucleic Acid (LNA) modification

Preparation of PCR reaction system

PCR reaction systems (total volume 30. mu.L) were prepared according to the following table.

Digital PCR working process

(3) micro-droplet detection: after the PCR was completed, the 8-row tube and the droplet detection chip (Xinyi manufacturing technology (Beijing) Co., Ltd.) were placed in a jig, 430. mu.L and 500. mu.L of detection oil were added to the oil holes, respectively, and the chip was covered with a rubber pad and placed in a chip analyzer (Xinyi manufacturing technology (Beijing) Co., Ltd.) for droplet detection. The chip analyzer comprises 5 fluorescence channels (FAM/VIC/ROX/Cy5/Cy5.5), 5 lasers with the wavelengths of 473 nm, 532 nm, 580 nm, 640 nm and 710nm are used as excitation light sources to excite a plurality of fluorescent dyes in micro-droplets, and finally fluorescence excited in the micro-droplets is collected through a fluorescence detection light path.

(4) Signal processing and data analysis: the method comprises the steps that each of tens of thousands of micro-droplets prepared by a droplet generation chip and a sample preparation instrument is an independent PCR reactor, most of the micro-droplets do not contain a target gene to be detected or contain at least one target gene to be detected, after PCR amplification, FAM, VIC, ROX, Cy5 and Cy5.5 fluorescence signal detection is carried out on each micro-droplet by a chip analyzer, the peak height of the micro-droplet signal is recorded, and the droplet containing the target gene to be detected can detect a corresponding fluorescence signal. And (3) digitizing the fluorescence intensity in the micro-droplets through a fluorescence classification threshold, judging the micro-droplets with stronger fluorescence as '1' (positive), judging the micro-droplets with weaker fluorescence as '0' (negative), counting the number of '1' and '0', and calculating the total copy number of the target genes marked by FAM, VIC, ROX, Cy5 and Cy5.5 in the template through Poisson distribution model correction.

Fifthly, calculating absolute copy number and judging result standard

(1) Absolute copy number calculation and determination

The method sets two reference genes so as to avoid error results caused by the abnormity of the specific reference genes and improve the detection reliability. Knowing that the internal reference genes (RPS27A and RPP30) are 2 copies of genes, the absolute copy number of the target gene can be obtained by calculating the ratio of the total copy number of the target gene (SMN1 exon 7, SMN1 exon 8, SMN2 exon 7) and the internal reference genes, and the calculation formula is as follows,

wherein x is SMN1 exon 7 or SMN1 exon 8 or SMN2 exon 7.

The ratios and their corresponding absolute copy numbers are shown in the following table.

Ratio of	Absolute number of copies
		0-0.1	0
0.3-0.7	1
		0.8-1.2	2
1.3-1.7	3
		1.8-2.2	4
2.3-2.7	5
		2.8-3.2	6
Others	Can not determine

From the above calculation formulas and corresponding tables, each target gene (SMN1 exon 7, SMN1 exon 8, SMN2 exon 7) was able to derive two sets of absolute copy numbers: absolute number of copies_{Reference gene 1}And absolute copy number_{Reference gene 2}。

Determining the absolute copy number of the target gene at either a1 or a2 if a1 is equal to a2 and is within the reference value range in the above table; if A1 or A2 are not equal, wherein one of A1 or A2 refers to a range of values in the above table, determining the absolute copy number of the target gene as the value; otherwise, the absolute copy number of the target gene cannot be determined.

(2) Result interpretation standard of sample to be tested

According to the final absolute copy numbers of the SMN1 exon 7 and the SMN1 exon 8, the SMA disease or carrying condition of the sample to be detected can be judged in an assisted manner, and the interpretation standards are shown in the following table.

While the absolute copy number of exon 7 of SMN2 can be used as a reference for determining the severity of patients, about 80% of SMA type I patients have 1 or 2 copies of the SMN2 gene, about 82% of SMA type II patients have 3 copies of the SMN2 gene, about 96% of SMA type III patients have 3 or 4 copies of the SMN2 gene, and about 100% of SMA type IV patients have 4 or 6 copies of the SMN2 gene.

Sixth, results and discussion

The results of the tests for normal persons, SMA carriers and SMA patients are shown in the following table, and two-dimensional graphs of the test results are shown in fig. 3, 4 and 5. The results show that normal human SMN1 exon 7, SMN1 exon 8 and SMN2 exon 7 are 2 copies, SMA carrier SMN1 exon 7 and SMN1 exon 8 are 1 copy, SMN2 exon 7 is 2 copies, SMA patient SMN1 exon 7 and SMN1 exon 8 are 0 copies, SMN2 exon 7 is 4 copies.

The capacity of simultaneously and one tube in the SMA detection has important significance on the SMN1 exon 7, the SMN2 exon 7 and the SMN1 exon 8. Wherein, the copy number detection of the SMN1 exon 7 and exon 8 is used for SMA carrier screening and diagnosis, and the copy number detection of the SMN2 exon 7 can assist in SMA typing and guiding medication. The three targets are detected by one tube, so that the detection time is effectively shortened, the operation steps are simplified, and the detection result is more accurate based on the digital PCR technology. It should be noted that the type of the sample to be tested is not particularly limited, and may be blood, dried blood spots, other body fluids, tissues or buccal swabs, and the like, and may be selected according to the needs. In addition, the 5 '-end labeled fluorophore of the detection probe used may be FAM, FITC, Cy3, VIC, HEX, ROX, TET, NED, Cy5, Cy5.5, and the 3' -end labeled may be MGB, BHQ-1, BHQ-2, BHQ-3.

It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Sequence listing

<110> Qinghua university

Xinyi Manufacturing Technology (Beijing) Co., Ltd.

<120> digital PCR kit for detecting spinal muscular atrophy and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 29

<212> DNA