CN114606306B

CN114606306B - Probe composition, reagent, kit and detection method for detecting mucopolysaccharidosis VII type

Info

Publication number: CN114606306B
Application number: CN202011406750.0A
Authority: CN
Inventors: 张帅
Original assignee: Jiangsu Yuanlong Medical Technology Co ltd
Current assignee: Jiangsu Yuanlong Medical Technology Co ltd
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2024-07-02
Anticipated expiration: 2040-12-04
Also published as: CN114606306A

Abstract

The invention relates to the field of genetic disease gene detection, in particular to a probe composition for detecting mucopolysaccharidosis VII type, and the targeting sequence of the gene related to the mucopolysaccharidosis VII type aiming at the probe composition is selected from a plurality of sites on a GUSB gene. The invention solves the problem that the conventional mucopolysaccharidosis VII type diagnosis method in the prior art can not carry out GUSB gene mutation pre-pregnancy carrier screening and prenatal diagnosis. The kit can detect all GUSB gene mutation sites at one time, can make up for the defect of poor timeliness in the detection of MPS VII by the existing diagnosis method, can be used for screening and prenatal diagnosis of a pregnant carrier, and improves the quality of birth population.

Description

Probe composition, reagent, kit and detection method for detecting mucopolysaccharidosis VII type

Technical Field

The invention relates to the field of genetic disease gene detection, in particular to a probe composition, a reagent, a kit and a detection method for detecting mucopolysaccharidosis VII type.

Background

Mucopolysaccharidoses (Mucopolysaccharidosis, MPS) are a group of progressive multisystem affected diseases induced in vivo by mucopolysaccharide hydrolysis enzyme gene mutation in lysosomes, in which mucopolysaccharide cannot be degraded, and include 7 subtypes, 11 lysosomal enzymes encoded by 11 genes are involved, and except MPS II type is X-linked recessive inheritance, the rest belong to autosomal recessive inheritance.

Mucopolysaccharidosis type VII (MPSVII, also known as Sly syndrome) is a very rare subtype with a prevalence of only 1/300000 to 1/2000000. Mucopolysaccharidosis type VII is a degradation disorder of dermatan sulfate, heparan sulfate and chondroitin sulfate due to beta-glucuronidase deficiency, deposition in lysosomes causing progressive impairment of many tissue and organ functions. The beta-glucuronidase coding gene is GUSB, is located in autosome 7q11.21 and consists of 11 introns and 12 exons. More than 60 different types of mutations have been reported to date for mucopolysaccharidosis type VII, of which 78.6% are missense mutations, 12.6% are nonsense mutations, 5.8% are deletion mutations, 2.9% are splice site mutations, and these mutations are distributed throughout the GUSB gene without mutation hot spot regions, and lack complete epidemiological data.

MPSVII clinical diagnosis is difficult, and the existing enzymology detection is a gold standard for diagnosis, but is easily interfered by other similar chemical substances, so that misdiagnosis and missed diagnosis are caused. MPSVII are complex phenotypes, classified as light, intermediate and severe, and patients, with sporadic and varying symptoms have been reported. In addition, MPSVII is very similar to clinical phenotype of MPSI, and for further diagnosis, bone X-ray examination, electrocardiogram, skull CT, echocardiogram, glycosaminoglycan electrophoresis analysis, ophthalmic examination and the like are required to be carried out on suspected patients, so that the process is complicated, and time and labor are wasted.

From a genetic perspective, the recurrence risk of MPS VII is 25% when the parents of MPS VII patients recuperate, and it is clinically recommended that all MPS VII patients and family members thereof should undergo genetic counseling to determine the GUSB gene mutation carrier condition. In addition, high risk fetuses are subjected to prenatal diagnosis and intrauterine intervention of GUSB gene mutations to reduce birth in infants.

Enzyme replacement therapy has made great progress in patients who have been born, and some drugs have been used for clinical treatment with good prognosis. Hematopoietic stem cell transplantation can also treat MPS VII, and studies have demonstrated that the less the age at surgery, the lighter the organ before transplantation suffers and the less residual symptoms. As can be seen, the effective method of MPS VII is to detect GUSB gene in combination with enzyme activity assay to confirm diagnosis in neonatal period and receive treatment early.

Traditional GUSB gene diagnosis is to perform microsatellite DNA labeling and linkage analysis, but cannot find mutation sites of microcrack and sporadic patients, so that the diagnosis is inconvenient for pre-pregnancy GUSB gene detection and prenatal diagnosis. Therefore, the current conventional diagnostic method cannot be the best choice for clinical diagnosis of MPS VII, nor can the current conventional diagnostic method perform screening and prenatal diagnosis of presoaked carriers of GUSB gene mutations.

Disclosure of Invention

In view of the above-described drawbacks of the prior art, an object of the present invention is to provide a probe composition for detecting mucopolysaccharidosis type VII, for solving the problem that the conventional diagnosis method of mucopolysaccharidosis type VII in the prior art cannot be performed for screening and prenatal diagnosis of a carrier before the GUSB gene mutation, and at the same time, to provide a reagent for detecting mucopolysaccharidosis type VII and a kit for detecting mucopolysaccharidosis type VII; in addition, the present invention provides a method for detecting a gene associated with mucopolysaccharidosis type VII. The kit can detect all GUSB gene mutation sites at one time, can make up for the defect of poor timeliness in the detection of MPS VII by the existing diagnosis method, can be used for screening and prenatal diagnosis of a pregnant carrier, and improves the quality of birth population.

To achieve the above-mentioned objects and other related objects,

In a first aspect of the invention, there is provided a probe composition for detecting mucopolysaccharidosis type VII, said probe composition being directed against mucopolysaccharidosis type VII-related gene targeting sequences selected from a number of sites on the GUSB gene;

the sites include at least one of c.1084G>A、c.1876T>C、c.1520G>A、c.1618G>A、c.328C>T、c.1820G>C、c.112T>G、c.448G>A、c.1818G>C、c.88C>T、c.1337G>A、c.1120C>T、c.406G>A、c.935C>A、c.1304G>C、c.1145G>A、c.728T>C、c.155C>T、c.398G>C、c.1586A>G、c.307C>T、c.1391+618_1391+619delTC、c.530C>T、c.295G>A、c.266A>G、c.1653+151A>G、c.1715G>A、c.1523A>G、c.1135A>G、c.1016A>G、c.455A>G、c.148G>T、c.1477-70C>T、c.1091C>T、c.581+1G>A、c.1775delT、c.1693C>G、c.867G>A、c.1874_1875delGA、c.1457_1460delACTA、c.959A/G>C、c.959A>G、c.959A>C、c.-12G>A、c.454G>A、c.646C>T、c.1831C>T、c.1144C>T、c.1617C>T、c.526C>T、c.1061C>T、c.1429C>T、c.1244C>T、c.1856C>T、c.1051C>T、c.1244+1G>A、c.1222C>T、c.1521G>A、c.1069C>T、c.1484A>G、c.1881G>T、c.442C>T、c.1730G>T、c.1050G>C.

The probe composition can effectively, rapidly and accurately detect the type VII of the mucopolysaccharidosis, not only can simplify the diagnosis process and reduce the detection cost, but also can improve the detection timeliness and the diagnosis accuracy and solve the problems of difficult diagnosis, case emission and the like. In addition, the probe composition can also be used for screening a carrier before pregnancy and diagnosing before birth, and the birth of an infant is reduced through high-quality and high-efficiency genetic diagnosis screening.

In one embodiment of the invention, the site comprises c.1084G>A、c.1876T>C、c.1520G>A、c.1618G>A、c.328C>T、c.1820G>C、c.112T>G、c.448G>A、c.1818G>C、c.88C>T、c.1337G>A、c.1120C>T、c.406G>A、c.935C>A、c.1304G>C、c.1145G>A、c.728T>C、c.155C>T、c.398G>C、c.1586A>G、c.307C>T、c.1391+618_1391+619delTC、c.530C>T、c.295G>A、c.266A>G、c.1653+151A>G、c.1715G>A、c.1523A>G、c.1135A>G、c.1016A>G、c.455A>G、c.148G>T、c.1477-70C>T、c.1091C>T、c.581+1G>A、c.1775delT、c.1693C>G、c.867G>A、c.1874_1875delGA、c.1457_1460delACTA、c.959A/G>C、c.959A>G、c.959A>C、c.-12G>A、c.454G>A、c.646C>T、c.1831C>T、c.1144C>T、c.1617C>T、c.526C>T、c.1061C>T、c.1429C>T、c.1244C>T、c.1856C>T、c.1051C>T、c.1244+1G>A、c.1222C>T、c.1521G>A、c.1069C>T、c.1484A>G、c.1881G>T、c.442C>T、c.1730G>T、c.1050G>C.

The probe composition covers all GUSB gene mutation sites, so that on one hand, the defect of poor timeliness of the existing diagnostic method for MPS VII detection can be overcome, and the standardization degree and the diagnostic accuracy of MPSVII detection are improved; on the other hand, a more effective detection method is provided for screening and prenatal diagnosis of the MPS VII pre-pregnant carrier, so that more GUSB genetic mutation detection problems can be solved, high-quality and high-efficiency genetic diagnosis screening is realized, and birth of the MPS VII infant is reduced.

In an embodiment of the present invention, the nucleotide sequence of the probe corresponding to c.1084G > A is shown as SEQ ID No.1;

the nucleotide sequence of the probe corresponding to c.1876T > C is shown as SEQ ID No.2;

The nucleotide sequence of the probe corresponding to c.1520G > A is shown as SEQ ID No.3;

the nucleotide sequence of the probe corresponding to c.1618G > A is shown as SEQ ID No.4;

the nucleotide sequence of the probe corresponding to c.328C > T is shown as SEQ ID No.5;

the nucleotide sequence of the probe corresponding to c.1820G > C is shown as SEQ ID No.6;

the nucleotide sequence of the probe corresponding to c.112T > G is shown as SEQ ID No.7;

The nucleotide sequence of the probe corresponding to c.448G > A is shown as SEQ ID No.8;

the nucleotide sequence of the probe corresponding to c.1818G > C is shown as SEQ ID No.9;

The nucleotide sequence of the probe corresponding to c.88C > T is shown as SEQ ID No.10;

the nucleotide sequence of the probe corresponding to c.1337G > A is shown as SEQ ID No.11;

the nucleotide sequence of the probe corresponding to c.1120C > T is shown as SEQ ID No.12;

The nucleotide sequence of the probe corresponding to c.406G > A is shown as SEQ ID No.13;

The nucleotide sequence of the probe corresponding to c.935C > A is shown as SEQ ID No.14;

the nucleotide sequence of the probe corresponding to c.1304G > C is shown as SEQ ID No.15;

The nucleotide sequence of the probe corresponding to c.1145G > A is shown as SEQ ID No.16;

The nucleotide sequence of the probe corresponding to c.728T > C is shown as SEQ ID No.17;

the nucleotide sequence of the probe corresponding to c.155C > T is shown as SEQ ID No.18;

the nucleotide sequence of the probe corresponding to c.398G > C is shown as SEQ ID No.19;

the nucleotide sequence of the probe corresponding to c.1586A > G is shown as SEQ ID No.20;

the nucleotide sequence of the probe corresponding to c.307C > T is shown as SEQ ID No.21;

The nucleotide sequence of the probe corresponding to c.1391+618_1391+619delTC is shown as SEQ ID No.22; the nucleotide sequence of the probe corresponding to c.5306 > T is shown as SEQ ID No.23;

the nucleotide sequence of the probe corresponding to c.5G > A is shown as SEQ ID No.24;

the nucleotide sequence of the probe corresponding to c.266A > G is shown as SEQ ID No.25;

the nucleotide sequence of the probe corresponding to c.1653+151A > G is shown as SEQ ID No.26;

the nucleotide sequence of the probe corresponding to c.1715G > A is shown as SEQ ID No.27;

The nucleotide sequence of the probe corresponding to c.1523A > G is shown as SEQ ID No.28;

the nucleotide sequence of the probe corresponding to c.1135A > G is shown as SEQ ID No.29;

the nucleotide sequence of the probe corresponding to c.1016A > G is shown as SEQ ID No.30;

the nucleotide sequence of the probe corresponding to c.455A > G is shown as SEQ ID No.31;

The nucleotide sequence of the probe corresponding to c.148G > T is shown as SEQ ID No.32;

the nucleotide sequence of the probe corresponding to c.1477-70C > T is shown as SEQ ID No.33;

the nucleotide sequence of the probe corresponding to c.1091C > T is shown as SEQ ID No.34;

the nucleotide sequence of the probe corresponding to c.581+1G > A is shown as SEQ ID No.35;

the nucleotide sequence of the probe corresponding to c.1775delT is shown as SEQ ID No.36;

The nucleotide sequence of the probe corresponding to c.1693C > G is shown as SEQ ID No.37;

the nucleotide sequence of the probe corresponding to c.867G > A is shown as SEQ ID No.38;

the nucleotide sequence of the probe corresponding to the c.1874_1875delGA is shown as SEQ ID No.39;

The nucleotide sequence of the probe corresponding to c.1457_1460delACTA is shown as SEQ ID No.40;

the nucleotide sequence of the probe corresponding to c.959A/G > C is shown as SEQ ID No.41;

the nucleotide sequence of the probe corresponding to c.959A > G is shown as SEQ ID No.42;

the nucleotide sequence of the probe corresponding to c.959A > C is shown as SEQ ID No.43;

the nucleotide sequence of the probe corresponding to the c-12G > A is shown as SEQ ID No.44;

the nucleotide sequence of the probe corresponding to c.454G > A is shown as SEQ ID No.45;

The nucleotide sequence of the probe corresponding to c.646C > T is shown as SEQ ID No.46;

the nucleotide sequence of the probe corresponding to c.1831C > T is shown as SEQ ID No.47;

the nucleotide sequence of the probe corresponding to c.1144C > T is shown as SEQ ID No.48;

The nucleotide sequence of the probe corresponding to c.1617C > T is shown as SEQ ID No.49;

the nucleotide sequence of the probe corresponding to c.526C > T is shown as SEQ ID No.50;

The nucleotide sequence of the probe corresponding to c.1061C > T is shown as SEQ ID No.51;

the nucleotide sequence of the probe corresponding to c.1429C > T is shown as SEQ ID No.52;

the nucleotide sequence of the probe corresponding to c.1244C > T is shown as SEQ ID No.53;

The nucleotide sequence of the probe corresponding to c.1856C > T is shown as SEQ ID No.54;

the nucleotide sequence of the probe corresponding to c.1051C > T is shown as SEQ ID No.55;

the nucleotide sequence of the probe corresponding to c.1244+1G > A is shown as SEQ ID No.56;

The nucleotide sequence of the probe corresponding to c.1222C > T is shown as SEQ ID No.57;

the nucleotide sequence of the probe corresponding to c.1521G > A is shown as SEQ ID No.58;

The nucleotide sequence of the probe corresponding to c.1069C > T is shown as SEQ ID No.59;

the nucleotide sequence of the probe corresponding to c.1484A > G is shown as SEQ ID No.60;

the nucleotide sequence of the probe corresponding to c.1881G > T is shown as SEQ ID No.61;

The nucleotide sequence of the probe corresponding to c.4472C > T is shown as SEQ ID No.62;

the nucleotide sequence of the probe corresponding to c.1730G > T is shown as SEQ ID No.63;

The nucleotide sequence of the probe corresponding to c.1050G > C is shown as SEQ ID No.64.

In one embodiment of the present invention, in the probes corresponding to SEQ ID No. 1-64, the probe with the head end A/T carries an allophycocyanin fluorescent group at the tail end and an phycoerythrin fluorescent group at the head end G/C.

In a second aspect of the invention there is provided a reagent for detecting mucopolysaccharidosis type VII, said reagent comprising a probe composition as described above.

In a third aspect of the invention, there is provided a kit for detecting mucopolysaccharidosis type VII, said kit comprising the probe composition described above.

The kit can detect all GUSB gene mutation sites at one time, can make up for the defect of poor timeliness in the detection of MPS VII by the existing diagnosis method, can be used for screening and prenatal diagnosis of a pregnant carrier, and improves the quality of birth population.

In one embodiment of the present invention, the kit includes a plurality of probes having nucleotide sequences of SEQ ID Nos. 1 to 64.

The kit covers all GUSB gene mutation sites, so that the defect of poor timeliness of the existing diagnosis method for detecting MPS VII can be overcome, and the standardization degree and diagnosis accuracy of MPSVII detection are improved; on the other hand, a more effective detection method is provided for screening and prenatal diagnosis of the MPS VII pre-pregnant carrier, so that more GUSB genetic mutation detection problems can be solved, high-quality and high-efficiency genetic diagnosis screening is realized, and birth of the MPS VII infant is reduced. The invention can realize three purposes of one box and remarkably improve the sanitation and economy benefits.

In an embodiment of the invention, the number of probes with the nucleotide sequence SEQ ID No.1～2、SEQ ID No.4～6、SEQ ID No.9、SEQ ID No.12～16、SEQ ID No.19～20、SEQ ID No.22、SEQ ID No.24～26、SEQ ID No.29～30、SEQ ID No.32、SEQ ID No.34～36、SEQ ID No.45～49、SEQ ID No.53～54、SEQ ID No.56～57、SEQ ID No.59、SEQ ID No.61～62 in the kit is two, and the number of the remaining probes is one. To increase the accuracy and detection rate of the results at the corresponding sites, some site probes are repeated and the probes are randomly placed at different positions on the chip.

In one embodiment of the present invention, the kit further comprises a denaturing mixture, an amplification mixture, a fragmentation mixture, a precipitation mixture, and a hybridization mixture.

In a third aspect of the invention, there is provided a method of detecting a gene associated with mucopolysaccharidosis type VII, comprising: the DNA to be detected is put into an array chip scanner CNVPLUS ^TM, the probes are loaded on the chip of the microarray chip scanner, and reagents adopted in CNVPLUS ^TM detection comprise dye mixtures, stable mixtures and connection mixtures.

As described above, the probe composition, reagent, kit and detection method for detecting mucopolysaccharidosis type VII of the present invention have the following beneficial effects:

1. The probe composition can effectively, rapidly and accurately detect the type VII of the mucopolysaccharidosis, not only can simplify the diagnosis process and reduce the detection cost, but also can improve the detection timeliness and the diagnosis accuracy and solve the problems of difficult diagnosis, case emission and the like. In addition, the probe composition can also be used for screening a carrier before pregnancy and diagnosing before birth, and the birth of an infant is reduced through high-quality and high-efficiency genetic diagnosis screening.

2. The kit covers all GUSB gene mutation sites, so that the defect of poor timeliness of the existing diagnosis method for detecting MPS VII can be overcome, and the standardization degree and diagnosis accuracy of MPSVII detection are improved; on the other hand, a more effective detection method is provided for screening and prenatal diagnosis of the MPS VII pre-pregnant carrier, so that more GUSB genetic mutation detection problems can be solved, high-quality and high-efficiency genetic diagnosis screening is realized, and birth of the MPS VII infant is reduced. The invention can realize three purposes of one box and remarkably improve the sanitation and economy benefits.

Drawings

FIG. 1 is a flow chart of DNA detection in example 2 of the present invention;

FIG. 2 is the result of DNA fragmentation quality control in step three of example 2;

FIG. 3 is a graph showing genotyping results in example 2, and FIG. 3a is an example of positive results for locus genotyping in example 2; FIG. 3b is an example of a negative result of the position genotyping in example 2.

Detailed Description

Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.

The chip design method comprises the following steps: through ClinVar, HGMD, gnomAD and databases disclosed by the national part of clinical laboratories, the related sites of GUSB are counted in combination with literature reports, all mutation sites reported by Chinese people are confirmed, and the sites are synthesized into corresponding probes and entrusted to be processed into chips.

Screening of probes:

(1) All the site sequences design a pair of primers according to the sequences of the front 35bp and the rear 35bp of the site for PCR verification;

(2) PCR verifies that the sites with correct bands only synthesize corresponding probes;

(3) Sites without bands or band abnormalities will be re-designed with a pair of primers for verification, or sites that are not verified will be removed from the chip design.

To increase the accuracy and detection rate of the results at the corresponding sites, some site probes are repeated and the probes are randomly placed at different positions on the chip.

The chip probe numbers and sequence information are as follows: (chromosome position 7q11.21)

Table 1

Example 1

The specific use method of the kit comprises the following steps:

1. DNA amplification

The denaturing mixture (Denaturation Master Mix) was prepared according to the following system: (all reagents are independently developed and prepared by purchasing raw materials, and currently, a registration certificate is applied for, and the tentative name is CNVPLUS ^TM as a matched detection reagent of a chip scanner)

Table 2

Composition of the components	1×
		Denaturing liquid (Denat Soln X)	2μl
Water and its preparation method	18μl

An amplification mixture (Amplification Master Mix) was prepared according to the following system: mixing the components of the amplification mixed solution by vortex, then turning a tube for 2 times, and mixing by vortex again to obtain the amplification mixed solution.

TABLE 3

S1, taking 20 mu L of DNA sample (5 ng/. Mu.L), adding 20 mu L of the denaturation mixture, and incubating for 10min at room temperature.

S2, adding 130 μl of neutral solution (Neutral Soln), covering a sealing membrane, shaking, mixing, and centrifuging.

S3, slowly adding 230 μl of amplification mixed solution along the tube wall, and repeatedly blowing up and down; the sealing film is covered, and the mixture is stirred and mixed for 2 times and centrifuged at 1000rpm for 1min.

S4, placing the sample in an incubator at 37 ℃ for incubation for 23+/-1 h, placing the sample in an incubator at 65 ℃ for incubation for 20min, and transferring the sample to the incubator at 37 ℃ for continuous incubation for 45min.

2. Fragmentation and precipitation

The fragmenting mixture (Fragmentation Master Mix) was configured according to the following system:

Table 4

The precipitation mixture (Precipitation Master Mix) was prepared according to the following system:

Table 5

Composition of the components	1×
		Precipitation solution 1 (Precip Soln 1)	238μl
Precipitation solution 2 (Precip Soln 2)	2μl

S5, transferring the amplified sample in the step S4 to room temperature by an incubator at 37 ℃, adding 57 μl of the fragmented mixed solution, covering a sealing film, shaking, mixing and centrifuging for 2 times.

S6, rapidly transferring the sample to an incubator at 37 ℃ for incubation for 30min, then adding 19 μl of stop solution, covering a sealing membrane, shaking, mixing and centrifuging.

And S7, adding 240 mu l of the precipitation mixed solution at room temperature, covering a sealing film, vibrating, uniformly mixing and centrifuging.

S8, adding 600 μl of isopropanol, uniformly mixing up and down by a pipette to ensure sufficient mixing, covering a sealing film, and transferring the sample to an environment of-20 ℃ overnight (16-24 h).

3. Drying, resuspension and quality control

Hybridization mixture (Hybridization Master Mix) was prepared according to the following system:

Table 6

Composition of the components	1×
		Hybridization Buffer (Hyb Buffer)	70.5μl
Hybridization solution 1 (Hyb Soln 1)	0.5μl
		Hybridization solution 2 (Hyb Soln 2)	9μl

S9, taking out the sample from the temperature of minus 20 ℃, and centrifuging at 3200rpm at 4 ℃ for 40min.

S10, carefully tearing off the sealing film, and inverting the sample on the waste liquid barrel to enable the liquid to flow out, and then lightly inverting the sample on dust-free paper for 5min.

S11, placing the sample in a hybridization furnace at 37 ℃ for 20min, and airing.

S12, adding 35 mul of heavy suspension buffer solution (Resusp Buffer, a product of Thermo filter product No. 901466 can be selected) into the sample, covering a sealing film, and placing the sample into a shaker for shaking (TITER PLATE SHAKERS-4PL shaking at a speed of 9 for 10min or Jitterbug shaking at a speed of 7 for 10 min).

S13, after the sample in the step 12 is completely resuspended, 80 μl of hybridization mixture is added, and the sealing film is covered, vibrated, mixed and centrifuged.

S14, taking 33 mu l of nuclease-free water into a PCR plate, mixing the water into the mixed solution of 3 mu l S to prepare Quality Control diluent, covering a sealing film, vibrating, mixing uniformly and centrifuging.

And (3) quality detection:

① . 10. Mu.l of the QC dilution sample was taken into an OD plate (96-well UV STAR PLATE, E & K SCIENTIC P/N25801), and 90. Mu.l of ultrapure nuclease-free water (Nuclease free wate, ultrapure MB Grade (USB, P/N71786)) was added to the OD plate (Optical Density), and the mixture was blown up and down to mix the sample, and the absorbance was read.

② . 120 Μl of 1000-fold diluted gel-loading dye is taken and added into a gel sample plate, 3 μl of the QC diluent is mixed, a sealing film is covered, the mixture is vibrated, mixed uniformly and centrifuged, and the gel is removed for quality control.

Wherein the Gel-loading dye is Invitrogen^TM TrackIt Cyan/Orange Loading Buffer(Invitrogen P/N10482-028);25bp Invitrogen Ladder(Invitrogen P/N 10488-022); Gel electrophoresis system Invitrogen E-Gel ^TM 48 agarose gels 4%, G8008-04.

4. Denaturation and hybridization

S15, placing the hybridized mixed solution at room temperature for 5min, after short shaking, centrifuging at 1000rpm for 30S, and placing at least 25min at room temperature.

S16, placing the hybridization plate in a PCR instrument, and starting a denaturation program (Denature program): denaturation at 95℃for 10min and denaturation at 48℃for 3min, cooling.

S17, after the denaturation procedure is finished, the hybridization plate is taken out and placed in a preheated 96-well metal tank, 105. Mu.l of the denatured hybridization solution is carefully transferred to a hybridization plate (CNVPLUS ^TM Consumables Kit) by a pipette, and no bubbles are generated.

S18, loading the hybridization tray into a microarray chip processing instrument, and continuously hybridizing for 23.5-24 hours.

5. CNVPLUS Equipment reagent preparation

Dye mixture a (STAIN A MASTER Mix) was configured according to the following system: the tube was gently turned over 10 times to mix and placed on ice in the dark.

Table 7

Composition of the components	1×
		Dye diluent (Wash A)	201.6μl
Dyeing Buffer (Stain Buffer)	4.2μl
		Colorant 1-A (Stain 1-A)	2.1μl
Colorant 1-B (Stain 1-B)	2.1μl

Dye mixture B (Stain B Master Mix) was formulated according to the following system: the tube was gently turned over 10 times to mix and placed on ice in the dark.

Table 8

The stabilization mixture (Stabilization Master Mix) was configured according to the following system: mix well at maximum speed vortex for 3s and place on ice.

Table 9

Composition of the components	1×
		Water and its preparation method	93.19μl
Stabilizing diluent (Stabilize Diluent)	10.50μl
		Stable liquid (Stabilize Soln)	1.31μl

The connection mixture A (Ligation A Master Mix) was configured according to the following system: mix well at maximum speed vortex for 3s and place on ice.

Table 10

Composition of the components	1×
		Connection buffer (Ligate Buffer)	66.15μl
Connecting liquid 1 (Ligate Soln 1)	13.12μl
		Connecting liquid 2 (Ligate Soln 2)	3.15μl

The connection mixture B (Ligation B Master Mix) was configured according to the following system: the tube was gently turned over 10 times to mix and placed on ice in the dark.

Table 11

Composition of the components	1×
		Ligation mixture A	82.42μl
Buffer 1 (Probe Mix 1)	10.5μl
		Buffer solution 2 (Probe Mix 2)	10.5μl
Ligase (Ligation Enzyme, preservation temperature-20 ℃ C.)	1.58μl

S19, adding the mixture into a corresponding scanning disc respectively: wherein 2 dye discs of dye mixture A (105. Mu.l per well), 1 dye disc of dye mixture B (105. Mu.l per well), 1 dye disc of stabilizing mixture (105. Mu.l per well), 1 dye disc of connecting mixture B (105. Mu.l per well) avoid the generation of bubbles.

S20, covering the scanning disc with a corresponding cover, and then directly loading the scanning disc into a CNVPLUS ^TM microarray chip analysis instrument.

S21, 150 μl Hold Buffer (fixed Buffer) is added into each hole of each scanning disk to avoid contacting the bottom of the scanning disk. The cut-out angle of the cover is aligned with the cut-out edge of the scan disk, covering the scan disk, and then placed on top of the table.

The CNVPLUS ^TM microarray chip processor is chip detection equipment, the chip is integrated into a 96-well plate type, one plate comprises 96 identical chips, and at most 96 samples can be detected in a single experiment.

The reagents of example 1 may be selected from the following products:

Table 12

Example 2

The chip is used for verifying GUSB positive sites

Step one, collecting a sample: after informed consent, peripheral blood was collected from the patients for a total of 12 cases including 7 cases for male patients and 5 cases for female patients as verification samples.

Step two, sample preparation: the verification sample is firstly subjected to DNA extraction, and quality control (such as quality detection of S14) is performed by using gel electrophoresis, a Nanodrop spectrophotometer and the like, so that each DNA sample is ensured to be free from degradation, free from impurities and high in purity. Samples which do not meet any condition need to be purified and the like, wherein the Optical Density (OD) 260/280nm ratio is 1.8-2.0 and the OD 260/230nm ratio is 1.5-2.0.

Step three, detection reaction: the sample with qualified quality is firstly amplified in whole genome, and the amplified product is then subjected to enzyme digestion, precipitation recovery, resuspension and other steps to obtain DNA fragments (the fragment length is 25-125bp, as shown in figure 2), hybridization reaction reagent is added into the heavy suspension, and the DNA fragments are transferred to a hybridization plate and then are hybridized by a machine. After hybridization, adding the prepared fluorescent reagent (dye mixture A and dye mixture B) for connection reaction, and washing and imaging scanning by CNVPLUS ^TM microarray chip processor to complete detection reaction.

Fourth, data analysis and interpretation: after the scanning is completed, each sample can obtain a CEL format file, original fluorescent signal data of each probe on the chip is recorded, and analysis is carried out on each CEL file by utilizing analysis interpretation software, wherein the analysis comprises chip quality control, sample quality control, probe sequence identification, sample comparison and genotyping analysis, so that a result file of DNA site information of the corresponding sample can be obtained.

The analysis principle is as follows: each probe is designed to be fixed on a chip and is distributed with corresponding coordinates, and the probes can be specifically combined with DNA mark fragments containing matching sites after hybridization. After combination, four types of bicolor probes are combined, the head ends of the bicolor probes respectively carry bases A, T, G, C, the tail ends of the bicolor probes carry fluorescent groups, the tail ends of the probes with the head ends of A/T carry allophycocyanin fluorescent groups, and the tail ends of the probes with the head ends of G/C carry phycoerythrin fluorescent groups. After hybridization, the probe and the hybridization sequence are connected by using DNA ligase, and then two groups are promoted to emit fluorescence signals through enzyme-linked reaction, and a scanner in the instrument can capture the fluorescence signals and generate CEL files. The software automatically performs quality control and genotyping analysis on each coordinate fluorescence signal data recorded in the CEL file (the genotyping result is shown in fig. 3, fig. 3a is an example of positive results of the genotyping of the loci in example 2, and fig. 3b is an example of negative results of the genotyping of the loci in example 2), and then outputs the result file. The result contains the mutation site and copy number variation information of the gene detected by each sample, and the information is subjected to further medical interpretation analysis to obtain clear association of the site and the disease.

Step five, analyzing results:

the site information of the sample was validated as follows (this site information was determined by other molecular diagnostic methods).

Table 13

The software analysis result after scanning by the microarray chip processor of the invention:

Table 14

Note that: normal indicates that the site is not detected as a mutation; affilected indicates that heterozygous or homozygous mutation was detected and was consistent with the patient site.

In conclusion, the product provided by the invention has the advantages that all the 12 positive GUSB mutation sites are detected, and the detection rate reaches 100%. Further analyzing the accuracy of the result, finding that the detection result of the MPS-04 sample is Normal, namely that the corresponding mutation is not detected, and the actual detection result is heterozygous mutation, the positive verification accuracy can reach 91.7%, and the universality and the effectiveness of the probe are fully illustrated.

It should also be noted that the above-listed examples are only one specific embodiment of the present invention, and only some of the identified GUSB positive sites are selected, and other GUSB sites may be detected. In theory, other people in the art can synchronously detect GUSB loci of all reported Chinese people by using one experiment from the disclosure of the invention, and the design concept is considered to be the protection scope of the invention.

In conclusion, the kit can detect all GUSB gene mutation sites at one time, can make up for the defect of poor timeliness in the detection of MPS VII by the existing diagnosis method, can be used for screening and prenatal diagnosis of a pregnancy carrier, and improves the quality of birth population. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

SEQUENCE LISTING

<110> Jiangsu Yuanlong medical science and technology Co., ltd

<120> A probe composition, reagent, kit, and detection method for detecting mucopolysaccharidosis type VII

<130> 2020.11.20

<160> 64

<170> PatentIn version 3.3

<210> 1

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 1

aagcaggttg aagtccttca ccagcagcgg ccagtcgaag cccttccctc ggatctagga 60

gatagcagag c 71

<210> 2

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 2

gtggggatac ctggtttcat tggcaatctt ccagtatctc tctcgcaaaa ggaacgctgc 60

actttttggt t 71

<210> 3

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 3

gctgaatcaa ctccaggtgc ccgtagtcgt gataccaaga gtagtagctg ttcaaacaga 60

tcacatccac a 71

<210> 4

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 4

ctggtgaaac cctgcaatcg tttctgctcc atactcgctc tgaataatgg gcttctgata 60

cttcttatac c 71

<210> 5

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 5

cagcaccact cttgtgcgca ggtcctgggt ccatcactcc ggcaggatca cctcccgttc 60

gtaccacacc c 71

<210> 6

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 6

ctgcactttt tggttgtctc tgccgagtga agatcccctt tttattcccc agcactctcg 60

tcggtgctac a 71

<210> 7

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 7

ggcgcggaag ctccagaggc cgtccagctc cttgcactcc cgcgacgggc tctcctgggg 60

gtacagcatc c 71

<210> 8

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 8

gggccccacc tggaccaggt tgctgatgtc ggcctcgaag gggaggtagc ccccctcatg 60

ctctagcgtg t 71

<210> 9

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 9

gcactttttg gttgtctctg ccgagtgaag atcccctttt tattccccag cactctcgtc 60

ggtgctacaa a 71

<210> 10

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 10

cagctccttg cactcccgcg acgggctctc ctgggagtac agcatcccgc cctgcagccc 60

cagcgcgcag c 71

<210> 11

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 11

attctaggtg ggacgcaggc tcgttggcca cagaccacat cacgaccgcg gggtggttct 60

tgtccctacg c 71

<210> 12

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 12

gtggctggta cggaaagcgt tggcaccaag ccagcaaagc aggttgaagt ccttcaccag 60

cagcggccag t 71

<210> 13

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 13

gaggtagccc ccctcatgct ctagcgtgtc gaccccattc acccactgca gacacaggag 60

atacggggag g 71

<210> 14

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 14

cagggagtgt gtagaagtca gacacaggcc ccagtgacgt ctgtgcagtc agctgcacct 60

atgacagcca a 71

<210> 15

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 15

accacatcac gaccgcgggg tggttcttgt ccctacgcac cacttcttcc atcacctgca 60

tgtggtgatg c 71

<210> 16

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 16

gcatcacttc ctctgcatag gggtagtggc tggtacggaa agcgttggca ccaagccagc 60

gaagcaggtt g 71

<210> 17

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 17

ggttactgcc cttgacagag atctggtaat tcaccagccc tgcaagaaac aagagagacc 60

agggctgagg g 71

<210> 18

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 18

accactgctc ctcgaagccc cggcgtcggt tgtcaaagaa gtcggcgcgg aagctccaga 60

ggccgtccag c 71

<210> 19

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 19

ccccctcatg ctctagcgtg tcgaccccat tcacccactg cagacacagg agatacgggg 60

agggggctgc a 71

<210> 20

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 20

actcgctctg aataatgggc ttctgatact tcttacacca gttctcaaac tgggtggcca 60

gctgcagctg a 71

<210> 21

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 21

gtcctgggtc catcgctccg gcaggatcac ctcccattcg taccacaccc agccgacaaa 60

atgccgcaga c 71

<210> 22

<211> 70

<212> DNA

<213> Artificial sequence (Artifical)

<400> 22

cagctactcg ggaggctgag gctggagaat cacttacctg agcggcagag gttgcagtga 60

gctgagatca 70

<210> 23

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 23

ggtattggat ggtccctggt ggcagggtgg tggggatgag tgtgttgttg atggcgatag 60

tgattcggag c 71

<210> 24

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 24

tcgctccggc aggatcacct cccgttcgta ccacacccag ccgacaaaat gccgcagacg 60

ccagtcctgg c 71

<210> 25

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 25

accacaccca gccgacaaaa tgccgcagac gccagccctg gctgatgtca ttgaagctgg 60

agggaactgg c 71

<210> 26

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 26

ctgaggacca agtgagtggg gcacaggcct ggctgctggc ggtgagggaa cgtggacggg 60

gccgtgggag g 71

<210> 27

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 27

ctccaaccac gtattttctg cgtttttgat ccagacccag atggtactgc tctagcagac 60

ttttctggta c 71

<210> 28

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 28

gcagctgaat caactccagg tgcccgtagt cgtgacacca agagtagtag ctgttcaaac 60

agatcacatc c 71

<210> 29

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 29

ctctgcatag gggtagtggc tggtacggaa agcgtcggca ccaagccagc gaagcaggtt 60

gaagtccttc a 71

<210> 30

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 30

gcttgttgac accgtggaaa tagaaaggtt tcccactgat gaggaactgg ctcttggtga 60

cagccacagt g 71

<210> 31

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 31

agggcagggg ccccacctgg accaggttgc tgatgccggc ctcgaagggg aggtagcccc 60

cctcatgctc t 71

<210> 32

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 32

ctcctcgaag ccccggcgtc ggttgtcaga gaagtaggcg cggaagctcc agaggccgtc 60

cagctccttg c 71

<210> 33

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 33

agtagacagc atgactcagc attcacacac tgcggaggct cctctggcag agaaggtaag 60

ggggatgtaa t 71

<210> 34

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 34

gccagcgaag caggttgaag tccttcacca gcagcagcca gtcgaagccc ttccctcgga 60

tctaggagat a 71

<210> 35

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 35

ggtgggtgtg tgcaatggag gcaggatggt acccacttgg aggtgtcagt caggtattgg 60

atggtccctg g 71

<210> 36

<211> 70

<212> DNA

<213> Artificial sequence (Artifical)

<400> 36

atgagccaaa ctgccactta cactgttcag tcatgaatcg gcaaaattcc aaatgagctc 60

tccaaccacg 70

<210> 37

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 37

tttttgatcc agacccagat ggtactgctc tagcacactt ttctggtact cttcagtgaa 60

catcagaggt g 71

<210> 38

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 38

tacagatagg cagggcgttc gtgcatcagg tacggccacc agaggctgac acctggcacc 60

ttaagttggc c 71

<210> 39

<211> 70

<212> DNA

<213> Artificial sequence (Artifical)

<400> 39

tggggatacc tggtttcatt ggcaatcttc cagtatctct cgcaaaagga acgctgcact 60

ttttggttgt 70

<210> 40

<211> 70

<212> DNA

<213> Artificial sequence (Artifical)

<400> 40

tggggacccc caggctcacc cccttgtctg ctgcatagag ttgctcacaa aggtcacagg 60

ccgggagggg 70

<210> 41

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 41

tgacagccac agtgcggatc cccacaggga gtgtgcagaa gtcagacaca ggccccagtg 60

acgtctgtgc a 71

<210> 42

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 42

tgacagccac agtgcggatc cccacaggga gtgtgcagaa gtcagacaca ggccccagtg 60

acgtctgtgc a 71

<210> 43

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 43

tgacagccac agtgcggatc cccacaggga gtgtggagaa gtcagacaca ggccccagtg 60

acgtctgtgc a 71

<210> 44

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 44

ggcaaccgcc gacccccggg ccatgcttcc cggtcccccg ctcggccacc gtctgcggcg 60

ctaagaaaag c 71

<210> 45

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 45

gggcaggggc cccacctgga ccaggttgct gatgtcggcc tcgaagggga ggtagccccc 60

ctcatgctct a 71

<210> 46

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 46

gtaggtggtg ggtgtcgtgt acagaagtac agaccactgc agtccagcgt agttgaaaaa 60

gtcaaaatat g 71

<210> 47

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 47

caaaaggaac gctgcacttt ttggttgtct ctgccaagtg aagatcccct ttttattccc 60

cagcactctc g 71

<210> 48

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 48

catcacttcc tctgcatagg ggtagtggct ggtacagaaa gcgttggcac caagccagcg 60

aagcaggttg a 71

<210> 49

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 49

tggtgaaacc ctgcaatcgt ttctgctcca tactcactct gaataatggg cttctgatac 60

ttcttatacc a 71

<210> 50

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 50

ttggatggtc cctggtggca gggtggtggg ggtgaatgtg ttgttgatgg cgatagtgat 60

tcggagccgg g 71

<210> 51

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 51

ccccgacaag gacccaggag ccccaacaca cgtccacatc ctcatgcttg ttgacaccgt 60

ggaaatagaa a 71

<210> 52

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 52

tgcatagtta gagttgctca caaaggtcac aggccaggag gggtccaagg atttggtgtg 60

agcgatcacc a 71

<210> 53

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 53

tgggcaggcg gagcaggtgc acagcagaga ctcacagcag cgccaggccc acgccgggac 60

actcatcgat g 71

<210> 54

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 54

tggcaatctt ccagtatctc tctcgcaaaa ggaacactgc actttttggt tgtctctgcc 60

gagtgaagat c 71

<210> 55

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 55

gacccaggag ccccaacaca cgtccgcatc ctcatacttg ttgacaccgt ggaaatagaa 60

aggtttccca t 71

<210> 56

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 56

ctgggcaggc ggagcaggtg cacagcagag actcacggca gcgccaggcc cacgccggga 60

cactcatcga t 71

<210> 57

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 57

agcagagact cacggcagcg ccaggcccac gccggaacac tcatcgatga ccacaatccc 60

atagcggtca c 71

<210> 58

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 58

agctgaatca actccaggtg cccgtagtcg tgataccaag agtagtagct gttcaaacag 60

atcacatcca c 71

<210> 59

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 59

cttcaccagc agcggccagt cgaagccctt ccctcagatc taggagatag cagagccaag 60

tgacccctgt c 71

<210> 60

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 60

aagagtagta gctgttcaaa cagatcacat ccacacacgg agcctaggac cagagcagca 60

gagcccgttc a 71

<210> 61

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 61

actgagtggg gatacctggt ttcattggca atcttacagt atctctctcg caaaaggaac 60

gctgcacttt t 71

<210> 62

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 62

cacctggacc aggttgctga tgtcggcctc gaaggagagg tagcccccct catgctctag 60

cgtgtcgacc c 71

<210> 63

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 63

aattccaaat gagctctcca accacgtatt ttctgagttt ttgatccaga cccagatggt 60

actgctctag c 71

<210> 64

<211> 71

<212> DNA

<213> Artificial sequence (Artifical)

<400> 64

acccaggagc cccaacacac gtccgcatcc tcatgcttgt tgacaccgtg gaaatagaaa 60

ggtttcccat t 71

Claims

1. A probe composition for detecting mucopolysaccharidosis type VII, characterized in that the probe composition targets mucopolysaccharidosis type VII-related gene targeting sequences selected from several sites on the GUSB gene;

The positions are c.1084G>A、c.1876T>C、c.1520G>A、c.1618G>A、c.328C>T、c.1820G>C、c.112T>G、c.448G>A、c.1818G>C、c.88C>T、c.1337G>A、c.1120C>T、c.406G>A、c.935C>A、c.1304G>C、c.1145G>A、c.728T>C、c.155C>T、c.398G>C、c.1586A>G、c.307C>T、c.1391+618_1391+619delTC、c.530C>T、c.295G>A、c.266A>G、c.1653+151A>G、c.1715G>A、c.1523A>G、c.1135A>G、c.1016A>G、c.455A>G、c.148G>T、c.1477-70C>T、c.1091C>T、c.581+1G>A、c.1775delT、c.1693C>G、c.867G>A、c.1874_1875delGA、c.1457_1460delACTA、c.959A/G>C、c.959A>G、c.959A>C、c.-12G>A、c.454G>A、c.646C>T、c.1831C>T、c.1144C>T、c.1617C>T、c.526C>T、c.1061C>T、c.1429C>T、c.1244C>T、c.1856C>T、c.1051C>T、c.1244+1G>A、c.1222C>T、c.1521G>A、c.1069C>T、c.1484A>G、c.1881G>T、c.442C>T、c.1730G>T and c.1050G > C;

the nucleotide sequence of the probe corresponding to c.1084G > A is shown as SEQ ID No.1;

the nucleotide sequence of the probe corresponding to c.1391+618_1391+619delTC is shown as SEQ ID No.22;

the nucleotide sequence of the probe corresponding to c.5306 > T is shown as SEQ ID No.23;

the nucleotide sequence of the probe corresponding to c.1050G > C is shown as SEQ ID No.64;

The probe composition consists of the above probes.

2. A probe composition for detecting mucopolysaccharidosis type VII according to claim 1, characterized in that: in the probes with the nucleotide sequences of SEQ ID No. 1-64, the tail end of the probe with the head end of A/T carries allophycocyanin fluorescent groups, and the tail end of the probe with the head end of G/C carries phycoerythrin fluorescent groups.

3. A reagent for detecting mucopolysaccharidosis type VII, characterized in that the reagent comprises the probe composition of any of claims 1-2.

4. A kit for detecting mucopolysaccharidosis type VII, characterized in that it comprises a probe composition according to any one of claims 1-2.

5. A kit for detecting mucopolysaccharidosis type VII according to claim 4, wherein: the number of probes with the nucleotide sequence SEQ ID No.1～2、SEQ ID No.4～6、SEQ ID No.9、SEQ ID No.12～16、SEQ ID No.19～20、SEQ ID No.22、SEQ ID No.24～26、SEQ ID No.29～30、SEQ IDNo.32、SEQ ID No.34～36、SEQ ID No.45～49、SEQ ID No.53～54、SEQ ID No.56～57、SEQID No.59、SEQ ID No.61～62 in the kit is two, and the number of the rest probes is one.

6. A kit for detecting mucopolysaccharidosis type VII according to any of claims 4-5, characterized in that: the kit also comprises a denaturation mixed solution, an amplification mixed solution, a fragmentation mixed solution, a precipitation mixed solution and a hybridization mixed solution.