CN112708673B

CN112708673B - Application of PRDM9 transposon fusion as congenital megacolon disease marker

Info

Publication number: CN112708673B
Application number: CN202110329812.0A
Authority: CN
Inventors: 朱云; 张彦; 夏慧敏
Original assignee: Guangzhou Women and Childrens Medical Center
Current assignee: Guangzhou Women and Childrens Medical Center
Priority date: 2021-03-26
Filing date: 2021-03-26
Publication date: 2021-06-25
Anticipated expiration: 2041-03-26
Also published as: CN112708673A

Abstract

The invention relates to the field of biomedicine, in particular to application of PRDM9 transposon fusion as a congenital megacolon disease marker. The fusion site of the PRDM9 transposon fusion is selected from PRDM9-type I located at chr5: 23299411; PRDM9-type II-1 at chr5: 23262356; and PRDM9-type II-2 at chr5: 23245181. Compared with the traditional diagnosis method (barium enema and the like), the method has the advantages of simple and quick operation, no intervention, high flux, low cost and the like.

Description

Application of PRDM9 transposon fusion as congenital megacolon disease marker

Technical Field

The invention relates to the field of biomedicine, in particular to application of PRDM9 transposon fusion as a congenital megacolon disease marker.

Background

Hirschsprung disease (HSCR) is a birth defect disease of infantile enteric nerve dysplasia, and the pathological mechanism is that cells at the intestinal neural crest migrate and differentiate into enteric neurons to generate obstacle, so that the enteric nerve is deficient to generate persistent spasm, which is one of the common congenital intestinal tract diseases of infants. Early congenital megacolon is manifested as vomiting, abdominal distension, diarrhea and the like, which can lead to death of newborn infants or complications such as repeated enteritis after operation, intractable constipation and the like clinically, and seriously affect the growth, development and life quality of children patients.

The timely diagnosis and treatment of the congenital megacolon can reduce the risk of the congenital megacolon enteritis and obtain good prognosis. The diagnosis of the disease requires pathological sections of the diseased tissue after surgery. The preoperative diagnosis method mainly comprises barium enema, rectal biopsy and rectal manometry to judge whether to implement 'giant colon radical operation'. At present, barium enema is the most important diagnostic method, the principle is that no nerve segment stenosis and proximal dilatation exist in the intestinal tract of a congenital megacolon child patient, the colon is diagnosed as megacolon by the dilatation and the stenosis after the barium enema is carried out, but the method can only diagnose the child patient with typical intestinal tract morphology change, the sensitivity needs to be improved, and the diagnostic accuracy is about 80%. Rectal biopsy is to directly take a rectal tissue, detect whether ganglion cells are absent or not, have high accuracy, but have influence on the result by a sampling position, but the method is invasive and has very high price, and the method is generally not obvious when being applied to a barium enema or is not applicable to a child patient, for example, when the child patient has the possibility of Necrotizing Enterocolitis (NEC), the barium enema can cause intestinal perforation, and the rectal biopsy is not considered when the barium enema is not suitable for diagnosis. Rectal manometry is to determine the innervation abnormality of the intestinal nerves by detecting the lack of relaxation of the internal anal sphincter, is only an auxiliary diagnosis method, has more false positives and false negatives, and cannot be used for single detection.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a diagnostic marker of the Hirschsprung's disease and application thereof, and provides a new accurate and sensitive detection way for diagnosing the Hirschsprung's disease.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the first aspect of the invention relates to the application of a quantitative detection agent of PRDM9 transposon fusion in the preparation of a diagnostic reagent or a kit for the congenital giant colon;

the fusion site of the PRDM9 transposon fusion is selected from:

PRDM9-type I located at chr5: 23299411;

PRDM9-type II-1 at chr5: 23262356; and

PRDM9-type II-2 at chr5: 23245181.

Optionally, for use as described above, the quantitative detection agent is for performing any one of the following methods:

polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method.

Optionally, in the above-mentioned application, the quantitative detection agent is a primer.

Optionally, for the above-mentioned application, the primer includes at least one of a-c:

a. SEQ ID NO: 1-2;

b. SEQ ID NO: 3-4; and

c. SEQ ID NO: 5 to 6.

Optionally, the primers further comprise an internal reference primer for detecting a wild-type sequence corresponding to at least one of PRDM9-type I, PRDM9-type II-1 and PRDM9-type II-2.

Optionally, in the above application, the reference primer includes at least one of d-f:

d. SEQ ID NO: 7-8;

e. SEQ ID NO: 9-10; and

f. SEQ ID NO: 11 to 12.

Optionally, in the above-mentioned application, the quantitative detection reagent further comprises one or more of a DNA extraction reagent, dNTP, DNA polymerase, double-strand specific fluorescent dye and water.

Optionally, for use as described above, the test sample of the reagent or kit is selected from at least one of blood, tissue, cell sample.

A second aspect of the invention relates to a primer as defined in the application as described above.

A third aspect of the invention relates to a kit containing a quantitative detection agent as defined in the application as described above.

The invention has the beneficial effects that:

barium enema detection and rectal biopsy methods used for the congenital megacolon are invasive, and bring certain physical and psychological pains to newborn patients and parents who wish to obtain definite diagnosis of diseases. The congenital megacolon children are manifested by vomit, abdominal distension, constipation, enteritis and the like, the group of children with the symptoms is large, the children with the congenital megacolon accounts for a small amount, and the method has high requirements on instruments and equipment and high price and is not suitable for screening diseases. The rectal manometry method is noninvasive, but has high false positive and false negative rates, and cannot effectively and accurately diagnose the congenital megacolon diseases.

The invention utilizes DNA diagnosis technology, adopts PRDM9 transposon fusion site with high specificity to diagnose children's Hirschsprung's disease, the adopted product and method can make the diagnosis of Hirschsprung's disease meet the requirements of strong specificity and high sensitivity, and has the advantages of simple and rapid operation, no intervention, high flux, low cost, etc., thus effectively making up the deficiencies in the prior art and being an effective alternative or auxiliary detection means for diagnosing or screening Hirschsprung's disease. The highest AUC value of the method reaches 0.9666 when the method diagnoses the congenital megacolon, the optimal limit corresponds to the sensitivity of 84.38 percent and the specificity of 95.35 percent, and the accuracy is even better than that of the existing diagnosis method. And when the sensitivity of the method is 100%, the specificity is 76.74%, and the method is suitable for disease screening.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic diagram showing the screening procedure of a fusion genetic marker of a congenital megacolon transposon in one embodiment of the present invention;

FIG. 2 shows the transcriptome sequencing and differentially expressed transposon analysis of Hirschmannica in one embodiment of the present invention;

FIG. 3 is a graph showing that machine learning according to one embodiment of the present invention confirms that transposon fusion of PRDM9 is closely associated with the Hirschsprung's segment type;

FIG. 4 shows the dual fluorescent reporter gene detection of PRDM9 transposon fusion occurring in the transcriptional regulatory region in one embodiment of the invention;

FIG. 5 shows the quantitative PCR detection of PRDM9 transposon fusion and the evaluation of the diagnostic effect of PRDM9 transposon fusion on Hirschsprung's disease using the ROC curve in one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention relates to an application of a PRDM9 transposon fusion quantitative detection agent in the preparation of a diagnostic reagent or a kit for congenital megacolon;

the fusion site of the PRDM9 transposon fusion is selected from:

PRDM9-type I located at chr5: 23299411;

PRDM9-type II-1 at chr5: 23262356; and

PRDM9-type II-2 at chr5: 23245181.

The invention analyzes transposon fusion of differential expression by sequencing of complete transcriptome of nervous segments and non-nervous segments of tissues from patients with congenital megacolon, screens transposon fusion related to congenital megacolon subtype by machine learning, and further verifies the effect of DNA in the congenital megacolon queue by quantitative PCR technology and ROC curve analysis.

The research result of the invention finds that PRDM9 transposon fusion is an effective diagnostic marker of the congenital megacolon disease, wherein, 3 PRDM9 transposons positioned at the 23299411 th position and/or the 23262356 th position and the 23245181 th position of the human chromosome 5 are fused (PRDM 9-type I, PRDM9-type II-1, PRDM9-type II-2), and the sequences which are not fused are respectively shown as SEQ ID NO: 16-18, and the fusion sequence is SEQ ID NO: 13 to 15, which are the most effective markers found in the present invention.

Wherein, the PRDM9-type I, PRDM9-type II-1 and PRDM9-type II-2 can be used for the congenital megacolon. Preferably, the PRDM9-type I site fusion level appears to be low in the congenital megacolon patient DNA relative to healthy controls and bowel disease controls. Preferably, the PRDM9-type II-1, PRDM9-type II-2 site fusion levels are present at high levels in the DNA of the congenital megacolon patients relative to healthy controls and intestinal disease controls.

The term "marker" or "biochemical marker" as used herein refers to a molecule to be used as a target for analyzing a patient test sample.

In some embodiments, the quantitative detection agent is used to perform any one of the following methods:

In some embodiments, the polymerase chain reaction is selected from the group consisting of restriction fragment length polymorphism, single strand conformation polymorphism, Taqman probe, qPCR, competitive allele-specific PCR, and allele-specific PCR.

In some embodiments, the biomass spectrometry is selected from flight mass spectrometer detection.

In some embodiments, the quantitative detection agent is a primer.

In some embodiments, the quantitative detection agent is used to quantitatively detect the presence of SEQ ID NO: 13-15, or a fragment thereof.

In some embodiments, the primer bears a detectable label.

The term "label" as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect and that can be attached to a nucleic acid or protein. Labels include, but are not limited to, dyes; radiolabels, e.g.³²P; binding moieties such as biotin; haptens such as digoxin; a luminescent, phosphorescent, or fluorescent moiety; and a fluorescent dye alone or in combination with a portion of the emission spectrum that can be suppressed or shifted by Fluorescence Resonance Energy Transfer (FRET). Labels can provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Labels can be charged moieties (positive or negative) or alternatively, can be charge neutral. The label may comprise or be combined with a nucleic acid or protein sequence, provided that the sequence comprising the label is detectable. In some embodiments, the nucleic acid is detected directly (e.g., direct sequence read) without a label.

In some embodiments, the label is a fluorophore, colorimetric label, quantum dot, biotin, and other label molecules that can be used for detection (e.g., alkyne groups for raman diffraction imaging, cyclic olefins for click reactions, priming groups for polymer labeling), and can also be selected from polypeptide/protein molecules, LNA/PNA, unnatural amino acids and their analogs (e.g., peptidomimetics), unnatural nucleic acids and their analogs (nucleomimetics), and nanostructures (including inorganic nanoparticles, NV-centers, aggregation/assembly-induced emission molecules, rare earth ion ligand molecules, polyoxometalate, etc.).

In some embodiments, the primers include at least one of a-c:

a. SEQ ID NO: 1-2;

b. SEQ ID NO: 3-4; and

c. SEQ ID NO: 5 to 6.

In some embodiments, the primers further comprise an internal reference primer for detecting a wild-type sequence corresponding to at least one of PRDM9-type I, PRDM9-type II-1, and PRDM9-type II-2.

In some embodiments, the reference primer is used to quantitatively detect the presence of SEQ ID NO: 16-18.

In some embodiments, the reference primer comprises at least one of d-f:

d. SEQ ID NO: 7-8;

e. SEQ ID NO: 9-10; and

f. SEQ ID NO: 11 to 12.

In some embodiments, the quantitative detection agent further comprises one or more of a DNA extraction reagent, dntps, a DNA polymerase, a double strand specific fluorescent dye, and water.

In some embodiments, the double-stranded specific fluorescent dye quantification is selected from any one of ethidium bromide, SYBR Green, PicoGreen, RiboGreen.

In some embodiments, the water is typically nucleic acid and/or nuclease-free water. The Water may be Distilled Water (Distilled Water), Deionized Water (Deionized Water), or Reverse osmosis Water (Reverse osmosis Water).

In some embodiments, the DNA polymerase is selected from any of Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase, Klenow fragment.

In some embodiments, the kit further comprises a sample treatment reagent; further, the sample processing reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a sample nucleic acid extraction reagent.

In some embodiments, the test sample of the reagent or kit is selected from at least one of a blood, tissue, cell sample.

Among these, preferred test samples are blood, and more preferred are those derived from peripheral blood.

The invention also relates to a primer as defined above.

The invention also relates to a kit for the quantitative detection of an agent as defined above.

According to a further aspect of the present invention, it also relates to a diagnostic method for the colons congenital jue, which comprises quantitatively measuring the fusion site of said PRDM9 transposon fusion in a test sample using a quantitative detection agent as defined above.

Generally, the detection of the fusion site of the PRDM9 transposon fusion was concluded in comparison to a control group (e.g., a healthy human). The increase or decrease is usually significant, and determining whether the subject is significantly different from the initial state of the healthy population (baseline) can be performed using statistical methods well known in the art and confirmed using confidence intervals and/or p-values. In some embodiments, the confidence interval may be 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99% and the p value may be 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

Embodiments of the present invention will be described in detail with reference to examples.

The test results of the invention are all analyzed by statistics,tthe test was used to assess the difference between the two groups, machine learning to assess the difference in transposon fusions in clinical parameters such as gender, age, disease phenotype, etc., and the statistical method was altmann. p is a radical of<0.05 was used to indicate statistical significance and all p-values were tested using a two-sided test. Statistical analysis was performed using R and Graphpad 8.0 software.

The "transposon" of the present invention refers to a DNA sequence which can be independently copied or broken from the original position, inserted into another site after circularization, and has a regulatory effect on the subsequent gene.

The congenital megacolon disease is one of common congenital intestinal diseases of children, namely, the congenital megacolon disease is clinically divided into a short segment type, a common type, a long segment type and a full colon type according to increasing severity because the colon is lack of ganglion cells to cause continuous spasm of an intestinal canal, excrement is stagnated in the proximal colon, and the proximal colon is thickened and expanded. Short-segment lesions are positioned at the near and middle segments of the rectum and are not more than 6.5cm away from the anal canal; the common lesions were located at the proximal rectum end or distal end of the rectosigmoid colon, about 9cm from the anal canal; long segment lesions extend to the sigmoid or descending colon; the colon-wide lesion reaches the whole colon and the tail end of the ileum within 30cm from the ileocecal valve.

The "ROC curve" of the present invention is a curve of 1-specificity (false positive rate) and sensitivity (true positive rate) changes, reflecting the diagnostic capabilities of the classifiers. A good classifier has a ratio of true positive rate to false positive rate of greater than 1, away from the 45 degree line.

The "AUC" refers to the area under the ROC curve, is between 0.1 and 1, and is used for evaluating the quality of the classifier, and the closer to 1, the better the classifier is.

Example 1 screening procedure for genetic marker fusion by Hirschsprung's transposon

The invention proves that PRDM9 transposon fusion can be used for early diagnosis and screening of Hirschsprung's disease, fills the blank of Hirschsprung's disease in the field of early diagnosis, and the following is a specific embodiment of the invention.

The screening procedure of the fusion genetic marker of the congenital megacolon transposon used in this example is shown in FIG. 1. The specific steps comprise taking tissue samples of the congenital megacolon operation, selecting paired tissue samples without nerve segments and with nerve segments to carry out whole transcriptome sequencing, detecting transposon and genome chimeric sequences, searching for reads of chimeric paired-end, wherein half of the reads are transposon sequences, determining the insertion positions of the transposons, statistically analyzing the total reads number of the chimeric transcripts of each insertion site in the segments with ganglia and without nerve segments, normalizing by using the total sequencing number, and carrying out differential expression analysis by using limma R, thereby analyzing the transposon fusion sites with obvious changes. The found transposon fusion sites were divided into two types of no nerve segment down-regulation (type I) and up-regulation (type II), and the genes in the sample were divided into three types including those carrying I and II transposons (gene I, II) and those not carrying transposons (gene N) according to the type of transposon fusion sites carried by the genes in the sample. Meanwhile, disease phenotypes are collected, which are closely related to diseases and comprise disease subtypes (according to the increasing severity degree: short segment type, common type, long segment type and whole colon type), enteritis, virus infection, sex and age. And analyzing the relationship between the phenotype of the disease and the transposon type of the gene by using a machine learning method to predict a possible driving gene. The transposon fusion genes obtained from the screening were verified in the megacolon cohort by qPCR.

Example 2 blood DNA and tissue sample Collection and grouping

Megacolon cohort: the blood DNA samples were divided into the Hirschsprung's group (32 cases), the other enteropathy control group (16 cases), the healthy children group (27 cases), age ranged from 3 months to 3 years, sex men 3/4 were males, and age and sex matched for the disease and control groups. All samples were from the Guangzhou city women's Children medical center, and the healthy children group had blood samples left after physical examination. The blood collection mode is anticoagulation blood collection, and the DNA is extracted by centrifugally separating leucocytes. The colon tissue samples were diseased tissues surgically removed from 52 children with colons of congenital jugonella, including nerve segments and nerve-free segments.

Example 3 sequencing of Whole Hirschmannin transcriptome and analysis of differentially expressed transposon

In 52 cases of the congenital megacolon patients, paired tissue samples with nerve segments and without nerve segments were taken as surgical tissue samples, total RNA was extracted by RNAeasy Kit (Qiagen), and transcriptome sequencing was performed by constructing Library with illumina TruSeq RNA Library Prep Kit v2 to obtain sequencing reads of 6GB paired-end per sample.

The sequenced fastq files were deblocked with trimmatic-0.36, short and low quality sequences were removed, STAR was aligned to the human genome (hg19), HTSeq-count was used to calculate gene expression, and edgeR calculated log2 (cpm). Gene expression was normalized by voom, differentially expressed genes were calculated using limma R package, and multiple comparisons were made using FDR. Genes with significant differential expression were defined as P-value <0.05 and log2FC > 1. Gene expression values were converted to Z-score and a heat map of the differentially expressed genes was drawn using a pheatmap. DAVID (version 6.8) was used for the signal pathway enrichment analysis of differentially expressed genes, with the parameter set to EASE value <0.1 as cut-off.

And (3) analyzing transposon fusion sites, detecting the chimeric sequences by using the method as above, searching for the reads of the chimeric paired-end, wherein half of the reads are the reads of the transposon sequences, determining the insertion positions of the transposons, statistically analyzing the total reads of the chimeric transcripts of each insertion site in segments with ganglia and without nerves, normalizing by using the total sequencing number, and performing differential expression analysis by using limma R to obtain the differential transposon fusion sites. These differential transposon fusion sites fall into two classes, one down-regulated (type I) and one up-regulated (type II) at the nervousless segments (representing segments).

FIG. 2 shows transposon fusion sites differentially expressed in the Hirschmannica. There are significant changes in the expression of transposon fusion sites in the congenital megacolon, and these differential transposon fusion sites fall into two categories, one down-regulated (type I) in nerve-free segments and one up-regulated (type II).

Example 4 machine learning demonstrates that PRDM9 transposon fusion is closely related to the Hirschsprung's segment type

Found transposon fusion sites without nerve segment down-regulation (type I) and up-regulation (type II) are associated with the most adjacent genes, and the genes in the sample are classified into three types according to the type of transposon fusion sites carried by the most adjacent genes in the sample, including those carrying type I and type II transposon fusion sites (gene I, II), and those not carrying transposon fusion sites (gene N). Meanwhile, disease phenotypes are collected, which are closely related to diseases and comprise disease subtypes (according to the increasing severity degree: short segment type, common type, long segment type and whole colon type), enteritis, virus infection, sex and age. Phenotypic feature selection (feature selection) was performed by a random forest method, and transposon fusion genes associated with disease phenotypes were analyzed to predict possible driver genes. The transposon fusion gene which is most obviously related to the congenital megacolon subtype is selected by using a machine learning method.

The results are shown in fig. 3, which shows that PRDM9 transposon fusions, closely related to the grand congenital colon segment type (p = 0.001), with the p-value ranking first and the importance (importance) ranking first among all transposon fused genes. Specifically, PRDM9-type I fusion is more in short segment type; whereas PRDM9-type II fusion is associated with long segment types. These results suggest that transposon fusion of PRDM9 is a marker closely related to the severity of the colon congenital.

Example 5 Dual fluorescent reporter Gene detection PRDM9 transposon fusion occurs in the transcriptional regulatory region

The three transposon fusion positions of PRDM9 are chr 523299411, 23262356, 23245181, and the sequences and the comparison sequence are shown in Table I. The PRDM9 fusion transposon is located upstream of PRDM9 in a non-coding region and presumably affects the transcription of PRDM 9. The influence of transposon insertion on the expression of PRDM9 is determined by placing sequences fused by PRDM9 and three transposons (E1 + TE, E2+ TE, E3+ TE), and (E1 + TE + C1, E2+ TE + C2, E3+ TE + C3), two control sequences (E1 + C1, E2+ C2, E3+ C3) and (C1, C2, C3) into the nano luciferase reporter gene PNL3.1, using firefly luciferase as a control, and using transient transformation 293T and SKNSH cell lines to detect the intensity of the reporter gene by two channels. The results showed that PRDM9-type I fusion further activated the expression of the PRDM9 enhancer, and PRDM9-type II-1, PRDM9-type II-2 fusion inhibited the expression of the PRDM9 enhancer (see FIG. 4).

Example 6 quantitative PCR of PRDM9 transposon fusion

To verify the diagnostic effect of PRDM9 transposon fusion on the colons congenital, we collected blood cell DNA from colons patients (32 cases), other bowel disease controls (16 cases including anal stenosis and intestinal stenosis fistulizing children's blood DNA samples), healthy children controls (27 cases), and used quantitative PCR to quantify the level of PRDM9 transposon fusion in blood DNA. 2 pairs of primers were designed for each fusion site to detect DNA levels of wild type (wt) and fused type, respectively, where wild type was used as an internal reference and the primer sequences are shown in Table two below. The results showed that PRDM9-type I transposon fusion was significantly lower in the blood DNA of megacolon patients than in the healthy control group and the bowel disease control; while PRDM9-type II-2 was significantly higher in the megacolon than the healthy and bowel disease controls, PRDM9-type II-2 was also higher than the healthy controls, but also higher in the bowel disease control (fig. 5).

Table one: PRDM9 transposon fusion site sequence

Table two: primer design for PRDM9 transposon fusion site

Example 7 ROC Curve evaluation of the diagnostic Effect of PRDM9 transposon fusion on Hirschsprung's disease

The ROC curve was used to evaluate the diagnostic effect of PRDM9 transposon fusion on the colons hirsutus (fig. 5). PRDM9-type I AUC value is 0.7885, PRDM9-type II-1 is 0.8503, and PRDM9-type II-2 is 0.7791. The AUC of the 3 combined assays was 0.9666, with the best limit corresponding to a specificity of 95.35% and a sensitivity of 84.38%. When the method is used for disease screening, the specificity is 76.74 when the sensitivity is 100%. Thus, PRDM9 transposon fusion can effectively diagnose and screen the congenital megacolon and shows good effect.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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<213> artificial sequence

<400> 17

ttcaaaaaca caaaatattc tggggggatt gggttttatc ccggtttaaa gctgaaacca 60

ctttaaggca tgttgtggca atacagtgat ggcagggtgg ttctggaaac tgttaaatct 120

acagtgttca gctaacaaaa agaatgtttt actttttttt tttttttttt tttttttttt 180

tttgagacag 190

<210> 18

<211> 187

<212> DNA

<213> artificial sequence

<400> 18

tattcttttt tttttttttt tttttttttt ttttacaaat acagtgtttc ctcttccaat 60

tttgagaaga tttcttattt ttaaaggttt gtctcaaagt atatacatgc atatttttca 120

tttaaatcat ttttcctttt cttagctttg aaatatgtct ttcattatat attttcctga 180

aatgcct 187

Claims

The application of PRDM9 transposon fusion quantitative detection agent in the preparation of a diagnostic reagent or a kit for the congenital giant colon;

the fusion site of the PRDM9 transposon fusion is selected from: at least one of PRDM9-type I, PRDM9-type II-1 and PRDM9-type II-2; wherein, the corresponding sequences of the PRDM9-type I, PRDM9-type II-1 and PRDM9-type II-2 which are not fused are respectively shown as SEQ ID NO: 16-18, and the corresponding sequences after fusion are respectively shown as SEQ ID NO: 13-15;

the quantitative detection agent is used for detecting the expression level of PRDM9 transposon fusion.
2. The use of claim 1, wherein the fusion site of the PRDM9 transposon fusion is a combination of PRDM9-type I, PRDM9-type II-1 and PRDM9-type II-2.
3. The use according to any one of claims 1 to 2, wherein the quantitative detection agent is used to perform any one of the following methods:

polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method.
4. The use of claim 1, wherein the quantitative detection agent is a primer.
5. The use of claim 4, wherein the primer comprises at least one of a-c:

a. SEQ ID NO: 1-2;

b. SEQ ID NO: 3-4; and

c. SEQ ID NO: 5 to 6.
6. The use of claim 5, wherein the primers further comprise an internal reference primer for detecting a wild-type sequence corresponding to at least one of PRDM9-type I, PRDM9-type II-1, and PRDM9-type II-2.
7. The use of claim 6, wherein the reference primer comprises at least one of d-f:

d. SEQ ID NO: 7-8;

e. SEQ ID NO: 9-10; and

f. SEQ ID NO: 11 to 12.
8. The use of any one of claims 4 to 7, wherein the quantitative detection reagent further comprises one or more of a DNA extraction reagent, dNTPs, DNA polymerase, a double-strand specific fluorescent dye, and water.
9. The use according to any one of claims 1 to 2 and 4 to 7, wherein the test sample of the reagent or kit is at least one selected from the group consisting of blood, tissue and cell sample.
10. The diagnostic primer of the Hirschmannica is characterized in that the sequence of the primer comprises at least one of the following a-c:

a. SEQ ID NO: 1-2;

b. SEQ ID NO: 3-4; and

c. SEQ ID NO: 5 to 6.
11. The diagnostic primer for the congenital megacolon as claimed in claim 10, wherein said primer further comprises an internal reference primer for detecting a wild-type sequence corresponding to at least one of PRDM9-type I, PRDM9-type II-1 and PRDM9-type II-2.
12. The diagnostic primer for the congenital megacolon according to claim 11, wherein the sequence of the internal reference primer comprises at least one of the following d-f:

d. SEQ ID NO: 7-8;

e. SEQ ID NO: 9-10; and

f. SEQ ID NO: 11 to 12.
13. The kit for diagnosing the Hirschsprung's disease is characterized by comprising primers, wherein the sequences of the primers comprise at least one of the following a-c:

a. SEQ ID NO: 1-2;

b. SEQ ID NO: 3-4; and

c. SEQ ID NO: 5 to 6.
14. The kit for diagnosing the congenital megacolon according to claim 13, wherein said primers further comprise an internal reference primer for detecting a wild-type sequence corresponding to at least one of PRDM9-type I, PRDM9-type II-1 and PRDM9-type II-2.
15. The kit for diagnosing the congenital megacolon according to claim 14, wherein the sequence of the internal reference primer comprises at least one of the following d-f:

d. SEQ ID NO: 7-8;

e. SEQ ID NO: 9-10; and

f. SEQ ID NO: 11 to 12.
16. The kit for diagnosing the congenital megacolon according to any one of claims 13 to 15, further comprising one or more of a DNA extraction reagent, dntps, a DNA polymerase, a double strand-specific fluorescent dye, and water.