阅读说明：本技术 检测糖尿病肾病易感性的试剂盒及其使用方法 (Kit for detecting susceptibility to diabetic nephropathy and using method thereof ) 是由 吴慧娟 王红羽 黄洁波 蔡小凡 张志刚 许之珩 于 2020-08-14 设计创作，主要内容包括：一种检测糖尿病肾病易感性的试剂盒及其使用方法,其中,试剂盒包括如SEQ ID NO：1至SEQ ID NO：4所示的引物,可特异性扩增样本中ELMO1基因的SNP rs741301位点；试剂盒使用方法,包括步骤(1)提供样本；(2)提供如SEQ ID NO：1至SEQ ID NO：4所示的引物；(3)通过PCR,使用引物特异性扩增样本中的ELMO1基因的SNP rs741301位点,取得扩增产物；以及(4)根据扩增产物解读样本的糖尿病肾病易感性基因型。(A kit for detecting susceptibility to diabetic nephropathy and a use method thereof, wherein the kit comprises the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4, the primer can specifically amplify the SNP rs741301 site of the ELMO1 gene in the sample; a method of using a kit comprising the steps of (1) providing a sample; (2) providing a polypeptide as set forth in SEQ ID NO: 1 to SEQ ID NO: 4, or a primer thereof; (3) specifically amplifying SNP rs741301 locus of ELMO1 gene in a sample by PCR (polymerase chain reaction) by using primers to obtain an amplification product; and (4) interpreting the diabetic nephropathy susceptibility genotype of the sample according to the amplification product.)

1. The kit for detecting the susceptibility of the diabetic nephropathy is characterized by comprising primers designed aiming at the Single Nucleotide Polymorphism (SNP) rs741301 site of phagocyte motor protein-1 (ELMO1) gene, wherein the sequences of the primers are respectively shown as SEQ ID NO: 1 to SEQ ID NO: 4, wherein the sequence of the primer-specific amplification product comprises a base complementary to the rs741301 site.

2. The kit of claim 1, wherein the amino acid sequence set forth in SEQ ID NO: 2 and SEQ ID NO: 4, the sequence length of the amplified product is 187 bp.

3. The kit of claim 1, wherein the amino acid sequence set forth in SEQ ID NO: 2 and SEQ ID NO: 4, the base of the amplification product corresponding to the rs741301 locus is Guanine (Guanine).

4. The kit of claim 1, wherein the amino acid sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 3, the sequence length of the amplification product of the primer is 273 bp.

5. The kit of claim 1, wherein the amino acid sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 3, the base of the amplification product corresponding to the rs741301 locus is Adenine (Adenine).

6. The use method of the kit for detecting the susceptibility to the diabetic nephropathy is characterized by comprising the following steps:

(1) providing a sample;

(2) providing a polypeptide as set forth in SEQ ID NO: 1 to SEQ ID NO: 4, or a primer thereof;

(3) specifically amplifying a Single Nucleotide Polymorphism (SNP) rs741301 site of a phagocyte motor protein-1 (ELMO1) gene in the sample by using the primer through Polymerase Chain Reaction (PCR) to obtain an amplification product; and

(4) interpreting the sample for susceptibility genotype to diabetic nephropathy based on the amplification product.

7. The method of use of the kit of claim 6, wherein the amino acid sequence set forth in SEQ ID NO: 2 and SEQ ID NO: 4, the sequence length of the amplified product is 187 bp.

8. The method of use of the kit of claim 6, wherein the amino acid sequence set forth in SEQ ID NO: 2 and SEQ ID NO: 4, the base of the amplification product corresponding to the rs741301 locus is Guanine (Guanine).

9. The method of use of the kit of claim 6, wherein the amino acid sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 3, the sequence length of the amplification product of the primer is 273 bp.

10. The method of use of the kit of claim 6, wherein the amino acid sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 3, the base of the amplification product corresponding to the rs741301 locus is Adenine (Adenine).

Technical Field

The invention relates to the fields of molecular biology and medicine, in particular to a kit for evaluating the relevance of a gene and diabetic nephropathy by using Single Nucleotide Polymorphism (SNP) and a using method thereof.

Background

Diabetes Mellitus (DM) is a common chronic metabolic disease worldwide, and the long-term hyperglycemia of a patient easily causes irreversible chronic damage to substantive organs and tissues such as heart, brain, kidney, blood vessel wall and the like, wherein Diabetic Nephropathy (DN) is one of the most serious complications of Diabetes Mellitus. Literature studies have shown that diabetic nephropathy has become a leading cause of death in patients with Type 1Diabetes Mellitus (T1 DM); mortality is also second only to macrovascular complications in patients with Type 2Diabetes mellitis, T2 DM. In the united states, 40% of End Stage Renal Disease (ESRD) develops from diabetic nephropathy; in china, this proportion also accounts for 15-22% and is still rising, placing a heavy economic burden on the families and society.

In addition, diabetic nephropathy often begins more slowly, while nephropathy due to type 2diabetes begins more insidious, and when the patient visits with microalbuminuria in the early stages, it has already entered stage 3 (GFR decline) of the clinical stage of diabetic nephropathy, and may have progressed to an irreversible stage. Therefore, it is very important for the early prevention and diagnosis of diabetic nephropathy.

Diabetes is a typical metabolic disease, the occurrence and development of diabetic nephropathy and the living eating habits of individuals are inseparable, so that the control of intake and the maintenance of normal blood sugar, blood fat and blood pressure levels are of great significance for delaying the development of the diabetic nephropathy. In addition to extrinsic environmental factors, diabetic nephropathy is also genetically susceptible, i.e., because genetic differences lead to an increased risk of developing diabetic nephropathy in patients with specific genes. In recent years, there are many documents and investigations reporting that research is conducted on genetic susceptibility to diabetic nephropathy, and it has been found that phagocytosis has a certain correlation with the onset of diabetic nephropathy caused by Cell Motility gene-1 (ELMO1) and type 2 diabetes. The importance of the ELMO1 gene in the onset of diabetic nephropathy is confirmed by a plurality of foreign genetic character surveys of Japanese, American African and Egyptian populations and a few Chinese genetic character surveys, and the genetic character surveys have a sufficient theoretical basis and clinical evidence support. Therefore, the ELMO1 gene is used for genetic evaluation of diabetic nephropathy, and the aim of early prevention can be achieved.

More specifically, the ELMO1 gene study of Wu et al (J.Endocrinol. invest.36:298-302,2013) on Han nationality indicates that the SNP located in ELMO1 gene is important and critical for influencing the susceptibility of the subject to diabetic nephropathy, but Wu et al only conclude that the allele A at the rs741301 locus may have a predictive effect on diabetic nephropathy, and do not specifically understand the specific influence of the allele; hou et al (diabetes. Metab. Syndr.11:97,2019) indicated that rs741301 is an important SNP site of ELMO1, but Hou et al only concluded that allele G may be associated with diabetic nephropathy, and did not understand the specific effects of the allele in detail. It can be seen that the detection of SNP against the ELMO1 gene to assess susceptibility to diabetic nephropathy is a reliable direction, but lacks detailed understanding of rs741301 allele and is difficult to be used as a reliable detection index.

In addition, for SNP detection, the currently common methods include sequencing, Polymerase Chain Reaction (PCR), probe methods, etc., wherein reagents and instruments required by the methods related to sequencing are expensive and difficult to popularize and apply; the PCR amplification method applied in combination with RFLP (restriction fragment length polymorphism) usually requires a probe method, a DNA sequencing method and an enzyme digestion reaction to be matched for accurate detection; the probe method relies on hybridization (hybridization), and usually requires complex probe design and complex and expensive optical detection instrument to detect fluorescence accurately, which not only requires complex preparation steps and expensive detection cost, but also is not suitable for popularization and application.

More specifically, although the studies proposed by Wu et al and Hou et al investigated the relationship between the SNP characteristics of the ELMO1 gene and diabetic nephropathy, they lack rigorous screening for the study population and do not consider the characteristic of long latency of diabetic nephropathy, and thus patients who have just suffered from diabetes may not have the kidney affected yet, and are not as stringent as a control group, and should be excluded because they are very influential to the science of the study. Furthermore, the Wu et al study used mass spectrometry (MassArray) to analyze SNPs, which is economically expensive; hou et al, analyze SNP by using PCR with restriction enzyme fragment diversity (RFLP), specifically, using PCR to amplify (copy) a sequence fragment containing SNP, then using specific restriction enzyme to enzyme-cut the fragment of PCR amplification product, and then determining the genotype of SNP site according to the enzyme-cut map.

The above requirements indicate that the current common SNP detection method is obviously limited in application of clinical detection tools, so that it is a demand to be solved in the field obviously that the establishment of a diabetic nephropathy susceptibility SNP detection method is simple and convenient, has a simple instrument, high efficiency, low cost and is accurate.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention provides a kit for detecting susceptibility to diabetic nephropathy and a method for using the same, so as to solve the above-mentioned technical problems. The kit and the method for detecting the susceptibility of the diabetic nephropathy, provided by the invention, can accurately detect the susceptibility genotype of the diabetic nephropathy by using a well-designed primer to perform single-step PCR, have the characteristics of accuracy, rapidness, simplicity, convenience, economy and the like, are suitable for various clinical environments or health industries, are used as auxiliary tools for evaluating the susceptibility risk of the diabetic nephropathy, and can help diabetic patients to prevent the occurrence or development of the diabetic nephropathy in advance, and make disease management well, so that the life quality of the diabetic patients is improved.

In order to solve the technical problems, the technical scheme adopted by the invention is to provide a kit for detecting susceptibility to diabetic nephropathy, which comprises a primer designed aiming at the Single Nucleotide Polymorphism (SNP) rs741301 site of phagocyte motor protein-1 (ELMO1) gene, such as SEQ ID NO: 1 to SEQ ID NO: 4, specifically amplifying the rs741301 site of the SNP of the ELMO1 gene in the sample, so that the sequence of the amplification products comprises a base complementary to the rs741301 site.

In accordance with the above objects, the present invention provides a kit, comprising the nucleotide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 4, the sequence length of the amplified product is 187 bp.

In accordance with the above objects, the present invention provides a kit, comprising the nucleotide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 4, the base of the amplified product corresponding to the rs741301 site is Guanine (Guanine).

In accordance with the above objects, the present invention provides a kit, comprising the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3, the sequence length of the amplified product is 273 bp.

In accordance with the above objects, the present invention provides a kit, comprising the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3, the base of the amplified product corresponding to the rs741301 locus is Adenine (Adenine).

On the other hand, in order to solve the aforementioned technical problems, the technical scheme adopted by the invention is to provide a use method of a kit for detecting susceptibility to diabetic nephropathy, comprising the following steps: (1) providing a sample; (2) providing a polypeptide as set forth in SEQ ID NO: 1 to SEQ ID NO: 4, or a primer thereof; (3) specifically amplifying a Single Nucleotide Polymorphism (SNP) rs741301 site of a phagocyte motor protein-1 (ELMO1) gene in a sample by using a primer through a Polymerase Chain Reaction (PCR); and (4) interpreting the diabetic nephropathy susceptibility genotype of the sample according to the amplification product of step (3).

In accordance with the above objects, the present invention provides a kit for use in a method of using, the kit as set forth in SEQ ID NO: 2 and SEQ ID NO: 4, the sequence length of the amplified product is 187 bp.

In accordance with the above objects, the present invention provides a kit for use in a method of using, the kit as set forth in SEQ ID NO: 2 and SEQ ID NO: 4, the base of the amplified product corresponding to the rs741301 site is Guanine (Guanine).

In accordance with the above objects, the present invention provides a kit for use in a method of using, the kit as set forth in SEQ ID NO: 1 and SEQ ID NO: 3, the sequence length of the amplified product is 273 bp.

In accordance with the above objects, the present invention provides a kit for use in a method of using, the kit as set forth in SEQ ID NO: 1 and SEQ ID NO: 3, the base of the amplified product corresponding to the rs741301 locus is Adenine (Adenine).

Drawings

FIG. 1 is a schematic flow chart of the method for using the kit for detecting susceptibility to diabetic nephropathy of the present invention.

FIG. 2 shows an agarose gel electrophoresis of the amplification product of example 1 of the present invention.

Detailed Description

So that the manner in which the above recited features and advantages of the present invention can be understood and attained, a more particular description of the invention, briefly summarized above, may be had by reference to the appended drawings, in which, in order to facilitate understanding of the nature of the invention, features, and advantages thereof, may be had by reference to the appended claims, which are included to illustrate, by way of example, embodiments of the invention. The drawings referred to below are schematic representations, not necessarily drawn to scale, showing features of the invention. The description of the embodiments related to the present invention will not be repeated, except for those skilled in the art.

The invention provides a kit for detecting susceptibility to diabetic nephropathy, which comprises specially designed primers, wherein the sequences of four primers are respectively shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4, respectively. The aforementioned primers can be directed to the SNP site in phagocyte motor protein-1 (hereinafter referred to as ELMO1) gene by polymerase chain reaction (abbreviated as PCR): rs741301, detecting the exact base of the genotype, and evaluating the correlation of the onset of diabetic nephropathy by interpreting the exact genotype.

Because the research on rs741301 lacks detailed analysis at present, the invention firstly carries out rigorous case statistical analysis on rs741301 to refine the relevance between the rs741301 locus allele and diabetic nephropathy, thereby designing a reliable detection kit according to the relation.

With the approval of ethical committee of basic medical college of the university of Compound Dan, 88 diabetic nephropathy patients (case group) and 52 diabetic renal-free patients (control group) which totally meet the requirements were collected from Central Hospital of Putuo district, nephrology of Longhua hospital and endocrinology department of Shanghai. The clinical data collected for all cases and control patients were then collected for inclusion and exclusion and correlation analysis, including but not limited to the following: age, sex, clinical diagnosis, kidney wear diagnosis, blood pressure, respiratory rate, heart rate, diabetic age, blood glucose, postprandial blood glucose, glycated hemoglobin, renal function (uremic nitrogen [ BUN ] and creatinine [ SCR ]), 24h urine protein quantification and Urine Albumin Creatinine Ratio (UACR), presence or absence of hypertension complicated with diabetes, diabetic retinopathy, etc. In addition, whole blood was collected simultaneously for all the case group and control group patients collected and used for nucleic acid extraction for detection.

Clinical data and nucleic acid analysis show that, in terms of variation, allele A variation at the rs741301 locus is more common in diabetic nephropathy, while allele G variation is more common in diabetic nephropathy-free patients (P <0.05), which indicates that diabetic patients with allele A variation are more likely to develop diabetic nephropathy, and allele G has protective significance for development of diabetic nephropathy; as for the genotype, AA is more frequently found in diabetic nephropathy patients (P <0.05), and is in significant positive correlation with the increased risk of diabetic nephropathy, and also proves that the diabetic patients with A variation at the site have higher risk of developing diabetic nephropathy. The analysis result reflects the literature results published by Wu et al and Hou et al, and although the targeted SNP targets are the same and are the same at the rs741301 locus, the detailed analysis of the invention shows that the effects of alleles A and G at the rs741301 locus are obviously different, so that the detection kit provided by the invention obviously overcomes the relatively rough prediction effect defect of the previous literature, and is more accurate and practical in the detection application of predicting diabetic nephropathy.

The invention can accurately, effectively and quickly detect the rs741301 genotype (allele) only by using a single PCR, and is different from the prior PCR and RFLP combined application or detection method with similar defects, so the kit provided by the invention can further comprise reagents required by the PCR, and the selection of related reagents can be easily selected by a person skilled in the art according to requirements, and is not limited.

The sample to which the detection kit of the present invention is applied is mainly a human sample, and any sample containing an isolatable nucleic acid can be used in the kit of the present invention without particular limitation. Specifically, the following description will be applied to various aspects. According to the convenience of general medical practice, the preferred sample is human whole blood, which is easy to obtain in clinical detection work; on the other hand, due to the high sensitivity of PCR detection, the invention is suitable for conventional detection in general blood drawing and is more particularly suitable for trace samples (such as trace blood samples collected by acupuncture), so that the interference of the blood drawing on a testee can be reduced (such as blood drawing is changed into acupuncture blood drawing during blood drawing, and wounds are reduced), and the invention is particularly beneficial to diabetics. In addition to samples directly derived from human body, nucleic acids prepared by human beings are also suitable for use as samples in the kit of the present invention, such as nucleic acid sequences or cDNA copied from ELMO1 gene, and in principle, any nucleic acid sequence that may cover the genotype of rs741301 can be used as the sample of the present invention.

Specifically, in the kit provided by the invention, the nucleotide sequence shown as SEQ ID NO: 2 and SEQ ID NO: 4 are respectively a forward primer and a reverse primer corresponding to a specific sequence in the ELMO1 gene, and after PCR (specific amplification), an amplification product with the length of 187bp (base pair) is obtained, and the base corresponding to the rs741301 locus of the amplification product is Guanine (Guanine, hereinafter referred to as G). SEQ ID NO: 1 and SEQ ID NO: the primers shown in 3 are a forward primer and a reverse primer corresponding to another specific sequence in the ELMO1 gene respectively, and after PCR specific amplification, an amplification product with the length of 273bp is obtained, and the base corresponding to the rs741301 locus of the amplification product is Adenine (Adenine, hereinafter referred to as A).

According to the design principle of the above primer, the present invention further provides a method for using the kit for detecting susceptibility to diabetic nephropathy, please refer to fig. 1, which illustrates the following steps:

step S1 is to provide any type of sample, and the selection conditions are as described above, without any particular limitation. Step S2 is providing the sequence as set forth in SEQ ID NO: 1 to SEQ ID NO: 4, 4 primers in total. In step S3, four primers are used to specifically amplify the SNP rs741301 site of the ELMO1 gene in the sample by polymerase chain reaction (hereinafter referred to as PCR), and two amplification products are obtained. In step S4, the rs741301 site genotype of the sample is interpreted according to the status of the amplification product to determine whether it meets the susceptibility genotype of diabetic nephropathy, thereby evaluating the relationship between the genotype and the susceptibility of diabetic nephropathy.

The characteristics of the kit and the method of use according to the present invention are described below.