阅读说明：本技术 一种磺胺二甲嘧啶核酸适配体筛选方法、试剂盒及应用 (Sulfamethazine aptamer screening method, kit and application ) 是由 乐涛 寇启明 孙琦 吴平 于 2020-05-13 设计创作，主要内容包括：发明提供了一种磺胺二甲嘧啶核酸适配体的筛选方法及其检测试剂盒,其中,试剂盒包括基于氧化石墨烯的荧光适配体传感器；其中,基于氧化石墨烯的荧光适配体传感器包括FAM荧光基团标记的适配体和氧化石墨烯；所述FAM荧光基团标记的磺胺二甲嘧啶核酸适配体序列为核苷酸序列表记载的No 1s。磺胺二甲嘧啶荧光检测试剂盒在检测牛奶或鸡蛋的测试样品中磺胺二甲嘧啶的定量检测的应用。(The invention provides a screening method of sulfadimidine aptamer and a detection kit thereof, wherein the kit comprises a graphene oxide-based fluorescent aptamer sensor; the graphene oxide-based fluorescent aptamer sensor comprises an FAM fluorophore labeled aptamer and graphene oxide; the sequence of the sulfamethazine aptamer marked by the FAM fluorescent group is No1s recorded in a nucleotide sequence table. Application of the sulfadimidine fluorescence detection kit in quantitative detection of sulfadimidine in a test sample for detecting milk or eggs.)

1. A screening method of a sulfadimidine aptamer is characterized in that: the method comprises the following steps:

1) screening a sulfadimidine aptamer by adopting a non-immobilized GO-SELEX technology;

2) an aptamer core recognition region is obtained through sequence optimization, and is cloned and synthesized in vitro to prepare the aptamer with high affinity and specificity to the sulfadimidine.

2. The sulfadimidine fluorescence detection kit comprises a graphene oxide-based fluorescence aptamer sensor; the method is characterized in that: the graphene oxide-based fluorescent aptamer sensor comprises an FAM fluorophore labeled aptamer and graphene oxide; the sequence of the sulfamethazine aptamer marked by the FAM fluorescent group is one of No.1, No.2, No.3, No.4, No. 5, No.6, No1s or No 5s described in a nucleotide sequence table.

3. The sulfadimidine fluorescence detection kit of claim 2, characterized in that: the sequence of the sulfamethazine aptamer marked by the FAM fluorescent group is one of No.1, No 5, No1s or No 5s recorded in a nucleotide sequence table.

4. The sulfadimidine fluorescence detection kit of claim 3, characterized in that: the sequence of the sulfamethazine aptamer marked by the FAM fluorescent group is one of No1s or No 5s recorded in a nucleotide sequence table.

5. The sulfadimidine fluorescence detection kit of claim 4, characterized in that: the sequence of the sulfamethazine aptamer marked by the FAM fluorescent group is No1s recorded in a nucleotide sequence table.

6. The sulfadimidine fluorescence detection kit of claim 2, characterized in that: the fluorescent aptamer sensor based on graphene oxide is constructed on the basis of the aptamer with high affinity and specificity of sulfadimidine obtained by the method of claim 1.

7. Application of a sulfadimidine fluorescence detection kit in quantitative detection of sulfadimidine in a milk test sample.

8. Application of a sulfadimidine fluorescence detection kit in quantitative detection of sulfadimidine in an egg tissue test sample.

Technical Field

The invention relates to the field of biotechnology and food antibiotic residue detection. More specifically relates to a screening method, a kit and application of a sulfadimidine aptamer.

Background

Sulfamethazine, chemically known as 2- (p-aminophenylsulfonamide) -4, 6-dimethylpyrimidine, is a common sulfonamide drug, is a broad-spectrum antibacterial agent, can be used for clinical treatment, can also be used as a veterinary drug and a feed additive, is widely applied to feeding of food-borne animals, and can be transferred to animal-borne foods such as meat, eggs, milk and the like after entering the animal body through various administration routes. If the food is improperly used, residues can be easily generated in animal tissues and animal-derived foods and enter human bodies through food chains, so that the human health is harmed. And is discharged into the external environment through animal dung, urine and other ways, thus causing ecological toxicological pollution. In order to ensure the safety of animal-derived foods, the Maximum Residual Limit (MRL) of sulfonamides such as sulfadimidine in animal-derived foods is regulated by governments of various countries. The total amount of the sulfonamides in food and feed is regulated by the international food code commission (CAC), the World Health Organization (WHO) and the FDA (food and drug administration) in the United states to be not more than 0.1mg/kg, the maximum residual limit of the sulfonamides in animal-derived food in China is 100ng/mL, and the total amount of various sulfonamides is not more than 100 ng/mL. Therefore, a rapid, efficient and accurate sulfadimidine detection method is established, and the method has very important significance for ensuring the dietary health of human beings and reducing ecological pollution. At present, methods such as high performance liquid chromatography, chromatography-mass spectrometry combined technology, enzyme-linked immunosorbent assay and the like are mostly adopted for detecting the sulfadimidine drug, and although the methods have high selectivity and sensitivity, the use of the chromatography requires relatively expensive analytical instruments and professional technicians, and particularly has the defects of complicated sample pretreatment process, low extraction efficiency of low-content target substances, high sample detection cost, matrix interference, difficulty in realizing rapid detection and the like. The enzyme-linked immunosorbent assay needs to prepare antibodies, while the preparation of drug antibodies needs to synthesize complete antigens and immunize animals, the experimental period is long, and the biological activity is easily inactivated due to the influence of various factors. In recent years, the biosensor method for detecting the drugs is more and more concerned by researchers due to the advantages of simple operation, high detection speed, low price and the like. The biosensor constructed based on the aptamer has the advantages of high detection sensitivity, simplicity in operation and strong selectivity, and has a good application prospect in antibiotic drug detection. According to the invention, the sulfadimidine aptamer is screened and sequence optimized based on the graphene oxide, the fluorescent aptamer sensor based on the graphene oxide is constructed, the fluorescent aptamer sensor is successfully detected in actual samples of milk and eggs, the linear relation is good, and a foundation is laid for the detection and development of sulfadimidine products.

The aptamer is a single-stranded DNA or RNA fragment which is screened out in vitro by Exponential Enrichment of ligand by expression evolution (SELEX) and can be combined with various target molecules with high affinity and high specificity, can form a secondary or tertiary structure through self folding to ensure that the aptamer has strong affinity to specific targets (such as metal ions, small molecules, proteins, viruses, cells and the like), and is used as an element for molecular recognition, thereby being widely applied to medical clinical diagnosis and treatment.

At present, the report of applying the aptamer as a probe to the detection of the antibiotic detection residue in food is available, the detection method mainly comprises an immunoassay method and an electrochemical biosensor, and the detection of the sulfadimidine residue in food and environment must be enhanced in order to ensure the quality of animal-derived food and guarantee the food safety of human beings.

The invention content is as follows:

according to the invention, through a screening process of a non-fixed GO-SELEX technology, a nucleic acid aptamer of sulfadimidine is obtained, an aptamer core recognition region is obtained through sequence optimization, and is cloned and synthesized in vitro, the prepared nucleic acid aptamer has high affinity and specificity to sulfadimidine, a graphene oxide-based fluorescent aptamer sensor is constructed, and when sulfadimidine appears in a system, the nucleic acid aptamer is combined to the sulfadimidine through strong affinity and is not adsorbed on the surface of the graphene oxide through pi-pi accumulation. Due to the difference of sulfadimidine concentration, the combined fluorophore-labeled aptamer generates different fluorescence signals. In contrast, in the absence of sulfadimidine, graphene oxide can adsorb fluorophore-labeled aptamers through pi-pi stacking interactions, quenching the fluorescent signal. By utilizing the principle, the quantitative detection of the sulfadimidine is realized. The specific invention content is as follows:

the sulfadimidine fluorescence detection kit comprises a graphene oxide-based fluorescence aptamer sensor; the graphene oxide-based fluorescent aptamer sensor comprises an FAM fluorophore labeled aptamer and graphene oxide; the nucleic acid adapter sequence of the sulfadimidine marked by the FAM fluorescent group is one of No.1, No.2, No.3, No.4, No. 5, No.6, No1s or No 5s recorded in a nucleotide sequence table. Aptamer derivatives obtained by modifying and modifying ssDNA, and the like, also belong to the protection scope of the invention.

The sulfamethazine nucleic acid adaptor sequence marked by the FAM fluorescent group is one of No1s or No 5s recorded in a nucleotide sequence table.

The sulfamethazine nucleic acid adaptor sequence marked by the FAM fluorescent group is No1s recorded in a nucleotide sequence table.

1) Screening a sulfadimidine aptamer by adopting a non-immobilized GO-SELEX technology;

2) obtaining an aptamer core recognition region through sequence optimization, and cloning and synthesizing in vitro to prepare the aptamer with high affinity and specificity to the sulfadimidine;

a method for screening a sulfadimidine aptamer, comprising the steps of:

1) screening a sulfadimidine aptamer by adopting a non-immobilized GO-SELEX technology;

2) an aptamer core recognition region is obtained through sequence optimization, and is cloned and synthesized in vitro to prepare the aptamer with high affinity and specificity to the sulfadimidine.

The construction method of the graphene oxide-based fluorescence aptamer sensor comprises the following steps: the prepared aptamer with high affinity and specificity to sulfadimidine is used for constructing the graphene oxide-based fluorescent aptamer sensor.

Application of a sulfadimidine fluorescence detection kit in quantitative detection of sulfadimidine in a milk test sample.

Application of a sulfadimidine fluorescence detection kit in qualitative detection of sulfadimidine in an egg tissue test sample.

The beneficial technical effects of the invention are as follows: aiming at the defects that the traditional large instrument can not realize on-site rapid detection and is complex to operate, and the immunoassay method needs to prepare antibodies through experimental animals and has long periodicity, the invention establishes the graphene oxide aptamer sensor-based fluorescence detection method, can be used for rapid and high-sensitivity quantitative detection of sulfadimidine residues in food, and overcomes the defects of the detection method. Provides a new method for detecting antibiotics. The method has the advantages of simple detection operation, non-fixed target, short screening rounds and the like, and lays a foundation for food safety detection and product development.

Drawings

FIG. 1: (ii) aptamer affinity assay;

FIG. 2: specific analysis of fluorescent aptamer sensors;

FIG. 3: fluorescence calibration curves plotted at emission wavelength 520nm using different concentrations of standards.

Detailed Description