CN112611876B - Urine amino acid detection kit and method for aptamer-quadruplex - Google Patents
Urine amino acid detection kit and method for aptamer-quadruplex Download PDFInfo
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Abstract
The invention belongs to the field of medical detection, and particularly relates to a urine amino acid detection kit and method of a nucleic acid aptamer-quadruplex. The invention discloses a urine amino acid detection kit of a nucleic acid aptamer-quadruplex, which comprises a certain amino acid specific nucleic acid aptamer and a DNA G quadruplex sequence, and is characterized in that the certain amino acid specific nucleic acid aptamer is complementary with part of the DNA G quadruplex sequence, and forms a complementary binding sequence under the condition that no target amino acid exists, and the DNA G quadruplex sequence cannot form a G quadruplex. The method has simple operation and high analysis speed, can detect the concentration of the amino acid in the range of nm-mum in the reaction system, and is beneficial to clinical development and screening of gastric cancer high-incidence areas.
Description
Technical Field
The invention belongs to the field of medical detection, and particularly relates to a urine amino acid detection kit and method of a nucleic acid aptamer-quadruplex.
Background
Metabonomics is a new developed subject following genomics and proteomics, and is used for researching the occurrence and development of diseases, searching disease biomarkers, predicting disease prognosis and the like by accurately, qualitatively and quantitatively analyzing small molecule metabolites in human bodies, analyzing the relationship between the metabolites and physiological and pathological changes of the human bodies. Metabonomics will have a broad development prospect in clinical diagnosis, and the main application direction has four aspects: the application in clinical diagnosis, research application in etiology and pathology mechanism, application in clinical medication guidance and application in preclinical animal model screening.
Chinese patent (CN 201310556941.9) discloses that a group of raised small molecular amino acids can be used as markers for screening gastric cancer in urine of gastric cancer patients through a meteorological chromatography-mass spectrometry combined technology. The new discovery of urine small molecule biomarkers enables noninvasive gastric cancer screening and diagnosis. However, the amino acids found in the urine of gastric cancer patients at the early stage belong to small molecular substances, and high-grade precise instruments such as gas chromatography/mass spectrometry (GC/MS) analyzers are needed, and the instruments are not clinically and conventionally used instruments, so that the amino acids are difficult to clinically popularize and are more difficult to apply to the general survey of people in the high-incidence area of gastric cancer.
Disclosure of Invention
The invention aims to provide a kit and a method for detecting urine amino acid of a nucleic acid aptamer-quadruplex, so as to meet the requirement of noninvasive gastric cancer common screening.
The invention discloses a urine amino acid detection kit of a nucleic acid aptamer-quadruplex, which comprises a certain amino acid specific nucleic acid aptamer and a DNA G quadruplex sequence, and is characterized in that the certain amino acid specific nucleic acid aptamer is complementary with part of the DNA G quadruplex sequence, and forms a complementary binding strand under the condition that no target amino acid exists, and the DNA G quadruplex sequence cannot form a G quadruplex.
The technical principle of the invention is based on the fact that the binding capacity between the nucleic acid aptamer and target amino acid is larger than the interaction force between the nucleic acid aptamer and the complementary DNA G quadruplex sequence, and the nucleic acid aptamer sequence targeting certain amino acid is hybridized with the DNA G quadruplex sequence; then adding amino acid into the reaction system; at this time, the nucleic acid aptamer sequence specifically binds to the target amino acid, releasing the DNA G quadruplex sequence bound as described above; and adding heme molecules into the system, wherein heme and DNA G quadruplex sequences form DNAzyme with catalytic activity, and using the HRP activity of the DNAzyme, catalyzing the mixed solution reaction of hypersensitive chemiluminescence liquid A, B to generate a chemiluminescent signal or catalyzing the characteristic of DAB to generate brown chromogenic substances, wherein the formed chemiluminescent optical density or chromogenic intensity is in direct proportion to the content of target amino acid molecules (figure 1).
In a preferred embodiment, the amino acid is serine.
In a preferred embodiment, the nucleic acid sequence of the nucleic acid aptamer is represented by Seq ID No. 1.
In a preferred embodiment, the DNA G quadruplex sequence has the nucleic acid sequence shown in Seq ID No. 2.
In a preferred embodiment, the urine amino acid detection kit further comprises a hypersensitive chemiluminescence liquid or a DAB chromogenic liquid;
on the other hand, the invention also discloses a DNA G quadruplex sequence which is partially complementary and matched with the serine specific nucleic acid aptamer sequence, and the nucleic acid sequence of the DNA G quadruplex sequence is shown as the sequence of the Seq ID No. 2;
on the other hand, the invention also discloses the application of the DNA G quadruplex sequence in preparing a gastric cancer diagnosis reagent.
On the other hand, the invention also discloses a method for detecting single amino acid in urine, which comprises the following steps:
hybridizing an amino acid aptamer with a DNA G quadruplex sequence;
adding a urine sample to be detected;
adding a heme reagent;
adding a hypersensitive chemiluminescent solution or DAB chromogenic solution;
chemiluminescence or colorimetry is detected.
In a preferred embodiment, the amino acid is serine.
In a preferred embodiment, the nucleic acid sequence of the nucleic acid aptamer is represented by Seq ID No. 1.
In a preferred embodiment, the DNA G quadruplex sequence has the nucleic acid sequence shown in Seq ID No. 2.
The method has simple operation and high analysis speed, can detect the concentration of the amino acid in the range of nm-mum in the reaction system, and is beneficial to clinical development and screening of gastric cancer high-incidence areas.
Drawings
FIG. 1 is a schematic diagram of the basic principle of the present invention;
FIG. 2 is a regression curve of serine standard test results;
FIG. 3 is a graph showing the results of a clinical sample tested using the method of the present invention;
FIG. 4 is a chromatogram of the detection result of an amino acid analyzer of a urine sample from a gastric cancer patient (positive cases are shown by the detection method of the present invention);
FIG. 5 is a chromatogram of the results of amino acid analyzer detection of urine samples from healthy subjects (negative cases were shown by the detection method of the present invention).
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
1. Instrument and reagent
QT-1 vortex meter (Shanghai QiTex analysis instruments Co., ltd.)
Pure water device (MILLI-XQ, merry biotechnology Co., ltd., china)
Micro-shaking table 2211k (Tai cang city science and education equipment factory)
Full wavelength multifunctional enzyme-labeled instrument Safire 2 (Tecan, switzerland)
Aptamer sequences (Shanghai Biotechnology Co., ltd., purified by PAGE)
Taking serine aptamer as an example (Seq ID No.1:5'-TGGGACAACATCAGACAGGTCGTTTGATGTCAGATTGGCCAGGTC-3', shanghai Biotechnology Co., ltd., purification by PAGE)
DNA G quadruplex oligonucleotide sequence (Seq ID No.2:5'-CCC AGG TTT GGG TAG GGC GGG TTG GG-3', shanghai Biol.Co., ltd., HPLC purification)
Heme (Hemin, 51280-1G, sigma sigma, germany)
Bovine serum albumin BSA; tween 20, naCl, mgCl2, tris
Dimethyl sulfoxide DMSO (A503039, shanghai worker)
Super-sensitized chemiluminescent fluid A, B (MA 0186-L, meilun organism)
Amino acid (Purity > 98.5%, LR, BBI Life Sciences, china)
DAB reagent (Meilun organism, 3ml, MA0201)
2. Preparation of main reagents
2.1 hybridization solution of DNA molecules (Tris-HCl buffer)
2.42g of Tris (LOT #: O1008A, merenia, china), 17.55g of NaCl (MB 2471, merenia, china) and 1.00g of MgCl2.6H2 (LOT: M0304A, merenia) were dissolved in 500ml of ultrapure water, the pH was adjusted to 8.3 with 0.1M of HCl, and finally, the solution was diluted to 1000ml of Tris-HCl solution with ultrapure water and stored at room temperature.
2.2 heme (Hemin) stock solution
Heme Hemin is dissolved in DMSO to prepare 10 mu M solution for storage, and the solution is stored at 4 ℃ for standby.
2.3 super-sensitivity chemiluminescent fluid A, B
The solutions A and B were formulated in a 1:1 ratio.
2.4 amino-terminal gradient solutions
The amino acid (serine in this case) was dissolved in a predetermined amount of sterilized deionized ultrapure water and then subjected to a predetermined concentration gradient (0, 2.5, 5, 10, 20, 40, 80, 160. Mu.g/ml) and stored at 4 ℃.
2.5 amino acid nucleic acid aptamer
Here, taking serine aptamer as an example, the aptamer for amino acid was dissolved in sterile deionized ultrapure water to prepare a 50. Mu.M solution for use.
3. Sample to be measured
Subjects were collected 64 early morning mid-stream urine (36 of gastric cancer hospitalized patients, 28 of contemporaneous healthy physical examination subjects). The urine sample was placed in a 15ml holding tube and capped and centrifuged at 3000RPM for 5 minutes, the supernatant was pipetted into a 1.5ml EP tube for dispensing and stored in a deep freezer at-80℃for further use.
Example 1 urine serine detection
The serine-targeting aptamer solution was thoroughly mixed with the G quadruplex DNA in a 5:4 ratio by amount in Tris-HCl buffer. In the detection system, a concentration gradient standard is firstly set according to the possible concentration range of the amino acid to be detected in a sample. Taking serine as an example, 8 serine concentration gradient standards are 0, 2.5, 5, 10, 20, 40, 80, 160 μg/ml. 120. Mu.L (50. Mu.M) of the aptamer and 96. Mu.L of the G quadruplex DNA (50. Mu.M) were hybridized in 600. Mu.L of Tris-HCl solution (pH 8.3), and the mixture was placed on a vortex machine and reacted in a water bath at 37℃for 1 hour to give a complete reaction. After the reaction, 75. Mu.l of hybridization mixture was added to each 1.5ml centrifuge tube, 75. Mu.l of serine standard or sample to be tested at each concentration gradient was added, and the mixture was placed in a 37℃water bath to be thoroughly mixed and reacted for 1 hour. Then, 16. Mu.l of a heme solution (storage concentration: 10. Mu.M) was added to the centrifuge tube, and the mixture was thoroughly mixed to a final concentration of 1. Mu.M, and reacted in a water bath at 37℃for 3 hours under a dark condition. Then, 20. Mu.L of the prepared luminescence AB mixed solution was added to each tube and mixed well to obtain a luminescence catalytic substrate. Finally, each tube of the above liquid was added to three sub-wells of a 96-well plate in a volume of 50. Mu.L per well. The 96-well plate is placed in a Safire 2 full-wave multifunctional enzyme-labeled instrument for detection.
Dividing the detection mean value of three pairs of holes of the sample to be detected by the blank control mean value of the standard curve to perform normalization treatment, and performing difference comparison among different groups by using unpaired t-test.
In order to compare and analyze the sensibility of the urine amino acid detection system, the invention adopts a standard amino acid analysis instrument to carry out parallel detection. The main detection process is as follows: 500. Mu.l of urine and 500. Mu.l of pretreatment liquid (0.1M hydrochloric acid and 10% trichloroacetic acid) were added to a 1.5ml EP tube at a ratio of 1:2, thoroughly mixed, centrifuged at 12000RPM for 20 minutes at 4℃and then allowed to stand at 4℃for 30 minutes, and centrifuged at 12000RPM for 20 minutes at 4 ℃. Taking 1ml of supernatant after centrifugation, adding the supernatant into a sample bottle of a full-automatic free amino acid analyzer for machine detection (Hitachi, japan), and taking the supernatant as a reference for the subsequent detection result.
Experimental results:
1. standard curve
Detection of chemiluminescent values obtained as a function of 8 standard concentration gradients (0, 2.5, 5, 10, 20, 40, 80, 160 μg/ml) of the internal control showed that the increase with increasing serine concentration was substantially linear (FIG. 2).
2. Clinical sample test results
A total of 64 urine samples were tested (28 healthy physical examination subjects, 36 gastric cancer patients). In order to eliminate the difference between detection batches, the average value of the actual chemiluminescence detection value and the standard substance luminescence value of the same batch is divided for normalization processing. As shown in fig. 3, the detected serine values of urine samples of gastric cancer patients and healthy physical examination patients are significantly different, and P is less than 0.01.
3. Evaluation of the method
For evaluating the detection method of the invention, the same batch of samples are subjected to parallel analysis by an amino acid detector, and clinical diagnosis is used as a reference (all gastric cancer patients are confirmed by pathological diagnosis after operation) by using a gold standard.
The critical value (cut-off) of the detection result of the detection method is obtained by adopting a 95% confidence interval. Wherein the threshold value of the method of the invention is 4.28, above which positive is defined and below which negative is defined. The threshold value for the amino acid detector method was 50.94, above which positive was defined and below which negative was defined. The results of the typical case analysis by an amino acid detector are shown in FIG. 4 and FIG. 5.
A four-grid table (Table 1) between gastric cancer diagnosis and gold standard of the detection method is calculated, which shows that the sensitivity of the detection method is 77.78%, the specificity is 85.71% and the accuracy is 81.25%; the amino acid detector used as a reference detects the result and a four-grid table (table 2) of clinical diagnosis, the sensitivity is 25%, the specificity is 82.14%, and the accuracy is 50%; .
TABLE 1 four-grid Table of the detection method of the invention
TABLE 2 four-grid Table of amino acid Analyzer detection methods
The detection result using serine as a target molecule shows that the urine amino acid detection system established by the invention has higher sensitivity and is more beneficial to clinical development and screening of gastric cancer high-incidence areas.
The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.
Sequence listing
<110> Shanghai transportation university medical college affiliated Ruijin Hospital
<120> kit and method for detecting urine amino acid of aptamer-quadruplex
<141> 2020-12-30
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tgggacaaca tcagacaggt cgtttgatgt cagattggcc aggtc 45
<210> 2
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
cccaggtttg ggtagggcgg gttggg 26
Claims (2)
1. A urine amino acid detection kit of a nucleic acid aptamer-quadruplex comprises a certain amino acid specific nucleic acid aptamer and a DNA G quadruplex sequence, and is characterized in that the certain amino acid specific nucleic acid aptamer is complementary with a part of the DNA G quadruplex sequence, under the condition that no target amino acid exists, the certain amino acid specific nucleic acid aptamer and the DNA G quadruplex sequence form a complementary combined chain, the DNA G quadruplex sequence cannot form a G quadruplex, the amino acid is serine, the nucleic acid sequence of the nucleic acid aptamer is represented by a Seq ID No.1, and the nucleic acid sequence of the DNA G quadruplex sequence is represented by a Seq ID No. 2; the urine amino acid detection kit also comprises a hypersensitive chemiluminescence liquid or DAB chromogenic liquid and a heme reagent.
2. The urine amino acid detection kit according to claim 1, which is used for diagnosis of gastric cancer.
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| CN105821046A (en) * | 2016-05-24 | 2016-08-03 | 上海敬元投资有限公司 | Specific L-serine aptamer and application thereof |
| WO2016168656A1 (en) * | 2015-04-15 | 2016-10-20 | Ji Hoon Lee | Aptasensor and method of detecting target material |
| CN110186892A (en) * | 2019-06-26 | 2019-08-30 | 苏州健雄职业技术学院 | A kind of glutamine detection method based on dual-enzyme coupling |
| CN111500684A (en) * | 2020-05-14 | 2020-08-07 | 河北医科大学 | Method for detecting dopamine based on aptamer-G quadruplex nanowire |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016168656A1 (en) * | 2015-04-15 | 2016-10-20 | Ji Hoon Lee | Aptasensor and method of detecting target material |
| CN105821046A (en) * | 2016-05-24 | 2016-08-03 | 上海敬元投资有限公司 | Specific L-serine aptamer and application thereof |
| CN110186892A (en) * | 2019-06-26 | 2019-08-30 | 苏州健雄职业技术学院 | A kind of glutamine detection method based on dual-enzyme coupling |
| CN111500684A (en) * | 2020-05-14 | 2020-08-07 | 河北医科大学 | Method for detecting dopamine based on aptamer-G quadruplex nanowire |
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| G-quadruplex based biosensor: A potential tool for SARS-CoV-2 detection;Hui Xi 等;《Biosensors and Bioelectronics》;第167卷;第1-10页 * |
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