A method for preparing a fluorescent probe and its application in detecting tetracycline and pyrophosphate ions

Abstract

The present invention relates to a method for preparing a fluorescent probe, comprising the following steps: adding purslane powder to ethanol and mixing evenly, performing heating reaction, and then cooling to room temperature to obtain a suspension; centrifuging or filtering the suspension to remove insoluble matter to obtain a homogeneous solution; rotary evaporating the homogeneous solution in a water bath to obtain a solute; dissolving the solute in ultrapure water, and freeze-drying to obtain a carbon quantum dot fluorescent probe powder. The fluorescent probe preparation method of the present invention is simple, and can quickly and efficiently detect the content of TC and PPi. The prepared fluorescent probe has stable performance, strong anti-interference ability, high accuracy and sensitivity. The method realizes the detection of two targets on a CQDs sensing platform, thereby improving the efficiency of analysis and reducing resource consumption. At the same time, the PCR amplification reaction can be monitored.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and particularly relates to a preparation method of a fluorescent probe and application of the fluorescent probe in detecting tetracycline and pyrophosphate ions. Background Art

[0002] Tetracycline (TC) is the most commonly used antibiotic in the medical field. It has the advantages of low cost, good oral absorption, and effectiveness against a variety of bacteria in controlling and preventing bacterial infections or infectious diseases. However, only a small amount of TC can be absorbed and actually utilized by humans and animals, and most TC will be released into the environment with its metabolites. In addition, excessive use of TC will enhance the spread of antibiotic-resistant bacteria and antibiotic-resistant genes, and TC residues will also accumulate in the human body, causing gastrointestinal diseases, allergies, nephrotoxicity, and adverse reactions in the blood or central nervous system. At present, many countries have set the maximum residue limit of TC in meat and water at 0.1 mg L -1 Pyrophosphate ion (PPi) is one of the most important anions in biological systems and plays a vital role in a wide range of biological processes. The lack or excess of PPi in living cells can lead to abnormalities in humans. At the same time, PPi is also the main cause of ecological problems such as eutrophication.

[0003] At present, the methods for detecting TC and PPi include electrochemical method, spectrophotometry, immunoassay, capillary electrophoresis and high performance liquid chromatography. Although these methods have their own advantages, they also have certain defects, such as cumbersome steps, time-consuming and labor-intensive, complex and expensive instruments and devices, long detection cycle, high cost, weak anti-interference ability and low accuracy, which limit the application of the above detection methods in practice. Fluorescence method has the advantages of fast response, low cost and high sensitivity, and is favored by many researchers. Therefore, it is urgent to develop a method for rapid and efficient fluorescence detection of TC and PPi.

[0004] Carbon quantum dots (CQDs) have the characteristics of simple operation, fast response speed, good selectivity, and high detection sensitivity for various analytes. In particular, green CQDs with biomass as precursors not only have the advantages of sustainability, low cost, biocompatibility, and ease of use, but also have low toxicity to the human body. Therefore, green CQDs have been widely used to construct various fluorescent probes for the detection of various target analytes such as metal ions, amino acids, drugs, and environmental pollutants. However, the green precursor CQDs currently developed are mainly used for the detection of a single target in optical sensing. If a single CQDs sensing platform can be used to detect two or more targets, it will help improve analysis efficiency and reduce resource consumption.

[0005] In addition, polymerase chain reaction (PCR) is an important technology for amplifying DNA fragments in vitro and is widely used in biological research and clinical diagnosis. Scientists usually use real-time fluorescence PCR to monitor PCR amplification products, that is, using fluorescent dyes that can bind to double-stranded DNA (such as SYBR Green) to monitor the amplification of DNA in real time during the PCR reaction. However, traditional fluorescent dyes have problems such as high price, low signal-to-noise ratio, high autofluorescence, and poor fluorescence stability. Summary of the invention

[0006] The object of the present invention is to provide a method for preparing a fluorescent probe and its application in detecting tetracycline and pyrophosphate ions, which can simultaneously detect tetracycline and pyrophosphate ions;

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for preparing a fluorescent probe comprises the following steps:

[0009] The purslane powder is added to ethanol and mixed evenly, and then heated for reaction, and then cooled to room temperature to obtain a suspension; the suspension is centrifuged or filtered to remove insoluble matter to obtain a homogeneous solution; the homogeneous solution is rotary evaporated in a water bath to obtain a solute; the solute is dissolved in ultrapure water, and freeze-dried to obtain a carbon quantum dot fluorescent probe powder.

[0010] Preferably, the ratio of the purslane powder to ethanol is: 0.2 g: 9-11 ml.

[0011] Preferably, the heating reaction temperature is 190-210° C., and the reaction time is 9.5-10.5 h.

[0012] Another aspect of the present invention provides a fluorescent probe prepared according to the above-mentioned preparation method.

[0013] Another aspect of the present invention provides the use of the fluorescent probe described above in detecting tetracycline and pyrophosphate ions.

[0014] In another aspect, the present invention provides a method for detecting tetracycline using the fluorescent probe described above, comprising the following steps:

[0015] (1) Add the fluorescent probe to ultrapure water and fully dissolve it by ultrasound to obtain a CQDs stock solution;

[0016] (2) adding tetracycline powder to anhydrous ethanol and performing sonication to fully dissolve the powder to obtain a tetracycline stock solution;

[0017] (3) After mixing the CQDs stock solution and ultrapure water, a dilution solution was obtained. Under the excitation of 331 nm, the fluorescence value at 402 nm was recorded as F0. Then, different volumes of tetracycline stock solution were added to the dilution solution to obtain a mixed solution. The fluorescence value F of the mixed solution was recorded. The linear relationship between the tetracycline concentration and the CQDs fluorescence intensity was obtained by linear fitting, and the first linear equation was established;

[0018] (4) The CQDs stock solution and the sample to be tested are mixed, and the change in fluorescence intensity at 402 nm is measured and substituted into the first linear equation to obtain the tetracycline content in the sample.

[0019] Preferably, the concentration of the CQDs stock solution is 5 mg / ml; the concentration of the diluent is 0.0005 mg / ml; the concentration of the tetracycline stock solution is 1 mM; within the linear range of 0.5-9 μM, the first linear equation is: F0-F=451.812Log c (TC) +173.590, R 2 =0.9958.

[0020] In another aspect, the present invention provides a method for detecting pyrophosphate ions using the fluorescent probe described above, comprising the following steps:

[0021] (1) Add the fluorescent probe to ultrapure water and dissolve it completely by ultrasound to obtain a CQDs stock solution. 3+ The solutions were mixed to obtain CQDs / Fe 3+ Stock solution,

[0022] (2) adding sodium pyrophosphate powder into ultrapure water and vortex mixing to fully dissolve it to obtain a PPi stock solution;

[0023] (3) CQDs / Fe 3+ After the stock solution and ultrapure water were mixed, a dilution was obtained. Under the excitation of 331 nm, the fluorescence value F0 at 402 nm was recorded. Then, different volumes of PPi stock solution were added to the dilution to obtain a mixed solution. The fluorescence value F of the mixed solution was recorded. The relationship between PPi concentration and CQDs / Fe was obtained by linear fitting. 3+ The linear relationship of fluorescence intensity was used to establish the second linear equation;

[0024] (4) CQDs / Fe 3+ The stock solution and the sample to be tested are mixed, and the PPi content in the sample can be obtained by measuring the change in fluorescence intensity at 402 nm and substituting it into the second linear equation.

[0025] Preferably, the concentration of the CQDs stock solution is 5 mg / ml, Fe 3+The solution concentration is 10mM, CQDs / Fe 3+ CQDs stock solution and Fe 3+ The volume ratio of the solution is 210:200; the concentration of the diluent is 0.0005 mg / ml; the concentration of the PPi stock solution is 10 mM; within the linear range of 0.5-9 μM, the linear equation is: (F-F0) / F0=171.770Log c (PPi) -113.630, R 2 =0.9926.

[0026] In another aspect, the present invention provides a method for detecting PCR amplification of Mycobacterium tuberculosis using the fluorescent probe described above, comprising the following steps: adding the fluorescent probe to ultrapure water, dissolving it fully by ultrasound to obtain a CQDs stock solution, mixing the CQDs stock solution with Fe 3+ The solutions were mixed to obtain CQDs / Fe 3+ Stock solution, in CQDs / Fe 3+ The PCR amplification product of Mycobacterium tuberculosis was added to the stock solution and fluorescence detection was performed.

[0027] The advantages of this method are: the precursor purslane is widely distributed, cheap and easy to obtain, ethanol is a common reagent and easy to purchase. The preparation method of the fluorescent probe is simple, and the content of TC and PPi can be detected quickly and efficiently. The prepared fluorescent probe has stable performance, strong anti-interference ability, high accuracy and sensitivity.

[0028] Compared with other methods for detecting TC and PPi, this method has several advantages. First, it is fast, effective and easy to operate. Second, the method has stable performance, low detection cost and high sensitivity. Most importantly, this method realizes the detection of two targets on a single CQDs sensing platform, thereby improving the efficiency of analysis and reducing resource consumption.

[0029] At the same time, compared with fluorescent dyes for monitoring PCR amplification products, the carbon quantum dots prepared by the present invention, as a new type of fluorescent probe, have lower price, better signal-to-noise ratio, lower autofluorescence level and longer fluorescence stability, and thus become one of the ideal choices for monitoring the PCR reaction process. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 In the figure, a is the UV and fluorescence spectra of the prepared CQDs, and b is the excitation wavelength-dependent spectrum of CQDs.

[0031] Figure 2 Figure a shows the selectivity experimental results of CQDs on TC detection, and figure b shows the interference experimental results of CQDs on TC detection.

[0032] Figure 3 (a) is a curve diagram of the change of CQDs fluorescence intensity when CQDs solution is titrated by TC, and (b) is a linear diagram of TC concentration and CQDs fluorescence intensity.

[0033] Figure 4 a is the CQDs to Fe 3+ The selectivity experimental results of the detection are shown in Figure 2 (a), and (b) are the selectivity of CQDs to Fe 3+ The interference experiment results of the detection are shown in Figure 2, where c is Fe 3+ The reaction time diagram of the fluorescence quenching of CQDs solution, d is the reaction time of the fluorescence quenching of CQDs solution, and d is the reaction time of the fluorescence quenching of CQDs solution. 3+ Titration of CQDs solution, and curve of the change of CQDs fluorescence intensity.

[0034] Figure 5 For metal anions (NO2 - 、F - 、H2PO4 - 、SCN - 、HPO4 2- 、HCO3 - 、ClO4 - , Cl - ,I - 、NO3 2- Br - 、S2O3 2- 、CO3 2- and PPi) on CQDs / Fe 3+ Graph showing the fluorescence recovery effect of the complex.

[0035] Figure 6 a is the titration of CQDs / Fe using PPi 3+ Solution, CQDs / Fe 3+ Fluorescence intensity change curve, b is the PPi concentration and CQDs / Fe 3+ Linear plot of fluorescence intensity.

[0036] Figure 7 a is the detection of H37Ra PCR product by agarose gel electrophoresis, b is the detection of CQDs / Fe 3+ Diagram of the complex detecting PPi in PCR products. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0038] 1. Preparation and characterization of CQDs

[0039] Step 1, hydrothermal reaction: First, add 0.2 g of purslane powder into 10 mL of ethanol and mix thoroughly in a 50 mL beaker; then, transfer the mixture to a high-pressure reactor containing a polytetrafluoroethylene liner; place the reactor at 200°C and heat the reaction for 10 hours.

[0040] Step 2, purification: After the reactor is naturally cooled to room temperature (about 25°C), a dark brown suspension (crude product) is obtained; then the suspension is centrifuged at 4000r / min for 10min to remove insoluble matter and larger particles; then, the resulting homogeneous solution, i.e., the supernatant, is rotary evaporated in a 50°C water bath to remove ethanol and obtain the solute; finally, the solute is dissolved in ultrapure water and freeze-dried to obtain a green carbon quantum dot fluorescent probe, i.e., CQDs solid powder.

[0041] Step 3: Weigh 50 mg of CQDs solid powder into 10 ml of ultrapure water and sonicate for 15 min to completely dissolve it to obtain a CQDs stock solution with a concentration of 5 mg / ml.

[0042] The purslane powder used above was air-dried purslane purchased from Tong Ren Tang Co., Ltd., Taiyuan, China, and was obtained by crushing and sieving through a 200-mesh sieve.

[0043] The ethanol used above is commercially available analytical grade ethanol with a purity of 100%.

[0044] Characterization of properties Figure 1 The CQDs aqueous solution exhibits bright blue fluorescence under 365nm ultraviolet light and transparent light yellow under visible light (see Figure 1 a illustration). The UV-visible absorption of CQDs shows two weak peaks at 249 nm and 274 nm, respectively. The peak at about 250 nm may be caused by the π-π* transition of aromatic CC bonds and C=C bonds, and the peak at about 275 nm may be attributed to the n-π* transition of carbonyl C=O (see Figure 1 a). The maximum emission wavelength of CQDs is 402nm and the excitation wavelength is 331nm (see Figure 1 a). When the excitation wavelength changes from 280nm to 380nm, the emission band moves to the red side (from 379nm to 438nm), showing a typical excitation-dependent fluorescence phenomenon (see Figure 1 b).

[0045] 2. Anti-interference experiment of tetracycline TC detection

[0046] Step 1: weigh 50 mg of CQDs solid powder, add 10 ml of ultrapure water, and sonicate for 15 min to completely dissolve it to obtain a CQDs stock solution with a concentration of 5 mg / ml.

[0047] Step 2: Prepare 17 kinds of amino acid solutions ( L -Glutamine, L -Lysine, L - Cysteine, L -Proline, DL -serine, L -Valine, Glutathione, L -Isoleucine, L -Tryptophan, L -Tryptophan, glycine, L -Glutamate, L -Arginine, L -Alanine, DL -Aspartic acid, D -aspartic acid) and 6 drug solutions (chloramphenicol, TC, erythromycin, kanamycin, vitamin B1, vitamin B3), all with a concentration of 100mM.

[0048] Step 3: Take 20 μL of the above CQDs stock solution and each test solution into a quartz cup, then dilute with ultrapure water to a final volume of 2 L, mix well, incubate for 1 min, and record the fluorescence spectrum value at an excitation wavelength of 331 nm.

[0049] Step 4: TC and other test solutions (all 1 mM) were added to the CQDs solution. After incubation for 1 min, the fluorescence spectrum of each sample was monitored at an excitation wavelength of 331 nm to check whether the presence of other test solutions had a significant effect on the quenching effect of TC. Figure 2 .

[0050] The results of the anti-interference experiment of TC detection showed that the fluorescence dropped sharply after the addition of TC, while the 17 amino acids and other 5 drugs had no or almost no fluorescence changes ( Figure 2 a). Compared with the quenching effect of TC, the effect of coexisting substances is almost negligible ( Figure 2 b), indicating that CQDs have good anti-interference ability in TC detection.

[0051] 3. Linear equation of TC concentration and CQDs fluorescence intensity

[0052] Step 1: Fluorescence titration method was used, that is, 200 μL of CQDs stock solution was diluted to 2 L with ultrapure water, and under the excitation of 331 nm, the fluorescence value of CQDs at 402 nm was recorded as F0.

[0053] Step 2: Add different volumes of TC (1 mM) to the above solution, record the fluorescence value of the CQDs and TC mixture, which is F, and the change of fluorescence intensity is shown in Figure 3 a.

[0054] Step 3: The linear relationship between TC concentration and CQDs fluorescence intensity was obtained by linear fitting using Origin software. Figure 3 b.

[0055] The results show that as the TC concentration increases from 0 to 207 μM, the fluorescence intensity of the CQDs emission peak position (about 402 nm) decreases ( Figure 3 a). When the linear range is 0.5-9μM, the linear equation is: F0-F=451.812Log c (TC) +173.590,R 2 =0.9958, the minimum detection limit is 13.3nM ( Figure 3 b).

[0056] 4. Detection of TC content in actual samples

[0057] The actual samples were tap water (from the 302 laboratory of the Institute of Biomedicine of Shanxi University) and local river water (Fen River, Taiyuan, China).

[0058] Step 1: Add 20ul of CQDs stock solution to 2mL of ultrapure water. The concentration of CQDs is 5μg·mL. -1 , measure the fluorescence intensity at 402 nm and record it as F0.

[0059] Step 2: Centrifuge the water sample at 8000 rpm for 5 min, then filter it with a 0.22 μm filter membrane for later use; finally, add 20 μL of CQDs stock solution to 2 mL of tap water and river water respectively, and the concentration of CQDs is 5 μg mL -1 , measure the fluorescence intensity at 402nm, record it as F, calculate F0-F, and substitute it into the linear equation: F0-F=451.812Log c (TC) +173.590, the TC concentration in the actual sample was calculated, and the results are shown in Table 1.

[0060] Table 1. TC content in two actual samples

[0061]

[0062] The results show that the CQDs prepared by this method can be used to detect the content of TC in actual samples, and the relative standard deviation is less than 6.23% (see Table 1), with good reproducibility.

[0063] 5. TC spike recovery experiment in actual samples

[0064] Step 1: Weigh 50 mg of CQDs solid powder into a beaker, add 10 mL of ultrapure water, and sonicate to fully dissolve it to obtain a CQDs mother solution with a concentration of 5 mg / mL.

[0065] Step 2: Weigh 0.0222 g of tetracycline powder, add it to 50 ml of anhydrous ethanol, and sonicate to fully dissolve it to obtain a TC stock solution with a concentration of 1 mM.

[0066] Step 3: Add 20 μL of CQDs stock solution to 2 mL of tap water. The concentration of CQDs is 5 μg mL -1 , measure the fluorescence intensity at 402 nm and record it as F0.

[0067] Step 4: Add 20 μL of CQDs stock solution to 2 mL of tap water, and then add 5 μM, 10 μM, and 20 μM TC spiked stock solutions, respectively. At this time, the concentration of CQDs is 5 μg mL -1 , measure the fluorescence intensity at 402nm, recorded as F.

[0068] Step 5: Add 20 μL of CQDs stock solution to 2 mL of river water. The concentration of CQDs is 5 μg mL -1 , measure the fluorescence intensity at 402 nm and record it as F0.

[0069] Step 6: Add 20 μL of CQDs stock solution to 2 mL of river water, and then add 2.5 μM, 5 μM, and 10 μM TC spiked stock solutions, respectively. At this time, the concentration of CQDs is 5 μg mL -1 , measure the fluorescence intensity at 402nm and record it as F.

[0070] Step 7: Substitute F0-F into the linear equation to calculate the spiked recoveries of TC in the two actual samples.

[0071] Table 2. Results of spike recovery experiments for TC in two real samples

[0072]

[0073]

[0074] The results showed that the spiked recoveries of TC in the two actual samples were between 95.2% and 104.5%, and the relative standard deviation was less than 4.79% (see Table 2), indicating that the CQDs prepared by this method can be used for spiked detection of TC in actual samples, without interference from other substances in the actual samples, and have good recovery results and reproducibility.

[0075] 6. CQDs / Fe 3+ Preparation of complexes

[0076] Step 1: Weigh 16.22 mg of FeCl3 solid powder into a beaker, add 10 mL of ultrapure water, and sonicate to fully dissolve it to obtain a concentration of 10 mM FeCl3. 3+ Ion stock solution.

[0077] Step 2: Prepare 17 common cations (Na + , K + 、Ag + , Li + , Ca 2+ , Mn 2+ , Ba 2+ 、Zn 2+ , Hg 2+ 、Co 2+ Mg 2+ , Cu 2+ , Sr 2+ 、Cd 2+ , Fe 2+ 、Al 3+ and In 3+ ) and 13 anions (NO2 - 、F - 、H2PO4 - 、SCN - 、HPO4 2- 、HCO3 - 、ClO4 - , Cl - ,I - 、NO3 2- Br - 、S2O3 2- 、CO3 2- ), the concentration was 100mM.

[0078] Step 3: Take 20 μL of the above CQDs stock solution and each test solution in a quartz cup, then dilute with ultrapure water to a final volume of 2L, mix well, incubate for 1 min, and record the fluorescence spectrum value at an excitation wavelength of 331 nm. Observe the fluorescence quenching of CQDs by various ions. The experimental results are shown in Figure 4 a.

[0079] Step 4: Add Fe 3+ After incubation for 1 min with other test solutions (final concentration of 1 mM), the fluorescence spectrum of each sample was monitored at an excitation wavelength of 331 nm to check whether the presence of other test solutions had an effect on Fe 3+ The experimental results are shown in Figure 4 b.

[0080] Step 5: Fluorescence titration method was used, i.e., 210 μL of CQDs stock solution was diluted to 2 L with ultrapure water, and the fluorescence value of CQDs at 402 nm was recorded as F0 under the excitation of 331 nm. Then, different volumes of Fe 3+ Stock solution (10 mM), recording CQDs and Fe 3+ The fluorescence value F of the mixed solution is recorded, and the time of fluorescence change is recorded (see Figure 4 c) and fluorescence intensity changes (see Figure 4 d).

[0081] Results Figure 4 Add Fe 3+ After that, the fluorescence of CQDs dropped sharply ( Figure 4 a), can be completed in just 1 minute ( Figure 4 c), while after adding 17 cations and 13 anions, the fluorescence of CQDs remained almost unchanged ( Figure 4 b), indicating that CQDs have a strong affinity for Fe 3+ It has good anti-interference performance during detection. 3+ As the concentration increases from 0 to 1000 μM, the fluorescence intensity of the CQDs emission peak position (about 402 nm) gradually decreases ( Figure 4 d). Finally, CQDs / Fe 3+ The optimal preparation conditions for the stock solution were 210 μL CQDs stock solution (5 mg / ml) and 200 μL Fe 3+ The standard solution (10 mM) was mixed.

[0082] 7. CQDs / Fe 3+ Complex detection PPi experiment

[0083] Step 1: Weigh 0.223 g of sodium pyrophosphate powder, add it to 50 ml of ultrapure water, and vortex to mix it to fully dissolve it to obtain a PPi stock solution with a concentration of 10 mM.

[0084] Step 2: Prepare 14 metal anions (NO2 - 、F - 、H2PO4 - 、SCN - 、HPO4 2- 、HCO3 - 、ClO4 - , Cl - ,I - 、NO3 2- Br - 、S2O3 2- 、CO3 2- and PPi) solutions, both at a concentration of 100 mM.

[0085] Step 3: Take the above CQDs / Fe 3+ 20 μL of the stock solution and each test solution were placed in a quartz cup, and then diluted with ultrapure water to a final volume of 2 L. After mixing, the mixture was incubated for 1 min, and the fluorescence spectrum value at an excitation wavelength of 331 nm was recorded.

[0086] Step 4: CQDs / Fe 3+ PPi and other tested solutions (all 1 mM) were added to the solution. After incubation for 1 min, the fluorescence spectrum of each sample was monitored at an excitation wavelength of 331 nm to observe the effects of PPi and other tested solutions on CQDs / Fe 3+ The effect of the fluorescence intensity of the complex. Figure 5 .

[0087] The results show that: 3+ The introduction of PPi (50 μM) into the solution can restore the fluorescence, while other anions have little or no effect on the fluorescence recovery (see Figure 5 ), indicating that the CQDs / Fe prepared by this method 3+ The complex can be used to detect PPi.

[0088] 8. PPi concentration and CQDs / Fe 3+ Linear equation of fluorescence intensity of complex

[0089] Step 1: Fluorescence titration method was used to titrate 200 μL of CQDs / Fe 3+ The stock solution was diluted to 2 L with ultrapure water, and the fluorescence value of CQDs at 402 nm was recorded as F0 under excitation at 331 nm.

[0090] Step 2: Add different volumes of PPi (1 mM) to the above solution and record the CQDs / Fe 3+ The fluorescence value of the mixed solution of PPi is F, and the change of fluorescence intensity is shown in Figure 6 a.

[0091] Step 3: Linear fitting of PPi concentration and CQDs / Fe was obtained by Origin software. 3+ The linear relationship of fluorescence intensity is shown in Figure 6 b.

[0092] The results showed that as the PPi concentration increased from 0 to 365 μM, the fluorescence intensity at the emission peak position (about 402 nm) gradually recovered ( Figure 6 a). In the linear range of 0.5-95 μM, the linear equation is: (F-F0) / F0=171.770Log c (PPi) -113.630, R 2 =0.9926, the minimum detection limit is 34.9nM ( Figure 6 b).

[0093] IX. PPi content detection in actual samples

[0094] The actual sample was selected from PPi in the PCR product.

[0095] Step 1: Use a bacterial genomic DNA extraction kit to extract the DNA of Mycobacterium tuberculosis H37Ra.

[0096] Step 2: Amplify the 16S rRNA V3-V4 region of Mycobacterium tuberculosis H37Ra using universal primers 27F and 1492R, where primer 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, 1492R: 5′-GGTTACCTTGTTACGACTT-3′. The PCR reaction was performed in a 20 μL system: 1 μL DNA template, 1 μL of each primer, 10 μL of mixed buffer and 7 μL of nuclease-free water. The PCR reaction program was: 95°C 30s pre-denaturation, 95°C 15s, 56°C 30s, 72°C 15s, 35 cycles in sequence, and final extension at 72°C 10min.

[0097] Step 3: The final PCR product was detected by 1% agarose gel electrophoresis. Figure 7 a.

[0098] Step 4: Take 4 μL of the final PCR product and place it on CQDs / Fe 3+ The fluorescence changes of the complex solution were detected (see Figure 7 b) to confirm the generation of PPi in the PCR product.

[0099] Agarose gel electrophoresis results showed that the PCR reaction successfully amplified the 16s rRNA V3-V4 region fragment of the H37Ra strain, with a length of about 1000 bp (see Figure 7 a). CQDs / Fe 3+ The complex can perform sensitive fluorescence detection of PPi in the final PCR product, and the experimental results are consistent with the results of agarose gel electrophoresis (see Figure 7 b), prove that the CQDs / Fe prepared by this method 3+ The complex can be used to verify the success of PCR amplification of Mycobacterium tuberculosis.

Claims

1. A method for detecting PCR amplification of Mycobacterium tuberculosis using a fluorescent probe, characterized in that: The steps include: adding the fluorescent probe to ultrapure water, dissolving it fully by ultrasonication to obtain a CQDs stock solution, and mixing the CQDs stock solution with Fe 3+ The solutions were mixed to obtain CQDs / Fe 3+ Stock solution, in CQDs / Fe 3+ The PCR amplification product of Mycobacterium tuberculosis was added to the stock solution and fluorescence detection was performed; The method for preparing the fluorescent probe comprises the following steps: The purslane powder is added to ethanol and mixed evenly, and then heated for reaction, and then cooled to room temperature to obtain a suspension; the suspension is centrifuged or filtered to remove insoluble matter to obtain a homogeneous solution; the homogeneous solution is rotary evaporated in a water bath to obtain a solute; the solute is dissolved in ultrapure water, and freeze-dried to obtain a carbon quantum dot fluorescent probe powder.

2. The method according to claim 1, characterized in that The ratio of the purslane powder to ethanol is: 0.2 g: 9-11 ml.

3. The method according to claim 1, characterized in that The heating reaction temperature is 190-210° C., and the reaction time is 9.5-10.5 h.

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