CN114107503A - Composite locked nucleic acid magnetic bead probe for detecting miRNA marker, construction method and diagnostic reagent containing probe - Google Patents

Composite locked nucleic acid magnetic bead probe for detecting miRNA marker, construction method and diagnostic reagent containing probe Download PDF

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CN114107503A
CN114107503A CN202111442305.4A CN202111442305A CN114107503A CN 114107503 A CN114107503 A CN 114107503A CN 202111442305 A CN202111442305 A CN 202111442305A CN 114107503 A CN114107503 A CN 114107503A
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何露·德杰比
查克·特里里
周大明
穆罕默德·巴里
孔祥东
石彪
田荣
王德强
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Chongqing Institute of Green and Intelligent Technology of CAS
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Abstract

The invention provides a composite locked nucleic acid magnetic bead probe for detecting miRNA markers, a construction method and a diagnostic reagent containing the composite probe, wherein the composite locked nucleic acid magnetic bead probe comprises three probes, namely an LNA probe-486-5p probe marked by an FAM fluorescent group, an LNA probe-21 probe marked by a Cy5 fluorescent group and an LNA probe-210 probe marked by a TAMRA fluorescent group, in the same magnetic bead. The invention fixes the DNA probe molecule of the modified fluorescent mark on the magnetic bead through the connection of biotin-streptavidin. The DNA probe sequence is matched with a target miRNA and hybridized to form a double strand, under the action of DSN enzyme, the DNA probe in the heterozygosis double strand is hydrolyzed into fragments, fluorophore molecules are suspended in reaction liquid, but the miRNA is kept complete and hybridized with the DNA probe again to generate DSN enzyme digestion reaction. Repeating the steps, the number of the fluorescent groups of the final system is obviously amplified. The sensitive detection of the concentration of the target miRNA can be realized by testing the signal intensity of the fluorescent group in the supernatant. The kit can be used for conveniently and ultrasensitively detecting three different lung cancer markers miRNA in body fluid at the same time, and accurately diagnosing lung cancer.

Description

Composite locked nucleic acid magnetic bead probe for detecting miRNA marker, construction method and diagnostic reagent containing probe
Technical Field
The invention relates to the fields of molecular biology and nucleic acid chemistry, in particular to a composite locked nucleic acid magnetic bead probe for detecting miRNA markers, a construction method and a diagnostic reagent containing the probe.
Background
Cancer is the first cause of death among the many causes of death among the residents of large and medium cities in our country. The world health organization makes the latest authoritative conclusion that if cancer patients can be found early, the cure rate can reach more than 80%. Therefore, it is very necessary to perform early diagnosis of cancer. The early discovery and early treatment of the cancer can relieve the pain, spirit and economic burden of patients.
Among cancer patients, lung cancer accounts for more than 20%. Traditional early detection techniques for lung cancer include chest radiography, Low Dose Computed Tomography (LDCT), and lung biopsy. However, these techniques have several limitations in the early detection of lung cancer, including high false positive rates, invasiveness, potential over-diagnosis, excessive cost, or radiation-induced cancer. On the other hand, standard immunoassay methods have become mature techniques for detecting lung cancer biomarkers, but up to now, no tumor marker with 100% sensitivity and specificity has been found. The detection of single index and single factor is difficult to realize the early detection of tumor, the monitoring of disease course, the evaluation of prognosis treatment effect, etc. As with the traditional Elisa method, detection of only a single protein factor can be performed. To improve the accuracy and specificity of detection, multiple Elisa experiments are required to detect different protein factors. Taking 10 protein factor assays as an example, 10 Elisa kits are needed, at least 1mL of samples, and one week is needed to obtain the results. Neither from the point of view of manpower, financial resources nor time and sample size is a good choice. And errors in the results may also be caused by not detecting the 10 factors simultaneously. Furthermore, most of these immunoassays are singleplex assays and are not sensitive enough to detect low concentrations of biomarkers in the early stages of the disease. Therefore, it is necessary to develop a high-sensitivity early lung cancer detection technique.
In recent years, scientists have proposed the diagnosis of lung cancer by markers, and among them, many studies have been made on microribonucleic acid miRNA. miRNA is a non-coding RNA, typically 18-25 bases in length. Research shows that about 30% of human genes are regulated by miRNA, and the expression level of miRNA is closely related to the occurrence of various human diseases. The traditional method for quantitatively detecting miRNA mainly comprises Northern blotting, in situ hybridization, a microarray method, rolling circle amplification, reverse transcription polymerase chain reaction and the like. These methods can be broadly classified into probe direct hybridization methods that do not require sample amplification and detection methods based on sample miRNAs amplification. They each have advantages and disadvantages: the method without amplification is simple and convenient to operate, but has large requirements on the sample size and low sensitivity; amplification-based methods have high sensitivity but are limited by the inability to directly detect miRNAs levels and non-specific amplification. The reason for this is that the sequences of miRNAs are very short and these amplification means and the corresponding probe design are very elaborate and complex processes. Traditional PCR techniques are mostly judged indirectly by examining their precursors and do not reflect the true miRNAs levels. Furthermore, miRNAs are expressed in much lower amounts in body fluids than in tissues, and thus conventional techniques for detecting miRNAs in tissues are not necessarily suitable for the detection of body fluid miRNAs.
In practical clinical application, the content level of various miRNAs markers in body fluid needs to be detected simultaneously so as to accurately diagnose the lung cancer. This is not currently achievable with the prior art.
Disclosure of Invention
In view of this, in order to overcome the defects of the prior art, the invention provides a composite locked nucleic acid magnetic bead probe for detecting a miRNA marker and a construction method thereof, the probe is a magnetic bead probe which is compounded with three LNAs and has clear cross hybridization, and compared with standard serum, the invention can simultaneously detect the obvious difference of the expression levels of the three mirnas corresponding to the probe in a sample to be detected.
The invention provides a composite locked nucleic acid magnetic bead probe for detecting miRNA markers, which comprises three probes, namely an LNA probe-486-5p probe marked by an FAM fluorescent group, an LNA probe-21 probe marked by a Cy5 fluorescent group and an LNA probe-210 probe marked by a TAMRA fluorescent group, in the same magnetic bead.
The invention also provides a construction method of the composite locked nucleic acid magnetic bead probe for detecting the miRNA marker, which comprises the following steps:
(1) preparing locked nucleic acid probe molecules: synthesizing corresponding locked nucleic acid as capture probes LNA probe-486-5p, LNA probe-21 and LNA probe-210 according to the base sequences of the miRNA-486-5p, miRNA-21 and miRNA-210 molecules of the markers to be detected, respectively modifying FAM fluorescent groups and biotin molecules at two ends of the LNA probe-486-5p,
cy5 fluorescent group and biotin molecule are respectively modified at two ends of the LNA probe-21 probe,
TAMRA fluorescent group and biotin molecule are respectively modified at two ends of the LNA probe-210 probe,
mixing the three probes according to a molecular ratio of 1:1:1 to prepare an LNA probe mixture;
(2) the nucleic acid locking probe molecules are fixed on the magnetic beads: the probe mixture is preheated at 95 ℃, subjected to cross hybridization, mixed with streptavidin magnetic bead SMBs suspension, and cultured at room temperature;
(3) cleaning: firstly, washing the cultured composite locked nucleic acid magnetic bead probe solution with NaOH solution to remove non-specifically bound or free DNA probes, then repeatedly washing with TT buffer solution, adding the magnetic bead probes into the TT buffer solution for resuspension, then culturing at 80 ℃, removing unstable coupled molecules bound in the liquid, and finally washing with DEPC water to obtain the composite locked nucleic acid magnetic bead probe for detecting the miRNA marker.
Further, in the method for constructing the composite locked nucleic acid magnetic bead probe, in the step (2), the streptavidin magnetic bead suspension is prepared according to the following method: and (2) carrying out covalent binding on the superparamagnetic microspheres and streptavidin to form streptavidin magnetic bead SMBs, washing with 1 XBW buffer solution at room temperature, fixing the streptavidin magnetic beads with a permanent magnet, filtering out supernatant, and dispersing in 2 XBW buffer solution to obtain an SMBs suspension.
Wherein, the BW buffer (probe fixation and washing buffer) is prepared according to the following method: A0.5M NaCl, 20mM Tris-HCl, 1mM EDTA solution was prepared and then the pH was adjusted to 8.0.
Further, in the step (2), the LNA probe complex and the streptavidin magnetic beads are mixed in a ratio of 20pmol LNA to 20 μ g MBs streptavidin magnetic beads during the mixing process.
Further, in the step (2), the concentration of each LNA in the prepared complex locked nucleic acid magnetic bead probe solution is 1 μ M.
Further, in the step (2), the preheating is carried out for 5min at 95 ℃; the incubation was at 37 ℃ for 20 minutes.
Further, in the step (3), the concentration of the NaOH solution is 0.15M; the TT buffer solution is prepared according to the following method: adding 0.1% of Tris-HCl buffer solution to 250mM
Figure BDA0003383924810000031
Figure BDA0003383924810000031
20, and adjusting the pH to 8.0; the incubation was at 80 ℃ for 10 minutes.
The invention also provides an application of the composite locked nucleic acid magnetic bead probe in the aspect of detecting miRNA markers, which comprises the following steps:
step I: and (3) carrying out DSN enzyme digestion reaction:
reaction system: the kit comprises a composite locked nucleic acid magnetic bead probe, miRNA to-be-detected liquid, 1 XDSN main buffer solution, a ribonuclease inhibitor and DSN enzyme; incubation at 55 ℃ for 120 min in a thermocycler; hybridizing a sample miRNA to be detected with the probe to form a double strand, carrying out enzyme digestion reaction on the DSN and the double strand, hydrolyzing the DNA into fragments, and separating out a fluorescent group; the released miRNA is kept complete and hybridized with unreacted DNA again, a new enzyme digestion reaction is generated, and the fluorescent group in the solution is increased;
step II: fluorescence detection
After the reaction is completed, irradiating the liquid containing the fluorescent molecules with exciting light, detecting the fluorescence intensity of cy5 group at 649/670 wavelength, detecting the fluorescence intensity of TAMRA group at 545/580 wavelength and detecting the fluorescence intensity of FAM group at 483/520 wavelength; further obtaining the respective concentrations of the three miRNA markers in the miRNA test solution.
Further, in the step II, after the reaction in the step I is completed, in order to remove LNA molecules connected to the magnetic beads that do not participate in the reaction, the magnetic beads are fixed by strong magnets to extract supernatant, and then fluorescence detection is performed.
The invention also provides a diagnostic reagent containing the composite locked nucleic acid magnetic bead probe, and the diagnostic reagent contains 20 mu g of the composite locked nucleic acid magnetic bead probe, 1 XDSN main buffer solution, 20U ribonuclease inhibitor, 0.4U DSN enzyme and standard serum.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a simple, convenient and ultrasensitive composite locked nucleic acid magnetic bead probe capable of simultaneously detecting three different lung cancer markers miRNA in body fluid. The probe is based on a signal amplification mechanism of a DSN enzyme digestion reaction, is combined with a molecular fluorescence intensity test, can simultaneously detect the concentration of three cancer markers miRNA in body fluid, and can accurately diagnose lung cancer.
2. The invention modifies the lung cancer three marker probe molecules on the surface of the magnetic bead at the same time and eliminates cross hybridization, and then the invention is used for detecting the serum sample of the lung cancer patient, compared with the sample of healthy people, the invention can detect the obvious difference of the three miRNA expression levels corresponding to the probes.
3. The probe is bonded to the magnetic beads through biotin-streptavidin, the bonding force is very strong, and the probe molecules which do not participate in the reaction can be conveniently and effectively separated, so that the background signal is reduced.
4. The application method of the probe introduces double-strand specific nuclease (DSN) to amplify the intensity of a fluorescent signal, and reduces a background signal by virtue of magnetic beads. The specific detection principle is as follows: the modified fluorescence-labeled DNA probe molecules are fixed on magnetic beads through biotin-streptavidin connection, and DNA probe sequences are matched with target miRNA and hybridized to form double chains. Under the action of DSN enzyme, the DNA probe in the heterozygosis double strand is hydrolyzed into fragments, the fluorescent group molecules are suspended in the reaction liquid, but miRNA is kept intact and is hybridized with the DNA probe again to generate DSN enzyme digestion reaction. The steps are repeated, the cyclic process of hybridization, enzyme digestion and fluorescent group release of one miRNA molecule and a plurality of probes under the constant temperature condition can be realized, and the number of the fluorescent groups of the final system is obviously amplified. After the reaction is finished, fixing the magnetic beads and the probe molecules which do not participate in the reaction under the action of a magnetic field, separating and extracting supernatant containing fluorescent molecules, and testing the signal intensity of fluorescent groups in the supernatant to realize the sensitive detection of the target miRNA.
5. The detection technology has the advantage that the linear amplification of the detection signal can be realized on the premise of keeping the initial miRNA quantity in the sample unchanged. As the technology does not need PCR, non-specific amplification is avoided, thereby improving the specificity of detection.
6. Compared with the traditional separation method, the magnetic beads are used for separating complex components of a biochemical sample, so that separation and enrichment can be carried out simultaneously, the separation speed and the enrichment efficiency are effectively improved, and the sensitivity of analysis and detection is greatly improved; this method can be combined with other molecular detection amplification techniques, such as rolling circle amplification (prior art), hybridization chain reaction, etc.
7. The invention realizes the scheme of simultaneously detecting three miRNAs, and more kinds of miRNAs can be simultaneously detected. The detection result of the practical case shows that the difference between the miRNA marker expression level in the lung cancer patient body and the expression level in the healthy human body is more than 3 times, and the miRNA marker expression level of the patient can be effectively detected whether the miRNA expression level of the patient is improved or inhibited.
Drawings
FIG. 1 shows the hybridization of miRNA to be detected and magnetic microsphere DNA probe into double strand and the release of fluorescent group by DSN enzyme digestion reaction;
FIG. 2 is a process of detecting miRNA to be detected by the magnetic microsphere DNA probe constructed in the present invention;
FIG. 3 shows the change of fluorescence intensity of different miRNA in the detection process when the incubation temperature is changed from 40 ℃ to 65 ℃, wherein the miRNA concentration is 1 nM;
FIG. 4 is a calibration curve of the concentration of miRNA detected by the method. DSN 0.4U, miRNA 0.5fM to 1nM, reaction 2 hours at 55 ℃;
FIG. 5 shows the cross-reaction test results of miRNA and LNA at a concentration of 1 nM;
FIG. 6 shows that the composite probe of the present invention is used to detect serum samples of lung cancer patients and healthy human bodies at the same time, and shows that the miRNA-21, miRNA-210 expression level is increased and miRNA-486-5p expression level is inhibited in the lung cancer patients.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example selection of optimum incubation temperature
Preparation of DNA Probe
Synthesizing corresponding Locked Nucleic Acid (LNA) as a capture probe LNA probe-486-5p according to a base sequence of a to-be-detected marker miRNA486-5p molecule, and respectively modifying molecules such as FAM fluorescent groups and biotin at two ends.
Immobilization of DNA Probe molecules on magnetic beads
The superparamagnetic microsphere and high-purity streptavidin are covalently combined to form streptavidin magnetic beads (SMBs, streptavidin MBs), and a certain amount of SMBs is taken and used as 1 XB at room temperature&W buffer washing and filtering off supernatant with the aid of a permanent magnet, anddisperse to 2 XB&W buffer. Taking a proper amount of LNA detection molecules, preheating for 5 minutes at 95 ℃ to remove the cross hybridization phenomenon, and mixing with SMBs turbid liquid to prepare a test solution with the concentration of each LNA being 1 mu M. Slightly shaking the test solution, and incubating for 20 minutes at 37 ℃ to better couple biotin and streptavidin to form the magnetic microsphere probe. Washing with 0.15M NaOH solution filters out non-specifically bound or free DNA probe molecules. Followed by TT buffer (250mM Tris-HCl pH 8.0, 0.1%)
Figure BDA0003383924810000061
20) And washing the obtained microsphere probe twice, adding new TT buffer solution to resuspend the magnetic beads, incubating at 80 ℃ for 10 minutes, and pouring off the liquid to further remove streptavidin-biotin combined unstable coupling molecules. Finally, the composite magnetic microsphere probe was washed 3 times with DEPC water in preparation for the next DSN reaction.
miRNA detection and DSN enzyme digestion reaction
20 μ L of the reaction solution was placed in a sterile PCR tube containing 20 μ g of magnetic beads coupled with LNA probe, 2 μ L of miRNA-486-5p at a concentration of 1nM, 1 XDSN primary buffer, 20U of ribonuclease inhibitor, 0.4U of DSN enzyme. Followed by incubation at 40 ℃ for 120 minutes in a thermocycler. After the full hybridization and enzyme digestion reaction is completed, the supernatant is extracted, and the fluorescence intensity in the solution is tested.
4. Fluorescence detection
After the reaction is finished, in order to remove LNA molecules which are connected on the magnetic beads and do not participate in the reaction, the magnetic beads are fixed by strong magnets to extract supernatant, 649/670nm exciting light is used for irradiating liquid containing FAM fluorescent molecules, the fluorescence intensity depends on the quantity of the fluorescent molecules released by the enzyme digestion reaction of the DSN, and the concentration of miRNA in the liquid to be detected is further reflected.
5. Optimizing cultivation temperature
The above experiment was then repeated, except that the incubation temperatures in step 3 were selected to be 45 ℃, 50 ℃, 55 ℃, 60 ℃ and 65 ℃ respectively, and the remaining conditions were unchanged. For the same temperature variation range, the same tests were also performed on miRNA-21(649/670 wavelength detecting cy5) and miRNA-210(545/580 detecting TAMRA group), and the fluorescence intensity detection results are shown in FIG. 3, wherein the ordinate is relative fluorescence intensity and the abscissa is temperature, it can be seen that for each miRNA, the relative fluorescence intensity unit is the maximum when the incubation temperature is 55 ℃, which indicates that 55 ℃ is the optimal incubation temperature.
Among them, DSN master buffer is purchased from Evagen Joint Stock Company under the trade name: DSN master buffer.
EXAMPLE two minimum detection concentration test
Preparation of DNA Probe
Synthesizing corresponding Locked Nucleic Acid (LNA) as a capture probe LNA probe-486-5p according to a base sequence of a to-be-detected marker miRNA486-5p molecule, and respectively modifying molecules such as FAM fluorescent groups and biotin at two ends.
Immobilization of DNA Probe molecules on magnetic beads
The superparamagnetic microsphere and high-purity streptavidin are covalently combined to form streptavidin magnetic beads (SMBs, streptavidin MBs), and a certain amount of SMBs is taken and used as 1 XB at room temperature&W buffer was washed and the supernatant was filtered off with the aid of a permanent magnet and redispersed to 2 XB&W buffer. Taking a proper amount of LNA detection molecules, preheating for 5 minutes at 95 ℃ to remove the cross hybridization phenomenon, and mixing with SMBs turbid liquid to prepare a test solution with the concentration of each LNA being 1 mu M. Slightly shaking the test solution, and incubating for 20 minutes at 37 ℃ to better couple biotin and streptavidin to form the magnetic microsphere probe. Washing with 0.15M NaOH solution filters out non-specifically bound or free DNA probe molecules. Followed by TT buffer (250mM Tris-HCl pH 8.0, 0.1%)
Figure BDA0003383924810000071
20) And washing the obtained microsphere probe twice, adding new TT buffer solution to resuspend the magnetic beads, incubating at 80 ℃ for 10 minutes, and pouring off the liquid to further remove streptavidin-biotin combined unstable coupling molecules. Finally, the composite magnetic microsphere probe was washed 3 times with DEPC water in preparation for the next DSN reaction.
miRNA detection and DSN enzyme digestion reaction
20 μ L of the reaction solution was placed in a sterile PCR tube containing 20 μ g of magnetic beads coupled with LNA probe, 2 μ L of miRNA-486-5p at a concentration of 1nM, 1 XDSN primary buffer, 20U of ribonuclease inhibitor, 0.4U of DSN enzyme. Followed by incubation at 55 ℃ for 120 minutes in a thermocycler. After the full hybridization and enzyme digestion reaction is completed, the supernatant is extracted, and the fluorescence intensity in the solution is tested.
4. Fluorescence detection
After the reaction is finished, in order to remove LNA molecules which are connected on the magnetic beads and do not participate in the reaction, the magnetic beads are fixed by strong magnets to extract supernatant, exciting light is used for irradiating liquid containing fluorescent molecules, the fluorescence intensity depends on the number of the fluorescent molecules released by the enzyme digestion reaction of the DSN, and the concentration of miRNA in the liquid to be detected is further reflected.
5. MiRNA detection at different concentrations
The above experiment was then repeated, except that the miRNA-486-5p concentration in step 3 was gradually decreased from 1nM to 0.5fM, and the conditions were unchanged. In the same miRNA range, the same tests were performed on miRNA-21(649/670 wavelength detection cy5) and miRNA-210(545/580 detection TAMRA group), and the fluorescence intensity detection results are shown in fig. 4, wherein the ordinate is relative fluorescence intensity and the abscissa is miRNA concentration, and it can be seen that when the miRNA concentration is gradually increased from 0.5fM to 1nM, the relative fluorescence intensity unit value is also increased. In the range of 0.5fM to 100pM, the relative fluorescence intensity unit value varies linearly with the logarithm of the miRNA concentration lg (miRNA), and can be expressed by the following formula:
RFU％＝35.23lg([miRNA-21])+214.264
RFU％＝31.21lg([miRNA-210])+170.23
RFU％＝24.16lg([miRNA-486-5p])+173.778
for the above methods, we can measure the minimum concentrations of miRNA-486-5p, miRNA-21, miRNA-210 to 3fM, 120aM, 300aM, respectively.
EXAMPLE three Cross-reactivity test
Preparation of DNA Probe
According to the base sequence of miRNA486-5p molecules of the marker to be detected, corresponding locked nucleic acid is synthesized to be used as a capture probe LNA probe-486-5p, and molecules such as FAM fluorescent groups and biotin are respectively modified at two ends.
Immobilization of DNA Probe molecules on magnetic beads
The superparamagnetic microsphere and high-purity streptavidin are covalently combined to form streptavidin magnetic beads (SMBs, streptavidin MBs), and a certain amount of SMBs is taken and used as 1 XB at room temperature&W buffer was washed and the supernatant was filtered off with the aid of a permanent magnet and redispersed to 2 XB&W buffer. Taking a proper amount of LNA detection molecules, preheating for 5 minutes at 95 ℃ to clear the cross hybridization phenomenon, and mixing with SMBs turbid liquid to prepare a test solution with the concentration of each LNA being 1 mu M. Slightly shaking the test solution, and incubating for 20 minutes at 37 ℃ to better couple biotin and streptavidin to form the magnetic microsphere probe. Washing with 0.15M NaOH solution filters out non-specifically bound or free DNA probe molecules. Followed by TT buffer (250mM Tris-HCl pH 8.0, 0.1%)
Figure BDA0003383924810000081
20) And washing the obtained microsphere probe twice, adding new TT buffer solution to resuspend the magnetic beads, incubating at 80 ℃ for 10 minutes, and pouring off the liquid to further remove streptavidin-biotin combined unstable coupling molecules. Finally, the composite magnetic microsphere probe was washed 3 times with DEPC water in preparation for the next DSN reaction.
miRNA detection and DSN enzyme digestion reaction
20 μ L of the reaction solution was placed in a sterile PCR tube containing 20 μ g of magnetic beads coupled with LNA probe, 2 μ L of miRNA-486-5p at a concentration of 1nM, 1 XDSN primary buffer, 20U of ribonuclease inhibitor, 0.4U of DSN enzyme. Followed by incubation at 55 ℃ for 120 minutes in a thermocycler. After the full hybridization and enzyme digestion reaction is completed, the supernatant is extracted, and the fluorescence intensity in the solution is tested.
4. Fluorescence detection
After the reaction is finished, in order to remove LNA molecules which are connected on the magnetic beads and do not participate in the reaction, the magnetic beads are fixed by strong magnets to extract supernatant, exciting light is used for irradiating liquid containing fluorescent molecules, the fluorescence intensity depends on the number of the fluorescent molecules released by the enzyme digestion reaction of the DSN, and the concentration of miRNA in the liquid to be detected is further reflected. The unit RFU% of fluorescence intensity after reaction of miRNA486-5p with LNA-probe486-5p is about 450, as shown in FIG. 5.
5. Cross reaction test
And then repeating the experiment, changing the nucleic acid molecules for testing in the step 3 into miRNA-21 or miRNA210, keeping the conditions such as the concentration of the nucleic acid molecules, the culture temperature and the like unchanged, and respectively recording the obtained fluorescence intensity after the reaction is finished. The fluorescence intensity value of the obtained fluorescent molecule after the miRNA-21 or the miRNA210 reacts with the LNA probe-486-5p is less than 50.
Similarly, probe molecules LNA probe-21 and LNA probe-210 were synthesized and used to detect the three lung cancer marker nucleic acid molecules, and as can be seen from the results in FIG. 5, the fluorescence intensity obtained after the reaction of LNA probe-21 and miRNA-21 was 600, and the fluorescence intensity obtained after the reaction of LNA probe-210 and miRNA-210 was 470. When the probe molecule is not matched with the base sequence of the nucleic acid molecule to be detected, the relative fluorescence intensity unit value of the reaction solution is below 50.
The results show that in the detection method, the three lung cancer marker nucleic acid molecules miRNA have strong reaction with the corresponding probe molecules, and the cross reaction degree with the other two probe molecules is weaker.
Example four preparation of the inventive composite locked nucleic acid magnetic bead probe and detection of body fluid
1. Locked nucleic acid probe preparation
Synthesizing corresponding locked nucleic acid as capture probes LNA probe-486-5p, LNA probe-21 and LNA probe-210 according to the base sequences of the to-be-detected markers miRNA-486-5p, miRNA-21 and miRNA-210 molecules, respectively modifying FAM fluorescent group and biotin molecules at two ends of LNA probe-486-5p, respectively modifying cy5 fluorescent group and biotin molecules at two ends of LNA probe-21, and respectively modifying TAMRA fluorescent group and biotin molecules at two ends of LNA probe-210. The molecular ratio of the three probes is 1:1: 1.
2. Nucleic acid locking probe molecule is fixed on magnetic bead
The superparamagnetic microsphere and high-purity streptavidin are covalently combined to form streptavidin magnetic beads (SMBs, streptavidin MBs), and a certain amount of SMBs is taken and used as 1 XB at room temperature&W buffer cleaning with the aid of permanent magnetsFiltering off supernatant with iron, and re-dispersing to 2 XB&W buffer. Taking a proper amount of LNA detection molecules, preheating for 5 minutes at 95 ℃ to clear the cross hybridization phenomenon, and mixing with SMBs turbid liquid to prepare a test solution with the concentration of each LNA being 1 mu M. Slightly shaking the test solution, and incubating for 20 minutes at 37 ℃ to better couple biotin and streptavidin to form the magnetic microsphere probe. Washing with 0.15M NaOH solution filters out non-specifically bound or free DNA probe molecules. Followed by TT buffer (250mM Tris-HCl pH 8.0, 0.1%)
Figure BDA0003383924810000091
20) And washing the obtained microsphere probe twice, adding new TT buffer solution to resuspend the magnetic beads, incubating at 80 ℃ for 10 minutes, and pouring off the liquid to further remove streptavidin-biotin combined unstable coupling molecules. Finally, the composite magnetic microsphere probe was washed 3 times with DEPC water in preparation for the next DSN reaction.
miRNA detection and DSN enzyme digestion reaction
mu.L of reaction solution is taken and put into a sterile PCR tube, wherein 20 mu.g of magnetic beads of the composite probe which is coupled with LNA are contained, and 2 mu.L of serum, 1 XDSN main buffer solution, 20U of ribonuclease inhibitor and 0.4U of DSN enzyme are respectively extracted from healthy people or lung cancer patients. Followed by incubation at 55 ℃ for 120 minutes in a thermocycler. After the full hybridization and enzyme digestion reaction is completed, the supernatant is extracted, and the fluorescence intensity in the solution is tested.
4. Fluorescence detection
After the reaction is completed, in order to remove LNA molecules attached to the magnetic beads which do not participate in the reaction, the magnetic beads are fixed with strong magnets to extract supernatant, the liquid containing fluorescent molecules is irradiated with excitation light, the fluorescence intensity of the cy5 group is detected at 649/670 wavelength, the fluorescence intensity of the TAMRA group is detected at 545/580 wavelength, and the fluorescence intensity of the FAM group is detected at 483/520 wavelength; the fluorescence intensity depends on the quantity of fluorescence molecules released by the DSN enzyme digestion reaction, and the concentration of miRNA in the serum to be detected is further reflected.
The test results in FIG. 6 show that the relative fluorescence intensity of the probe molecules LNA probe-21 and LNA probe-210 after reacting with the serum of healthy human is below 50, and the relative fluorescence intensity after reacting with the serum of lung cancer patients is between 110 and 140, which indicates that miRNA-21 and miRNA-210 are over-expressed in the lung cancer patients; after the probe molecule LNA probe-486-5p reacts with the serum of healthy human, the relative fluorescence intensity is between 110-140, and after the probe molecule LNA probe-486-5p reacts with the serum of the lung cancer patient, the relative fluorescence intensity is between 50-160, which indicates that the expression of miRNA-486-5p in the body of the lung cancer patient is inhibited.
Example five diagnostic reagents of the invention comprising a complexed locked nucleic acid magnetic bead probe
The diagnostic reagent of the invention comprises 20 mu g of prepared composite locked nucleic acid magnetic bead probe, 1 XDSN main buffer solution, 20U ribonuclease inhibitor, 0.4U DSN enzyme and standard serum. The detection process of the fourth embodiment can be performed, and an accurate diagnosis can be made for lung cancer.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.

Claims (10)

1. The composite locked nucleic acid magnetic bead probe for detecting the miRNA marker is characterized by comprising three probes, namely an LNA probe-486-5p probe marked by an FAM fluorescent group, an LNA probe-21 probe marked by a Cy5 fluorescent group and an LNA probe-210 probe marked by a TAMRA fluorescent group, in the same magnetic bead.
2. The method for constructing the complex locked nucleic acid magnetic bead probe for detecting miRNA markers according to claim 1, wherein the method comprises the following steps:
(1) preparing locked nucleic acid probe molecules: synthesizing corresponding locked nucleic acid as capture probes LNA probe-486-5p, LNA probe-21 and LNA probe-210 according to the base sequences of the miRNA-486-5p, miRNA-21 and miRNA-210 molecules of the markers to be detected, respectively modifying FAM fluorescent groups and biotin molecules at two ends of the LNA probe-486-5p,
cy5 fluorescent group and biotin molecule are respectively modified at two ends of the LNA probe-21 probe,
TAMRA fluorescent group and biotin molecule are respectively modified at two ends of the LNA probe-210 probe,
mixing the three probes according to a molecular ratio of 1:1:1 to prepare an LNA probe compound;
(2) the nucleic acid locking probe molecules are fixed on the magnetic beads: preheating the probe mixture at 95 ℃ to remove cross hybridization, mixing the probe mixture with streptavidin magnetic bead SMBs suspension, and culturing at room temperature;
(3) cleaning: firstly, washing the cultured composite locked nucleic acid magnetic bead probe solution with NaOH solution to remove non-specifically bound or free DNA probes, then repeatedly washing with TT buffer solution, adding the magnetic bead probes into the TT buffer solution for resuspension, then culturing at 80 ℃, removing unstable coupled molecules bound in the liquid, and finally washing with DEPC water to obtain the composite locked nucleic acid magnetic bead probe for detecting the miRNA marker.
3. The method for constructing the locked nucleic acid magnetic bead probe according to claim 2, wherein in the step (2), the streptavidin magnetic bead suspension is prepared by the following method: and (2) carrying out covalent binding on the superparamagnetic microspheres and streptavidin to form streptavidin magnetic bead SMBs, washing with 1 XBW buffer solution at room temperature, fixing the streptavidin magnetic beads with a permanent magnet, filtering out supernatant, and dispersing in 2 XBW buffer solution to obtain an SMBs suspension.
4. The method for constructing a composite magnetic bead lock nucleic acid probe according to claim 3, wherein in the step (2), the LNA probe composite and the streptavidin magnetic bead are mixed at a ratio of 20pmol LNA to 20 μ g MBs streptavidin magnetic bead in the mixing process.
5. The method for constructing a locked nucleic acid magnetic bead probe according to claim 4, wherein in the step (2), the concentration of each LNA in the prepared locked nucleic acid magnetic bead probe solution is 1. mu.M.
6. The building method according to claim 5, wherein in the step (2), the preheating is preheating at 95 ℃ for 5 min; the incubation was at 37 ℃ for 20 minutes.
7. The constructing method according to claim 6, wherein in the step (3), the concentration of the NaOH solution is 0.15M; the TT buffer solution is prepared according to the following method: adding 0.1% of Tris-HCl buffer solution to 250mM
Figure FDA0003383924800000021
20, and adjusting the pH to 8.0; the incubation was at 80 ℃ for 10 minutes.
8. The use of the composite locked nucleic acid magnetic bead probe of claim 7 for detecting a miRNA marker, comprising the steps of:
step I: and (3) carrying out DSN enzyme digestion reaction:
reaction system: the kit comprises a composite locked nucleic acid magnetic bead probe, miRNA to-be-detected liquid, 1 XDSN main buffer solution, a ribonuclease inhibitor and DSN enzyme; incubation at 55 ℃ for 120 min in a thermocycler; hybridizing a sample miRNA to be detected with the probe to form a double strand, carrying out enzyme digestion reaction on the DSN and the double strand, hydrolyzing the DNA into fragments, and separating out a fluorescent group; the released miRNA is kept complete and hybridized with unreacted DNA again, a new enzyme digestion reaction is generated, and the fluorescent group in the solution is increased;
step II: fluorescence detection
After the reaction is completed, irradiating the liquid containing the fluorescent molecules with exciting light, detecting the fluorescence intensity of cy5 group at 649/670 wavelength, detecting the fluorescence intensity of TAMRA group at 545/580 wavelength and detecting the fluorescence intensity of FAM group at 483/520 wavelength; further obtaining the respective concentrations of the three miRNA markers in the miRNA test solution.
9. The application of the composite locked nucleic acid magnetic bead probe in detecting miRNA markers according to claim 8, wherein in step II, after the reaction in step I is completed, in order to remove unreacted LNA molecules attached to the magnetic bead, the magnetic bead is fixed by a strong magnet to extract supernatant, and then fluorescence detection is performed.
10. A diagnostic reagent comprising the locked nucleic acid magnetic bead probe of claim 7, wherein the diagnostic reagent comprises 20 μ g of the locked nucleic acid magnetic bead probe, 1 XDSN master buffer, 20U RNase inhibitor, 0.4U DSN enzyme and standard serum.
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