Molecular beacon probe composition, kit and application thereof

Abstract

The invention discloses a molecular beacon probe composition, a kit and application thereof, and relates to the technical field of molecular biology. A molecular beacon probe is introduced into an in-vitro transcription system, and the probe emits a fluorescence signal through specific binding with an in-vitro transcription product-RNA target sequence. In a real-time fluorescence monitoring system, the intensity of a probe fluorescence signal is in direct proportion to the RNA concentration transcribed in vitro, so that real-time quantitative detection of an in-vitro transcription product can be realized. In addition, specific probes are respectively arranged on the in-vitro transcription product RNA, and the yield of the in-vitro transcription full-length RNA can be obtained by comparing the fluorescence difference of the two probes. The sequence of the molecular beacon probe can be in specific complementary pairing with partial sequence of in-vitro transcription RNA, so that a target transcription product can be accurately recognized, non-specific binding is avoided, the reliability of data is ensured, and high-sensitivity quantitative analysis is provided.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a molecular beacon probe composition, a kit and applications thereof. Background Art

[0002] mRNA (messenger RNA) is an important molecule in cells, playing a critical role in the transmission of genetic information in organisms. In organisms, genetic information is transferred from DNA to mRNA through the transcription process, and mRNA is then translated to generate proteins, which in turn form various biological functions. In recent years, with the continuous deepening of research, people have found that DNA can also be transcribed into mRNA by constructing a suitable reaction system in vitro and optimizing the buffer, salt ions, NTP, RNA polymerase and other components in the system. The development of in vitro transcription system provides technical support for the functional research of RNA, and greatly promotes the development of disease diagnosis, materials, nucleic acid drugs and other fields using RNA as a carrier. Among them, RNA vaccine is one of the most eye-catching applications in recent years. For example, Pfizer and Moderna's COVID-19 vaccine is an mRNA vaccine. After entering the organism, the mRNA vaccine can use the organism's own translation system to produce antigenic proteins, thereby inducing the body to produce antibodies and immune responses, protecting individuals from infection with specific pathogens. In addition, mRNA can also be used as a drug for disease treatment, which introduces normal mRNA to compensate for missing or dysfunctional proteins to treat genetic diseases or certain cancers. Of course, mRNA can also be used to produce various enzymes or proteins, including ribozymes and industrial enzymes, which generate large-scale ribozyme or protein products through the translation process.

[0003] Existing in vitro transcribed mRNA quantitative methods mainly include qPCR, RT-qPCR, RNA-Seq, Northern Blot, ELISA, absorbance detection, etc. However, all of the above methods require the extraction of the transcribed RNA and cannot be analyzed in situ. mRNA is easily affected by factors such as environmental cleanliness, transcription system, purification strategy, etc. during in vitro transcription, purification, and detection. Not only is the process time-consuming and laborious, but the product is easily degraded, which has a great impact on subsequent operations. Therefore, it is urgent to develop a real-time detection method for in vitro transcription with fewer operating steps and processes, low detection complexity, rapidity, accuracy, and efficiency.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a molecular beacon probe composition, a kit and applications thereof to solve the above technical problems.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a molecular beacon probe composition, comprising: a first molecular beacon probe and a second molecular beacon probe, both of which have a stem-loop structure, the first molecular beacon probe comprising a first loop sequence and a first stem sequence, and the first loop sequence is complementary to a base at a first position on a target RNA, the first stem sequence comprises a complementary first upstream stem sequence and a first downstream stem sequence, and the first loop sequence is located between the first upstream stem sequence and the first downstream stem sequence;

[0008] The second molecular beacon probe includes a second loop sequence and a second stem sequence, and the second loop sequence is complementary to the base at the second position on the target RNA, the second stem sequence includes a complementary second upstream stem sequence and a second downstream stem sequence, and the second loop sequence is located between the second upstream stem sequence and the second downstream stem sequence;

[0009] The 5' ends of the first molecular beacon probe and the second molecular beacon probe are both provided with a fluorescent reporter group, and the fluorescent reporter groups of the first molecular beacon probe and the second molecular beacon probe are different; the 3' ends are both provided with a quenching group capable of quenching the fluorescent reporter group;

[0010] The length of the first loop sequence and the second loop sequence is 15-30 bp, the length of the first stem sequence and the second stem sequence is 4-8 bp, and the first position is located upstream of the second position.

[0011] In a second aspect, the present invention further provides a kit, which comprises: the above-mentioned molecular beacon probe composition and a transcription reaction solution.

[0012] In a third aspect, the present invention also provides an application of a molecular beacon probe composition or the above-mentioned kit in the quantitative detection of in vitro transcription products. The application is not for the purpose of disease diagnosis, and the application includes: mixing an RNA standard with the molecular beacon probe composition, performing a transcription reaction in a transcription reaction system, performing fluorescence quantification of the transcription reaction product, drawing a standard curve of the concentration of the RNA standard and the maximum fluorescence intensity, obtaining the maximum fluorescence intensity of the in vitro transcription process of the DNA sample to be tested, and obtaining the amount of the product after in vitro transcription of the DNA sample to be tested through the standard curve.

[0013] In a fourth aspect, the present invention also provides an application of a molecular beacon probe composition or the above-mentioned kit in detecting the yield of in vitro transcription products, the application comprising: mixing an RNA standard with the molecular beacon probe composition, performing a transcription reaction in a transcription reaction system, performing fluorescence quantification on the transcription reaction product, drawing a first standard curve of the maximum fluorescence intensity of the first RNA standard and the first molecular beacon probe, and drawing a second standard curve of the concentration of the second RNA standard and the maximum fluorescence intensity of the second molecular beacon probe; obtaining the maximum fluorescence intensity of the first molecular beacon probe and the second molecular beacon probe during the in vitro transcription of the DNA sample to be tested, obtaining the first product amount and the second product amount of the DNA sample to be tested after in vitro transcription through the standard curve of the first RNA standard and the standard curve of the second RNA standard, and obtaining the in vitro transcription product yield according to the ratio of the second product amount to the first product amount.

[0014] The present invention has the following beneficial effects:

[0015] The present invention provides a molecular beacon probe composition, which introduces a molecular beacon probe into an in vitro transcription system for real-time, rapid quantitative detection of products. The molecular beacon probe usually has a fluorescent group and a quenching group at both ends. When the probe is not bound to the target RNA, the probe is a stem-loop structure, and the fluorescent group and the quenching group are close to each other, and the probe does not emit fluorescence or the fluorescence signal is very low. When the probe is bound to the target RNA, the probe is a linear structure, and the fluorescent group and the quenching group are far apart, and the fluorescent group signal cannot be quenched, so that the probe emits fluorescence. A molecular beacon probe is added to the in vitro transcription system, and the probe emits fluorescence by specific binding to the transcription product RNA sequence, and the intensity of the fluorescence signal is proportional to the RNA yield, thereby realizing quantitative detection of in vitro transcription products.

[0016] Molecular beacon probes are designed to be fully complementary to specific RNA sequences, and can accurately identify target transcription products, avoid nonspecific binding, and ensure data reliability. The release of fluorescent signals after probe binding is proportional to RNA abundance, and can detect low-abundance RNA, thereby providing highly sensitive quantitative analysis. Combined with real-time PCR technology, molecular beacon probes can monitor changes in fluorescent signals in real time during the reaction process, achieve dynamic data collection, facilitate rapid acquisition of experimental results, and also avoid RNA loss and degradation. These advantages make molecular beacon probes an efficient and accurate technology for RNA quantitative analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 This is a schematic diagram of the standard curve results established using the VIC probe for standard RNA① in an embodiment of the present invention;

[0019] Figure 2 This is a schematic diagram of the standard curve results established using the FAM probe for standard RNA② in the embodiment of the present invention;

[0020] Figure 3 This is a result diagram of using the VIC probe to detect RNA products in the transcription product reaction system in an embodiment of the present invention;

[0021] Figure 4 This is a result diagram of detecting RNA products in the transcription product reaction system using a FAM probe in an embodiment of the present invention. DETAILED DESCRIPTION

[0022] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0023] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0024] Molecular beacon probes are probes that can specifically recognize and detect target nucleic acid sequences. Both ends of the probes are usually labeled with a fluorescent group that can emit fluorescence and a quenching group that prevents the emission of the fluorescent group. When the fluorescent group of the probe binds to the target nucleic acid sequence, it will emit fluorescence and be detected.

[0025] In the present invention, the term "promoter sequence" or "promoter" refers to a dsDNA sequence that RNA polymerase recognizes, binds to and initiates transcription, and may be a naturally occurring or artificially modified sequence that can be recognized by RNA polymerase.

[0026] In the present invention, the term "base complementary pairing" or "base complementarity" refers to the Watson / Crick base pairing rules, where the bases of each nucleotide residue in a nucleic acid molecule are A with T, A with U, 6m A and T, G and C, etc. are connected to each other by hydrogen bonds.

[0027] In the present invention, "transcription" and "in vitro transcription" refer to the process of synthesizing RNA under the catalysis of RNA polymerase using a determined strand of double-stranded DNA (the template strand is used for transcription, and the sense strand is not used for transcription) as a template and four ribonucleotides or analogs of A, U, C, and G as raw materials.

[0028] In a first aspect, the present invention provides a molecular beacon probe composition, comprising: a first molecular beacon probe and a second molecular beacon probe, both of which have a stem-loop structure, the first molecular beacon probe comprising a first loop sequence and a first stem sequence, and the first loop sequence is complementary to a base at a first position on a target RNA, the first stem sequence comprises a complementary first upstream stem sequence and a first downstream stem sequence, and the first loop sequence is located between the first upstream stem sequence and the first downstream stem sequence;

[0029] The second molecular beacon probe includes a second loop sequence and a second stem sequence, and the second loop sequence is complementary to the base at the second position on the target RNA, the second stem sequence includes a complementary second upstream stem sequence and a second downstream stem sequence, and the second loop sequence is located between the second upstream stem sequence and the second downstream stem sequence;

[0030] The 5' ends of the first molecular beacon probe and the second molecular beacon probe are both provided with a fluorescent reporter group, and the fluorescent reporter groups of the first molecular beacon probe and the second molecular beacon probe are different; the 3' ends are both provided with a quenching group capable of quenching the fluorescent reporter group;

[0031] The length of the first loop sequence and the second loop sequence is 15-30 bp, the length of the first stem sequence and the second stem sequence is 4-8 bp, and the first position is located upstream of the second position.

[0032] The molecular beacon probe has a fluorescent group and a quenching group at both ends. When the probe is not bound to the target RNA, the probe is a stem-loop structure, and the fluorescent group and the quenching group are close to each other, and the probe does not emit fluorescence or the fluorescence signal is very low. When the probe is bound to the target RNA, the probe is a linear structure, and the fluorescent group and the quenching group are far apart, and the fluorescent group signal cannot be quenched, so that the probe emits fluorescence. A molecular beacon probe is added to the in vitro transcription system, and the probe emits fluorescence by specific binding to the transcription product RNA sequence, and the intensity of its fluorescence signal is proportional to the yield of RNA, thereby achieving quantitative detection of in vitro transcription products.

[0033] The target RNA is the RNA molecule transcribed in vitro.

[0034] The length of the first ring sequence and the second ring sequence is 15bp, 16bp, 17bp, 18bp, 19bp, 20bp, 21bp, 22bp, 23bp, 24bp, 25bp, 26bp, 27bp, 28bp, 29bp or 30bp. The length of the second upstream stem sequence and the second downstream stem sequence is 4bp, 5bp, 6bp, 7bp or 8bp. The length of the first upstream stem sequence and the first downstream stem sequence is 4bp, 5bp, 6bp, 7bp or 8bp.

[0035] In a preferred embodiment of the present invention, the first position is located at the 5' end of the target RNA; such a position facilitates monitoring of the 5' end of the transcribed RNA molecule, helps monitor RNA molecules with missing partial transcribed fragments, and detects the integrity of the transcribed mRNA molecule.

[0036] In a preferred embodiment of the present invention, the second position is located at the 3' end of the target RNA. Such a position facilitates monitoring the 3' end of the transcribed RNA molecule, helps monitor RNA molecules with missing partial transcribed segments, and detects the integrity of the transcribed mRNA molecule.

[0037] In a preferred embodiment of the present invention, the first position is the region where the T7, T3, SP6, K1-5, K1E, K1F or K11 transcription promoter starts transcribing RNA. Preferably, the first position is the region where the T7 transcription promoter starts transcribing RNA.

[0038] In a preferred embodiment of the present invention, the bases on the first molecular beacon probe and the second molecular beacon probe are modified or unmodified. Those skilled in the art can set the modification groups of the bases on the molecular beacon probes as needed to improve the stability and detection sensitivity of the probes.

[0039] In a preferred embodiment of the present invention, the fluorescent reporter group is selected from FAM, VIC, HEX, JOE, ROX, Cy5, TET, CY3, Texas RED, LC RED460, FITC group, AMC group, Alexa Fluor group, Rhodamine B group, BODIPY group, Cy5.5 group, Cy7 group, ICG group, IR780 group, IR820 group or IRDye 800 group; the quencher group is selected from BHQ0, BHQ1, BHQ2, BHQ3, TAMRA or DABCYL.

[0040] In a preferred embodiment of the present invention, the fluorescent reporter group on the first molecular beacon probe is VIC, the fluorescent reporter group on the second molecular beacon probe is FAM; and the quencher group is selected from BHQ1.

[0041] FAM (6-carboxyfluorescein): FAM is a common water-soluble fluorescent dye with high quantum yield (high quantum efficiency) and good photostability. It is connected to the 3' end of the probe and can be easily detected. The excitation wavelength of FAM is usually around 485nm and the emission wavelength is around 518nm.

[0042] VIC: VIC is a fluorescent dye similar to FAM, but its spectral properties are slightly different. The excitation wavelength of VIC is around 530nm and the emission wavelength is around 570nm.

[0043] Correspondingly, the quencher group was selected as BHQ-1.

[0044] BHQ-1: BHQ-1 is a non-emitting fluorescent group, usually connected to the 5' end of the probe, to quench the natural fluorescence of FAM and other fluorophores. When BHQ1 binds to the fluorophore, it blocks the energy emission of the fluorophore, thereby reducing background fluorescence and improving the specificity and sensitivity of the detection.

[0045] In a preferred embodiment of the present invention, the first upstream stem sequence is the same as or different from the second upstream stem sequence, preferably the same.

[0046] In a preferred embodiment of the present invention, the first loop sequence is as shown in SEQ ID NO: 1; AATTTTTTCAAATACCAT.

[0047] In a preferred embodiment of the present invention, the first upstream stem sequence is CACGCT, and the first downstream stem sequence is AGCGTG;

[0048] The second loop sequence is as shown in SEQ ID NO: 2, GTGGTGGTGGTGGTGGTG; the second upstream stem sequence is CACGCT, and the second downstream stem sequence is AGCGTG.

[0049] In a second aspect, the present invention further provides a kit, which comprises: the above-mentioned molecular beacon probe composition and a transcription reaction solution;

[0050] In a preferred embodiment of the present invention, the transcription reaction solution includes at least one of NTPs, transcription reaction buffer, RNA polymerase, RNA enzyme inhibitor and water.

[0051] The kit also includes a positive control.

[0052] Positive controls include RNA Standard 1 and RNA Standard 2. RNA Standard 1:

[0053] 5'-GGAGAUAUACAUAUGGUAUUUGAAAAAAUUGAUAAAAAAUAGUUGG-3'; (SEQ ID NO: 3)

[0054] RNA Standard 2:

[0055] 5'-CAAGUAGCUCGAGCACCACCACCACCACUGAGAUCCGGCUGCU-3'. (SEQ ID NO: 4)

[0056] In a third aspect, the present invention also provides an application of a molecular beacon probe composition or the above-mentioned kit in the quantitative detection of in vitro transcription products. The application is not for the purpose of disease diagnosis, and the application includes: mixing an RNA standard with the molecular beacon probe composition, performing a transcription reaction in a transcription reaction system, performing fluorescence quantification of the transcription reaction product, drawing a standard curve of the concentration of the RNA standard and the maximum fluorescence intensity, obtaining the maximum fluorescence intensity of the in vitro transcription process of the DNA sample to be tested, and obtaining the amount of the product after in vitro transcription of the DNA sample to be tested through the standard curve.

[0057] The RNA standard includes RNA standard 1 and RNA standard 2. In an optional embodiment, only one RNA standard, such as RNA standard 2, may be provided. The maximum fluorescence intensity after the transcription reaction of RNA standard 2 and the second molecular beacon probe at different concentrations is detected. The corresponding standard curve is drawn, and the maximum fluorescence intensity of the in vitro transcription process of the DNA sample to be tested is further obtained by a fluorescence quantifier, and the product amount of the DNA sample to be tested after in vitro transcription is obtained by substituting the standard curve. The fluorescence value collected by qPCR increases with the increase of the mRNA yield of the transcription product.

[0058] The second molecular beacon probe binds to the terminal sequence of the product RNA, and the RNA yield can be obtained through the fluorescence value; the first molecular beacon probe is complementary to the front-end sequence of the transcription product RNA, and the ratio of the RNA yield corresponding to the maximum fluorescence value of the second molecular beacon probe to the RNA yield corresponding to the maximum fluorescence value of the first molecular beacon probe is calculated to obtain the in vitro transcription product yield.

[0059] Molecular beacon probes with fluorescent groups and quenching groups have the following advantages in detecting in vitro transcribed RNA yield:

[0060] High sensitivity: The design of the fluorescent group and the quenching group can significantly improve the sensitivity of the detection. When the molecular beacon probe binds to the target RNA, the distance between the fluorescent group and the quenching group changes, resulting in a significant increase in the fluorescent signal, making the detection more sensitive.

[0061] High specificity: Molecular beacon probes can be designed to specifically bind to target RNA sequences, thereby reducing interference from nonspecific signals. This high specificity ensures the accuracy and reliability of the test results.

[0062] Real-time monitoring: Molecular beacon probes are often used in real-time fluorescence quantitative PCR (qPCR), allowing real-time monitoring of in vitro transcribed RNA. This real-time monitoring can provide dynamic information on the changes in RNA production over time, thus facilitating dynamic tracking of experiments.

[0063] Simplified operation: The use of molecular beacon probes can reduce the need for additional markers and simplify experimental operations. Since the probe design already includes fluorescent and quenching groups, no additional fluorescent dyes or markers are required, reducing the number of experimental steps.

[0064] Improve detection accuracy: Since the enhancement or inhibition of the fluorescence signal is directly related to the probe binding, the detection results are more accurate and reliable, and can effectively avoid false positive or false negative results.

[0065] During detection, the molecular beacon probe combination is reacted in one reaction system. Compared with setting the first molecular beacon probe and the second molecular beacon probe in two reaction systems, detection errors caused by cross-system interference, different batches, operators and equipment can be avoided.

[0066] In a fourth aspect, the present invention also provides an application of a molecular beacon probe composition or the above-mentioned kit in detecting the yield of in vitro transcription products, the application comprising: mixing an RNA standard with the molecular beacon probe composition, performing a transcription reaction in a transcription reaction system, performing fluorescence quantification on the transcription reaction product, drawing a first standard curve of the maximum fluorescence intensity of the first RNA standard and the first molecular beacon probe, and drawing a second standard curve of the concentration of the second RNA standard and the maximum fluorescence intensity of the second molecular beacon probe; obtaining the maximum fluorescence intensity of the first molecular beacon probe and the second molecular beacon probe during the in vitro transcription of the DNA sample to be tested, obtaining the first product amount and the second product amount of the DNA sample to be tested after in vitro transcription through the standard curve of the first RNA standard and the standard curve of the second RNA standard, and obtaining the in vitro transcription product yield according to the ratio of the second product amount to the first product amount.

[0067] In a preferred embodiment of the present invention, the sample to be tested is derived from an animal, a plant, a microorganism or the environment, and the conditions for the transcription reaction are: incubation at 37°C ± 0.5°C for 1-2h;

[0068] In a preferred embodiment of the present invention, in the reaction system, the final concentration of the first molecular beacon probe and the second molecular beacon probe is 0.5-2 μM.

[0069] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0070] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0071] Example 1

[0072] 1. Preparation of Linearized Template

[0073] The plasmid was synthesized by entrusting Sangon Biotech (Shanghai) Co., Ltd.; the plasmid was linearized using Pst I fast-cutting enzyme, and the enzyme digestion reaction system was as follows:

[0074]

[0075] The above components were mixed and incubated at 37°C for 2 h. The product was purified using a gel recovery kit. The recovered template DNA (SEQ ID No. 5) had a concentration of 600 ng / μl as measured by a NanoDrop UV spectrophotometer and was stored at -20°C for later use.

[0076] SEQ ID No.5:

[0077]

[0078] 2. Establishment of standard curve

[0079] Experimental methods:

[0080] In this experiment, the in vitro transcription reaction system used was the Sangon B639253 High-yield T7 RNA in vitro transcription kit;

[0081] The sequence of the first molecular beacon probe (i.e., probe ①) used is:

[0082] 5'-VIC-CACGCTAATTTTTTCAAATACCATAGCGTG-BHQ-1-3',

[0083] The second molecular beacon probe sequence (i.e., probe ②) is:

[0084] 5'-FAM-CACGCTGTGGTGGTGGTGGTGGTGAGCGTG-BHQ-1-3'.

[0085] Accordingly, the standard RNA① sequence for the two sets of probes to be complementary is:

[0086] 5'-GGAGAUAUACAUAUGGUAUUUGAAAAAAUUGAUAAAAAAUAGUUGG-3';

[0087] The standard RNA② sequence is:

[0088] 5'-CAAGUAGCUCGAGCACCACCACCACCACCACUGAGAUCCGGCUGCU-3'.

[0089] (1) Experimental Materials

[0090]

[0091]

[0092] (2) Experimental design

[0093] According to the kit instructions, the reaction system is:

[0094]

[0095] The dilution system used to establish the standard curve is:

[0096]

[0097] Ensure that the amount of the two probes added is fixed, and use the standard RNA as a variable for gradient dilution; prepare the experimental group according to the following formula:

[0098]

[0099] The blank control group was prepared according to the following formula:

[0100]

[0101] Three parallel experiments were set up for each group to reduce errors.

[0102] Mix the above experimental groups and put them into the qPCR instrument. According to the in vitro transcription reaction conditions, set the program to react at 37°C for 2 hours and collect fluorescence.

[0103] (3) Results analysis

[0104] In the embodiment, the concentration of the standard RNA is used as the horizontal axis, and the maximum fluorescence value collected is used as the vertical axis to establish a standard curve between the two probes and the corresponding standard RNA.

[0105] The results of VIC fluorescence standard curve and FAM fluorescence standard curve refer to Figure 1 and Figure 2 As shown, the results show that the standard curve has good linearity and can be used for quantitative detection of transcription products.

[0106] Example 2

[0107] This example provides an in vitro transcription reaction system.

[0108] (1) According to the first molecular beacon probe and the second molecular beacon probe provided in Example 1, the following in vitro transcription reaction system for practical application is prepared. The sources of the substances in the system are the same as those in Example 1. The in vitro transcription efficiency of the template DNA is detected:

[0109]

[0110] Four parallel groups were set up for each group. The above components were mixed and incubated at 37°C for 2 h according to the above in vitro transcription reaction conditions, and the fluorescence during the process was collected.

[0111] (2) Results Analysis

[0112] After the results were processed, the four groups of FAM fluorescence values ​​and VIC fluorescence values ​​(Table 1) actually collected at the final stable time point were substituted into the corresponding standard curves to obtain the mass of the corresponding product relative to the standard RNA.

[0113] Table 1

[0114]

[0115]

[0116] In addition, the mass of the corresponding standard RNA calculated in the previous step can be used to obtain the molar amount of the corresponding standard RNA in the system, where the molar amount of the corresponding standard RNA ① is 0.029 nmol, and the molar amount of the corresponding standard RNA ② is 0.116 nmol. The yield of the full-length RNA is 24.946% (molar amount of standard RNA ① / molar amount of standard RNA ②).

[0117] Figure 3 The statistical result graph of VIC fluorescence values ​​collected at each time point is shown in the figure. Figure 4 Statistical results of FAM fluorescence values ​​collected at each time point.

[0118] In summary, by substituting the real-time fluorescence values ​​detected by the two probes in the in vitro transcription reaction system into the corresponding standard curve, the concentration of the real-time in vitro transcription reaction product RNA can be obtained. This method can effectively monitor the real-time yield of the product during the in vitro transcription reaction and reduce the loss of subsequent detection; since the two probes are respectively bound to the front and end of the transcript, the fluorescence values ​​detected by the two probes at a certain moment are substituted into the standard curve, and the yield of long-chain RNA at this moment can be obtained according to the corresponding RNA concentration ratio. This indicator can be further used to detect the properties of RNA polymerase.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A molecular beacon probe composition, characterized in that: It includes: A first molecular beacon probe and a second molecular beacon probe, wherein the first molecular beacon probe and the second molecular beacon probe both have a stem-loop structure, the first molecular beacon probe comprises a first loop sequence and a first stem sequence, and the first loop sequence is complementary to a base at a first position on a target RNA, the first stem sequence comprises a complementary first upstream stem sequence and a first downstream stem sequence, and the first loop sequence is located between the first upstream stem sequence and the first downstream stem sequence; The second molecular beacon probe includes a second loop sequence and a second stem sequence, and the second loop sequence is complementary to the base at the second position on the target RNA, the second stem sequence includes a complementary second upstream stem sequence and a second downstream stem sequence, and the second loop sequence is located between the second upstream stem sequence and the second downstream stem sequence; The 5' ends of the first molecular beacon probe and the second molecular beacon probe are both provided with a fluorescent reporter group, and the fluorescent reporter groups of the first molecular beacon probe and the second molecular beacon probe are different; the 3' ends are both provided with a quenching group capable of quenching the fluorescent reporter group; The lengths of the first loop sequence and the second loop sequence are 15-30 bp, the lengths of the first stem sequence and the second stem sequence are 4-8 bp, and the first position is located upstream of the second position.

2. The molecular beacon probe composition according to claim 1, characterized in that The first position is located at the 5' end of the target RNA; Preferably, the second position is located at the 3' end of the target RNA.

3. The molecular beacon probe composition according to claim 2, characterized in that: The first position is the region where the T7, T3, SP6, K1-5, K1E, K1F or K11 transcription promoter starts transcribing RNA.

4. The molecular beacon probe composition according to claim 1, characterized in that The bases on the first molecular beacon probe and the second molecular beacon probe are modified or unmodified; The fluorescent reporter group is selected from FAM, VIC, HEX, JOE, ROX, Cy5, TET, CY3, Texas RED, LC RED460, FITC group, AMC group, Alexa Fluor group, Rhodamine B group, BODIPY group, Cy5.5 group, Cy7 group, ICG group, IR780 group, IR820 group or IRDye 800 group; The quenching group is selected from BHQ0, BHQ1, BHQ2, BHQ3, TAMRA or DABCYL; Preferably, the fluorescent reporter group on the first molecular beacon probe is VIC, and the fluorescent reporter group on the second molecular beacon probe is FAM; and the quencher group is selected from BHQ1.

5. The molecular beacon probe composition according to claim 1, characterized in that The first upstream stem sequence is the same as or different from the second upstream stem sequence.

6. The molecular beacon probe composition according to claim 5, characterized in that: The first loop sequence is shown in SEQ ID NO: 1; Preferably, the first upstream stem sequence is CACGCT, and the first downstream stem sequence is AGCGTG; The second loop sequence is as shown in SEQ ID NO: 2; the second upstream stem sequence is CACGCT, and the second downstream stem sequence is AGCGTG.

7. A kit, characterized in that: It comprises: the molecular beacon probe composition according to any one of claims 1 to 6 and a transcription reaction solution; Preferably, the transcription reaction solution comprises at least one of NTPs, transcription reaction buffer, RNA polymerase, RNA enzyme inhibitor and water; Preferably, the kit further comprises a positive control.

8. Use of the molecular beacon probe composition according to any one of claims 1 to 6 or the kit according to claim 7 in quantitative detection of in vitro transcription products, characterized in that: The application is not intended for the diagnosis of a disease, and the application includes: mixing an RNA standard with the molecular beacon probe composition, performing a transcription reaction in a transcription reaction system, performing fluorescence quantification on the transcription reaction product, drawing a standard curve between the concentration of the RNA standard and the maximum fluorescence intensity, obtaining the maximum fluorescence intensity during the in vitro transcription process of the DNA sample to be tested, and obtaining the amount of the product after in vitro transcription of the DNA sample to be tested through the standard curve.

9. Use of the molecular beacon probe composition according to any one of claims 1 to 6 or the kit according to claim 7 in detecting the yield of in vitro transcription products, characterized in that: The application includes: mixing an RNA standard with the molecular beacon probe composition, performing a transcription reaction in a transcription reaction system, performing fluorescence quantification on the transcription reaction product, drawing a first standard curve of the maximum fluorescence intensity of the first RNA standard and the first molecular beacon probe, and drawing a second standard curve of the concentration of the second RNA standard and the maximum fluorescence intensity of the second molecular beacon probe; obtaining the maximum fluorescence intensity of the first molecular beacon probe and the second molecular beacon probe during the in vitro transcription of the DNA sample to be tested, obtaining the first product amount and the second product amount of the DNA sample to be tested after in vitro transcription through the standard curve of the first RNA standard and the standard curve of the second RNA standard, and obtaining the in vitro transcription product yield according to the ratio of the second product amount to the first product amount.

10. The use according to claim 8 or 9, characterized in that: The sample to be tested is derived from animals, plants, microorganisms or the environment, and the conditions of the transcription reaction are: incubation at 37°C ± 0.5°C for 1-2h; Preferably, in the reaction system, the final concentration of the first molecular beacon probe and the second molecular beacon probe is 0.5-2 μM.

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