Compositions, kits, uses thereof and methods

Abstract

The application discloses a composition, a kit, uses and methods thereof. The application provides a detection system based on RPA-Nfo amplification and nucleic acid flow layer chromatography technology, which can qualitatively detect isomer BCR1, BCR2 and BCR3 generated by recombination of PML gene at different breakpoint set areas, and can separately detect BCR1, BCR2 and BCR3. The detection system is simple to operate, can effectively shorten the detection time, has good accuracy, and is convenient for in-hospital rapid diagnosis and typing of acute promyelocytic leukemia (APL), and assists doctors in drug guidance.

Description

Technical Field

[0001] This article generally relates to the field of biotechnology, and in particular to a composition, kit, its use, and a method for detecting the PML / RARA fusion gene in a sample. Background Technology

[0002] Acute promyelocytic leukemia (APL) is a specific type of acute myeloid leukemia (AML-M3), characterized by the formation of the PML / RARA fusion gene, which is caused by the chromosomal translocation t(15;17)(q22;q12). The protein product of the PML / RARA fusion gene leads to cell differentiation arrest and insufficient apoptosis, and is a key molecular mechanism in the pathogenesis of APL. This fusion gene exhibits heterogeneity; PML gene recombination can occur in different breakpoint concentration regions, producing three main isoforms: BCR1 (L-type), BCR2 (V-type), and BCR3 (S-type).

[0003] Detection of the PML / RARA fusion gene is crucial for the diagnosis of APL, as it is highly correlated with the AML-M3 genotype. Traditional detection methods include cytomorphological assessment and molecular biology techniques, but these methods are limited by their time-consuming nature, operational complexity, and the need for specialized equipment. Therefore, developing a rapid, accurate, and low-cost detection method is of great significance for the early diagnosis and treatment of APL. Summary of the Invention

[0004] Based on this, this application provides a composition comprising:

[0005] Upstream bait oligonucleotide, wherein the upstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO:1-5;

[0006] Downstream decoy oligonucleotides, wherein the downstream decoy oligonucleotides are derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 6-8; and

[0007] The probe sequence is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or two sequences in SEQ ID NO: 9-10.

[0008] In another aspect, this application also provides a kit comprising the composition described herein.

[0009] On the other hand, this application also provides the use of the compositions or kits described herein in the early diagnosis and / or subtyping of acute promyelocytic leukemia (APL).

[0010] On the other hand, this application also provides a method for detecting the PML / RARA fusion gene in a sample, comprising:

[0011] RPA-Nfo amplification was performed on the test sample using the composition or kit described herein, followed by determination of the amplification products by electrophoresis, fluorescent probe method or lateral flow chromatography. The results were used to determine the presence and / or type of the PML / RARA fusion gene in the sample.

[0012] To address the issues of complex operation, long amplification time, and high cost, this application provides a method that utilizes the RPA-Nfo amplification system and nucleic acid flow chromatography to qualitatively detect isoforms BCR1, BCR2, and BCR3 generated from different breakpoint concentration regions of PML gene recombination. This method also allows for the separate detection of BCR1, BCR2, and BCR3. The system is simpler to operate, effectively shortens detection time, and offers good accuracy, facilitating rapid diagnosis and classification of acute promyelocytic leukemia (APL) within hospitals and assisting physicians in guiding medication use.

[0013] Compared with existing technologies, this application uses RPA technology to detect PML / RARA fusion genes, which improves the speed of clinical testing for AML leukemia, provides a simple and less demanding solution, increases the accessibility of PML / RARA fusion gene detection while ensuring high sensitivity for nucleic acid detection, and has high sample tolerance, good compatibility and wide range of applications.

[0014] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the embodiments described in the description and the accompanying drawings. Attached Figure Description

[0015] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0016] Figure 1This is a schematic diagram illustrating the binding of a probe sequence to a template and its cleavage by the Nfo enzyme in one embodiment of this application.

[0017] Figure 2 The results for Example 2 of this application, using probe P1 with upstream primers F11, F12, and F13 and downstream primers R1, R2, and R3, and the detection results using BCR3 type standard, are shown. From left to right, the combinations are F11+R1, F12+R1, F13+R1, F11+R2, F12+R2, F13+R2, F11+R3, F12+R3, and F13+R3, a total of 9 combinations. The top and bottom images show repeated experiments of the same combination.

[0018] Figure 3 The results of the detection using BCR3 standard are shown in Example 2 of this application, where probe P1 is combined with upstream primer F14 and downstream primers R1, R2, R3, and R4. From left to right, the combinations are P1+F14+R1, P1+F14+R2, P1+F14+R3, and P1+F14+R4, a total of four combinations.

[0019] Figure 4 In Example 2 of this application, probe P1 was used in combination with upstream primers F11, F12, F13, and F14 and downstream primer R4, and detection was performed using BCR3 standard; and probe P2 was used in combination with upstream primers F21, F22, and F23 and downstream primer R4, and detection was performed using BCR1 standard. From left to right, the combinations are P1+F11+R4, P1+F12+R4, P1+F13+R4, P1+F14+R4, P2+F21+R4, P2+F22+R4, and P2+F23+R4, for a total of 7 combinations.

[0020] Figure 5 The results for using negative standards are shown in Example 2 of this application, where probe P1 is combined with upstream primers F11, F12, and F13 and downstream primers R1, R2, and R3. From left to right, the combinations are F11+R1, F12+R1, F13+R1, F11+R2, F12+R2, F13+R2, F11+R3, F12+R3, and F13+R3, a total of 9 combinations. The top and bottom images show repeated experiments of the same combination.

[0021] Figure 6The results for Example 2 of this application, using probe P1 with upstream primers F11, F12, and F13 and downstream primers R1, R2, and R3, and the detection results using BCR1 and BCR2 standards, are shown. From left to right, the combinations are F11+R1, F12+R1, F13+R1, F11+R2, F12+R2, F13+R2, F11+R3, F12+R3, and F13+R3, a total of 9 combinations. The upper part shows the results using BCR1 standard, and the lower part shows the results using BCR2 standard.

[0022] Figure 7 The images show the detection results using BCR1 and BCR2 standards in Example 2 of this application, where probe P2 is combined with upstream primers F21 and F22 and downstream primers R1, R2, and R3. From left to right, the combinations are F21+R1, F22+R1, F21+R2, F22+R2, F21+R3, and F22+R3, a total of six combinations. The images above and below show the tests using BCR1 and BCR2 standards.

[0023] Figure 8 The images show the test results of negative standards in Example 2 of this application, using probe P2 with upstream primers F21 and F22 and downstream primers R1, R2, and R3. From left to right, the combinations are F21+R1, F22+R1, F21+R2, F22+R2, F21+R3, and F22+R3, a total of 6 combinations. The top and bottom images show the test results of negative standards.

[0024] Figure 9 The results of the detection using BCR3 standard are shown in Example 2 of this application, which uses probe P2 in combination with upstream primers F21 and F22 and downstream primers R1, R2, and R3. From left to right, they are F21+R1, F22+R1, F21+R2, F22+R2, F21+R3, and F22+R3, a total of 6 combinations. Detailed Implementation

[0025] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application pertains. When a quantity, concentration, or other value or parameter is expressed as a range, preferred range, or preferred upper and lower numerical limits, it should be understood that this is equivalent to specifically disclosing any range by combining any pair of upper or preferred values ​​with any lower or preferred value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range.

[0026] When used with a numerical variable, the terms "about" or "approximately" usually mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within the 95% confidence interval of the mean) or within ±10% of the specified value, or a wider range.

[0027] The expression "comprising," or similar expressions such as "including," "containing," and "having," is open-ended and does not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0028] The expression "at least one" or "one or more" indicates 1, 2, 3, 4, 5, 6, 7, 8, 9 or more kinds.

[0029] For the diagnosis of acute promyelocytic leukemia (APL), recombinase polymerase amplification (RPA) technology offers a novel solution. RPA is a novel isothermal nucleic acid amplification technique that enables rapid detection of the target within 10-30 minutes at 37-42℃. It boasts advantages such as high sensitivity, high specificity, low cost, and low instrument dependence, making it particularly suitable for grassroots and on-site point-of-care testing. RPA technology relies on the participation of multiple enzymes and proteins. The recombinase protein forms a complex with the primer, searches for homologous sequences in double-stranded DNA, and inserts the primer into the homologous site through strand displacement activity, achieving exponential amplification of the target region on the template.

[0030] The advantages of RPA technology lie in its high detection sensitivity, capable of amplifying trace amounts of nucleic acid template (down to a single copy) to detectable levels, typically without the need for nucleic acid purification. Furthermore, RPA technology exhibits high sample tolerance, making it suitable for amplifying complex samples, such as unpurified blood or nasal swabs, which only require pretreatment with heat or alkali to promote nucleic acid release. RPA technology offers diverse detection methods, including electrophoresis, fluorescent probe assays, and lateral flow chromatography strip assays.

[0031] In one aspect, this application provides a composition comprising:

[0032] Upstream bait oligonucleotide, wherein the upstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO:1-5;

[0033] Downstream decoy oligonucleotides, wherein the downstream decoy oligonucleotides are derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 6-8; and

[0034] The probe sequence is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or two sequences in SEQ ID NO: 9-10.

[0035] In some embodiments, the compositions described herein include:

[0036] Upstream decoy oligonucleotide, wherein the upstream decoy oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of any one or more sequences in SEQ ID NO:1-5;

[0037] Downstream decoy oligonucleotides, wherein the downstream decoy oligonucleotides are derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 6-8; and

[0038] The probe sequence is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or two sequences in SEQ ID NO: 9-10.

[0039] In some embodiments, the compositions described herein include:

[0040] Upstream decoy oligonucleotide, wherein the upstream decoy oligonucleotide is selected from one or more sequences shown in SEQ ID NO:1-5;

[0041] Downstream decoy oligonucleotides, wherein the downstream decoy oligonucleotides are selected from one or more sequences shown in SEQ ID NO:6-8; and

[0042] The probe sequence is selected from one or two of the sequences shown in SEQ ID NO:9-10.

[0043] In some embodiments, the compositions described herein include:

[0044] The upstream decoy oligonucleotide, the downstream decoy oligonucleotide, and the probe sequence are provided. The upstream decoy oligonucleotide is selected from one or more sequences shown in SEQ ID NO:1-3, the downstream decoy oligonucleotide is selected from one or more sequences shown in SEQ ID NO:6-8, and the probe sequence is the sequence shown in SEQ ID NO:9; or

[0045] The upstream decoy oligonucleotide, the downstream decoy oligonucleotide, and the probe sequence are provided. The upstream decoy oligonucleotide is selected from one or two of the sequences shown in SEQ ID NO:4-5, the downstream decoy oligonucleotide is selected from one or more of the sequences shown in SEQ ID NO:6-8, and the probe sequence is the sequence shown in SEQ ID NO:10.

[0046] In all respects disclosed elsewhere in this document, the decoy oligonucleotide may be referred to as a “primer,” “oligonucleotide primer,” or “nucleic acid primer.” The primer may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The primer may contain one or more non-natural nucleotides. Non-natural nucleotides may be, for example, deoxyinosine. The primer may be a forward primer. The primer may be a reverse primer. The primer length may be from about 25 to about 50 nucleotides. The primer length may be at least 25, 30, 35, 40, 45, or 50 or more nucleotides. The primer length may be at most 50, 45, 40, 35, 30, or 25 nucleotides. The primer length may be about 25, 30, 35, 40, 45, or 50 nucleotides.

[0047] In this study, optionally, the upstream and downstream primers are 30-35 nucleotides in length; the amplicon obtained by amplification using the upstream and downstream primers is 80-400 bases in length; multiple Gs should be avoided at the 5' end of the primers, and the 3' end should contain as many Gs and Cs as possible; palindromic structures and primer dimers should be avoided; and the GC content should be 40%-60%.

[0048] A primer pair may consist of a forward primer (upstream primer) and a reverse primer (downstream primer). The forward primer may be configured to hybridize with a first region (e.g., the 3' end) of the nucleic acid sequence, while the reverse primer may be configured to hybridize with a second region (e.g., the 5' end) of the nucleic acid sequence, thereby being configured to amplify the nucleic acid sequence under conditions sufficient for nucleic acid amplification. Different primer pairs may be configured to amplify different target nucleic acid sequences.

[0049] The probe is 45-50 bp in length, with tetrahydrofuran (THF) modification at least 30 bp from the 5' end and at least 15 bp from the 3' end. The THF substituted bases are complementary template nucleotides, and the substituted bases can be A, G, C or T. The upstream primer can have a 10-20 bp overlap with the probe.

[0050] THF is an abbreviation for tetrahydrofuran. In nucleic acid probes, it specifically refers to a synthetically produced abasic site analog, a deoxyribose substitute with a tetrahydrofuran ring structure. THF replaces the deoxyribose in the DNA strand with a tetrahydrofuran ring (a five-membered ring ether structure), forming a stable "base-free" site (i.e., an abasic site) to mimic the natural abasic site (AP site). This structure cannot pair with any bases but can be recognized and cleaved by specific endonucleases (such as Nfoase). In the RPA-Nfo amplification system, THF is a core component of probe design.

[0051] In some implementations, this application provides primer and probe combinations as shown in the table below:

[0052]

[0053]

[0054] In some embodiments, the upstream decoy oligonucleotides shown in SEQ ID NO:1-3, the downstream decoy oligonucleotides shown in SEQ ID NO:6-8, and the probe sequence shown in SEQ ID NO:9 are used to detect the BCR3 type PML / RARA fusion gene in the sample; the upstream decoy oligonucleotides shown in SEQ ID NO:4-5, the downstream decoy oligonucleotides shown in SEQ ID NO:6-8, and the probe sequence shown in SEQ ID NO:10 are used to detect the BCR1 type and BCR2 type PML / RARA fusion genes in the sample.

[0055] In some embodiments, the 5' end of the probe contains a label, which is a hapten; optionally, the label is a fluorescein or a derivative thereof (e.g., 6-FAM and FITC), digoxin (DIG), Texas red, dansyl sulfonyl, Cascade blue, or dinitrophenol.

[0056] In some implementations, the 5' end of the upstream or downstream primer described herein contains a label, preferably biotin.

[0057] In some embodiments, the labels described herein include fluorescein or derivatives thereof, digoxin, Texas red, dansyl sulfonyl, Cascade blue, or dinitrophenol (DNP). Many fluorescein derivatives are known, but preferred fluorescein derivatives include fluorescein amides such as 6-FAM or fluorescein isothiocyanate (FITC). Optionally, the labels include fluorescent compounds or derivatives thereof, fluorogenic compounds or derivatives thereof, or steroids or derivatives thereof. Optionally, the labels include fluorescent compounds containing aromatic groups or fluorogenic compounds containing aromatic groups. The labels according to this application optionally include biotin.

[0058] In some embodiments, the 3' end of the probe described herein contains a polymerase blocking group, wherein the polymerase blocking group optionally includes a hydrocarbon spacer, a reverse deoxynucleotide, phosphorylation, a primary amine, or a dideoxynucleotide.

[0059] In some implementations, the probes and primers described herein can be modified using any modification methods known in the art for RPA-Nfo amplification detection systems.

[0060] In some implementations, the 5' end of the probe described herein and the 3' end of the downstream primer may be modified with a combination of FAM, DIG, and blocking groups.

[0061] In another aspect, this application also provides a kit comprising the composition described herein.

[0062] In some implementations, the kit may also contain one or more of the following: recombinase, single-stranded DNA-binding protein (SSB), strand displacement DNA polymerase (e.g., Bsu), specific endonuclease (e.g., Nfo enzyme), dNTPs, metal cations, protein protectants, and buffers.

[0063] In some implementations, the recombinase is selected from the group consisting of RecA, DrRecA, Rad51, RadA, or UvsX recombinase or homologs thereof.

[0064] Recombinases play a central role in homologous recombination in a variety of organisms. Such recombinases have been described in archaea, bacteria, eukaryotes, and viruses. The recombinase according to this application is optionally a recombinase that promotes homologous recombination. Optionally, the recombinase according to this application is a recombinase capable of targeting oligonucleotides (such as primers or oligonucleotide probes) to a target DNA sequence (optionally in double-stranded DNA). Optionally, the recombinase is capable of pairing oligonucleotides (such as primers or oligonucleotide probes) with a complementary DNA sequence (optionally in double-stranded DNA). Optionally, the recombinase according to this application is a recombinase selected from the group consisting of RecA, DrRecA, Rad51, RadA, or UvsX recombinases or homologs thereof. The RecA family of recombinases is encoded by several organisms. Optionally, the recombinase is RecA of *Escherichia coli* or *Deinococcus radiodurans*. Optionally, the recombinase is UvsX recombinase of bacteriophage T4. Optionally, the recombinase is Rad51 and its cofactor. Optionally, the recombinase is DrRecA from *Radiata spp.*

[0065] Endonuclease IV (Nfo), also known as N-nucleotide endonuclease, is a multifunctional endonuclease. Its primary function is to recognize apurinyl / pyrimidine dehydrogenase sites (AP sites) in the DNA strand and cleave the phosphodiester bond at the 5' end of the AP site, producing a 3'-hydroxyl and 5'-deoxyribose phosphate terminus (dRP). In addition, this enzyme also possesses 3'-diesterase activity, releasing glyceraldehyde-3-phosphate, intact deoxyribose 5'-phosphate, and phosphate from the 3' end of DNA; and 3'-5' exonuclease activity, removing nucleotides from the DNA strand from the 3' to 5' direction.

[0066] In some implementations, the kit may also include instructions for use of any of the aforementioned substances described herein. The kit may provide detection reagents, including primers, probes, a solid support with binding probes, and other detection reagents, along with appropriate instructions and other necessary reagents for the detection as described herein. Typically, the kit contains in a separate container: a combination of primers and probes (either already bound to a solid support; or separately, with reagents to bind them to the matrix), control formulations (positive and / or negative), labeling reagents—when required for the detection form, and signal-generating reagents (e.g., enzyme substrates)—if the labeling does not directly generate a signal. Instructions for performing the detection (e.g., written, magnetic tape, VCR, CD-ROM, etc.) are typically included in the kit. Depending on the specific detection applied, the kit may also contain other packaging reagents and materials (i.e., elution buffers, etc.). Standard detections, as described herein, can be performed using these kits. Instructions are generally recorded on a suitable recording medium. For example, instructions may be printed on a substrate such as paper or plastic. Similarly, instructions may be included as a package insert in the kit, on the label of the container of the kit or its components (e.g., attached to the packaging or repackaging), etc. In other embodiments, the instructions exist as an electronic storage data file on a suitable computer-readable storage medium, such as a CD-ROM, disk, etc., including the same medium on which the program is stored. The kit may also include instructions for using the kit. The kit may also include buffers, dNTPs, and controls (e.g., positive and negative control nucleic acids) for carrying out the method. Primers in this reagent may be detectably labeled or unlabeled.

[0067] On the other hand, this application also provides the use of the compositions or kits described herein in the early diagnosis and / or subtyping of acute promyelocytic leukemia (APL).

[0068] On the other hand, this application also provides a method for detecting the PML / RARA fusion gene in a sample, comprising:

[0069] RPA-Nfo amplification was performed on the test sample using the composition or kit described herein, followed by determination of the amplification products by electrophoresis, fluorescent probe method or lateral flow chromatography. The results were used to determine the presence and / or type of the PML / RARA fusion gene in the sample.

[0070] In some embodiments, the presence or absence of the amplification product is used to determine the presence of the PML / RARA fusion gene in the sample. In some embodiments, the BCR3 type PML / RARA fusion gene in the sample is detected using the upstream decoy oligonucleotides shown in SEQ ID NO:1-3, the downstream decoy oligonucleotides shown in SEQ ID NO:6-8, and the probe sequence shown in SEQ ID NO:9; the BCR1 and BCR2 type PML / RARA fusion genes in the sample are detected using the upstream decoy oligonucleotides shown in SEQ ID NO:4-5, the downstream decoy oligonucleotides shown in SEQ ID NO:6-8, and the probe sequence shown in SEQ ID NO:10.

[0071] The RPA-Nfo amplification system is a detection system within recombinase polymerase amplification (RPA) technology. It combines the specific probe cleavage function of endonuclease IV (Nfo) and is primarily used for endpoint detection (such as lateral flow chromatography strip detection). Its core feature is the visualization and analysis of highly specific amplification products through probe design, making it suitable for rapid on-site detection. During amplification, after the probe binds to the target sequence, the Nfo enzyme cleaves the debasement site, releasing the labeled group. For example, in lateral flow chromatography strip detection, the biotin-labeled amplification product binds to the antibody on the strip, forming a visible detection line. The RPA-Nfo system is typically used in conjunction with lateral flow chromatography strips (LFD), and the results are determined by visually observing the colored bands on the strip.

[0072] Methods for performing RPA-Nfo amplification reactions to amplify the target DNA sequence according to the invention are well known in the art. These well-known methods include how and when to contact the sample according to the invention with the primers, dNTPs, enzymes, probes, and other reaction components required for amplification. Additionally, various methods for detecting the amplification products according to the invention after or during amplification, also known as real-time detection, such as real-time PCR or real-time RPA, are well known in the art.

[0073] In some implementations, the sample to be tested can be a biological sample. The sample can be derived from a biological sample. Biological samples can be, for example, blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces, or tears. Biological samples can also be fluid samples. Fluid samples can be blood or plasma.

[0074] The primers and probes in this application are designed based on different isoform sequences of the PML / RARA fusion gene, which can be used for rapid qualitative detection of the common PML / RARA fusion gene in acute promyelocytic leukemia (APL / AML-M3 type). Combined with nucleic acid chromatography strips, it can complete rapid nucleic acid detection and typing, making the operation simpler and more convenient, without the need for additional instruments for result interpretation.

[0075] This application describes several embodiments, but these descriptions are exemplary and not limiting, and it will be apparent to those skilled in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature or element of any embodiment may be used in combination with, or may replace, any feature or element of any other embodiment.

[0076] This application includes and contemplates combinations of features and elements known to those skilled in the art. The embodiments, features, and elements disclosed in this application can also be combined with any conventional features or elements to form unique inventive solutions. Any feature or element of any embodiment can also be combined with features or elements from other inventive solutions to form another unique inventive solution. Therefore, it should be understood that any feature shown and / or discussed in this application can be implemented individually or in any suitable combination. Therefore, the embodiments are not limited except by the limitations imposed by the appended claims and their equivalents. Furthermore, various modifications and changes can be made within the scope of the appended claims.

[0077] Furthermore, in describing representative embodiments, the specification may have presented methods and / or processes as a specific sequence of steps. However, the method or process should not be limited to the specific order of steps described herein, to the extent that it does not depend on such a specific order. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation of the claims. Moreover, the claims concerning the method and / or process should not be limited to the steps performed in the written order, and those skilled in the art will readily understand that these orders can be varied and still remain within the spirit and scope of the embodiments of this application.

[0078] Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental materials in the following embodiments without specified sources are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, then generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer are followed. Unless otherwise defined or stated, all technical and scientific terms used in this application have the same meaning as those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of this application.

[0079] Example

[0080] The materials used in the embodiments are shown below:

[0081] Example 1. Primer and probe design

[0082] The genomic sequence of the PML / RARA fusion gene was downloaded from NCBI. The breakpoints in the fusion gene were analyzed using the DNAStar software package. Based on the structural characteristics of different BCR1, BCR2, and BCR3 sequences, RPA primers and nfo probes were designed within the corresponding conserved regions. Two sets of probes, seven sets of upstream primers, and four sets of downstream primers were designed, as detailed in the table below:

[0083]

[0084]

[0085] Example 2. Primers and probes for the detection and typing of the PML / RARA fusion gene.

[0086] RPA reaction reagents were prepared using different sets of primers and probes for the corresponding genotypes. At the same time, the PML / RARA fusion gene standards of the corresponding types were detected. The color development effect of test strips with different primer-probe combinations of the same type was observed, and the better primer-probe combinations were screened out.

[0087] The markings are as follows:

[0088] The probe's 5' end is labeled with FAM or DIG, its 3' end with a C3 spacer, and its middle end with THF; the downstream primer's 5' end is labeled with biotin.

[0089] P1:

[0090] [5'FAM]-GAGCCTGCAAGCTGCCGTGCGCACCGATGG[THF]TTCGACGAGTTCAAG(SEQ ID NO:9)-[3'C3spacer]

[0091] P2:

[0092] [5'DIG]-CAGTGCCCCAGGAAGGTCATCAAGATGGAGTC[THF]GAGGAGGGGAAGG AG(SEQ IDNO:10)-[3'C3spacer]

[0093] R1: [5'-Biotin]-CTCTTCAGAACTGCTGCTCTGGGTCTCAATG (SEQ ID NO:6)

[0094] R2: [5'-Biotin]-GTCCTGACAGACAAAGCAAGGCTTGTAGATGC (SEQ ID NO:7)

[0095] R3: [5'-Biotin]-GTAGCCTGAGGACTTGTCCTGACAGACAAAGC (SEQ ID NO:8)

[0096] R4: [5'-Biotin]-GGGTGGCGAGGGAGGGCTGGGCACTATCTC (SEQ ID NO:12)

[0097] 1. Sample preparation

[0098] BCR1, BCR2, and BCR3 standards were serially diluted to 1×10⁻⁶ using enzyme-free water. 5 1 copy / mL, store at -20℃ for later use.

[0099] 2. Reagent preparation

[0100] Remove each reagent component and place them on the table to equilibrate to room temperature. Gently vortex to mix and then briefly centrifuge. Prepare the corresponding amplification reagents using each set of primers and probes, as follows:

[0101] Prepare RPA mixture according to the table below.

[0102]

[0103] The lyophilized microspheres (purchased from Weifang Anpu Future Biotechnology Co., Ltd., catalog number WLN8203KIT; its main components include: T4 UvsX protein, T4 UvsY protein, T4 gp32, DNA polymerase, dNTPs, PEG 20000, dithiothreitol, creatine phosphate, creatine kinase, adenosine triphosphate (ATP), Tris, potassium acetate, and magnesium acetate) and other components were dissolved and mixed thoroughly (the lyophilized microspheres are solid and contain all components of the RPA reaction system except for primers, probes, and MgOAc; they can be dissolved after adding enzyme-free water; the lyophilized microspheres can be stored at room temperature) and thoroughly mixed (shaking). Finally, the mixture was briefly centrifuged and the reaction was started.

[0104] 3. Amplification detection

[0105] Incubate at 39℃ for 20 min. After the reaction is complete, add diluent to 100 μL and add to a nucleic acid test strip (Weifang Anpu Future Biotechnology Co., Ltd., catalog number WLFS8201). Let stand for 5 min and observe the results.

[0106] 4. If only the C line appears on the test strip, the result is negative. If both the T line and the C line appear on the test strip, the result is positive. If the C line does not appear, the test result is invalid and needs to be retested.

[0107] 5. Results Analysis

[0108] After the reaction, observe the amplification results of each primer and probe group (see...). Figures 2-9 ).

[0109] Figure 2 The results show the detection of probe P1 with upstream primers F11, F12, and F13 and downstream primers R1, R2, and R3 using BCR3 type standards. From left to right, the combinations are F11+R1, F12+R1, F13+R1, F11+R2, F12+R2, F13+R2, F11+R3, F12+R3, and F13+R3, a total of 9 combinations. The top and bottom images show repeated experiments of the same combination. The first 7 combinations clearly detected the BCR3 subtype standard. The positive results of the F12+R3 and F13+R3 combinations were not obvious. Therefore, the first 7 combinations are the preferred options.

[0110] Figure 3 The results for Example 2 of this application, using probe P1 with upstream primer F14 and downstream primers R1, R2, R3, and R4, and the detection results using BCR3 standard, are shown. From left to right, the combinations are P1+F14+R1, P1+F14+R2, P1+F14+R3, and P1+F14+R4, a total of four combinations. It is evident that the upstream primer F14 does not yield satisfactory positive results.

[0111] Figure 4 In Example 2 of this application, probe P1 was used in combination with upstream primers F11, F12, F13, and F14 and downstream primer R4, and detection was performed using BCR3 standard; and probe P2 was used in combination with upstream primers F21, F22, and F23 and downstream primer R4, and detection was performed using BCR1 standard. From left to right, the combinations are P1+F11+R4, P1+F12+R4, P1+F13+R4, P1+F14+R4, P2+F21+R4, P2+F22+R4, and P2+F23+R4, a total of 7 combinations. It is evident that downstream primer R4 does not yield satisfactory positive results.

[0112] Figure 5 The results show the detection of probe P1 with upstream primers F11, F12, and F13 and downstream primers R1, R2, and R3 using negative standards (human normal genome extracts). From left to right, the combinations are F11+R1, F12+R1, F13+R1, F11+R2, F12+R2, F13+R2, F11+R3, F12+R3, and F13+R3, a total of 9 combinations. The top and bottom images show replicate experiments of the same combination, and no false positives were observed.

[0113] Figure 6 The results show the detection of probe P1 with upstream primers F11, F12, and F13 and downstream primers R1, R2, and R3 using BCR1 and BCR2 standards. From left to right, the combinations are F11+R1, F12+R1, F13+R1, F11+R2, F12+R2, F13+R2, F11+R3, F12+R3, and F13+R3, a total of nine combinations. The top part shows the results using BCR1 standards, and the bottom part shows the results using BCR2 standards. It is evident that F13+R3 produces false positives when detecting BCR1, while the other combinations do not produce false positives.

[0114] Figure 7 The results show the detection of probe P2 with upstream primers F21 and F22 and downstream primers R1, R2, and R3 using BCR1 and BCR2 standards. From left to right, the combinations are F21+R1, F22+R1, F21+R2, F22+R2, F21+R3, and F22+R3, a total of 6 combinations. The top and bottom images show the tests using BCR1 and BCR2 standards, respectively. All 6 combinations effectively detected BCR1 and BCR2 standards.

[0115] Figure 8The results are obtained using negative standards (human normal genome extracts) for the combination of probe P2 with upstream primers F21 and F22 and downstream primers R1, R2, and R3. From left to right, the combinations are F21+R1, F22+R1, F21+R2, F22+R2, F21+R3, and F22+R3, a total of 6 combinations. The top and bottom images show the replicate experiments of the negative standard test results, and no false positives were observed.

[0116] Figure 9 The results are obtained using BCR3 standards for the combination of probe P2 with upstream primers F21 and F22 and downstream primers R1, R2, and R3. From left to right, the combinations are F21+R1, F22+R1, F21+R2, F22+R2, F21+R3, and F22+R3, a total of 6 combinations, with no false positives observed.

[0117] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A composition, characterized in that, include: Upstream decoy oligonucleotide, said upstream decoy oligonucleotide being derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 1-5; Downstream decoy oligonucleotides, wherein the downstream decoy oligonucleotides are derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 6-8; and The probe sequence is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or two sequences in SEQ ID NO: 9-10.

2. The composition according to claim 1, characterized in that, The composition comprises: Upstream decoy oligonucleotide, said upstream decoy oligonucleotide being derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 1-5; Downstream decoy oligonucleotides, said downstream decoy oligonucleotides being derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or more sequences in SEQ ID NO: 6-8; and The probe sequence is derived from at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any one or two sequences in SEQ ID NO: 9-10.

3. The composition according to claim 2, characterized in that, The composition comprises: Upstream decoy oligonucleotide, wherein the upstream decoy oligonucleotide is selected from one or more sequences shown in SEQ ID NO:1-5; Downstream decoy oligonucleotides, wherein the downstream decoy oligonucleotides are selected from one or more sequences shown in SEQ ID NO:6-8; and The probe sequence is selected from one or two of the sequences shown in SEQ ID NO:9-10.

4. The composition according to any one of claims 1-3, characterized in that, The composition comprises: An upstream decoy oligonucleotide, a downstream decoy oligonucleotide, and a probe sequence, wherein the upstream decoy oligonucleotide is selected from one or more sequences shown in SEQ ID NO:1-3, the downstream decoy oligonucleotide is selected from one or more sequences shown in SEQ ID NO:6-8, and the probe sequence is the sequence shown in SEQ ID NO:9; or The upstream decoy oligonucleotide, the downstream decoy oligonucleotide, and the probe sequence are provided. The upstream decoy oligonucleotide is selected from one or two of the sequences shown in SEQ ID NO:4-5, the downstream decoy oligonucleotide is selected from one or more of the sequences shown in SEQ ID NO:6-8, and the probe sequence is the sequence shown in SEQ ID NO:

10.

5. The composition according to any one of claims 1-4, characterized in that, The probe has a label at its 5' end, which is a hapten; optionally, the label is a fluorescein or a derivative thereof (e.g., 6-FAM and FITC), digoxin, Texas red, dansyl sulfonyl, Cascade blue, or dinitrophenol.

6. The composition according to any one of claims 1-5, characterized in that, The probe contains a polymerase blocking group at its 3' end, optionally including a hydrocarbon spacer, a reverse deoxynucleotide, phosphorylation, a primary amine, or a dideoxynucleotide.

7. A reagent kit, characterized in that, The kit comprises the composition according to any one of claims 1-6.

8. The reagent kit according to claim 7, characterized in that, The kit also contains one or more of the following: recombinase, single-stranded DNA-binding protein (SSB), strand displacement DNA polymerase (e.g., Bsu), specific endonuclease (e.g., Nfo enzyme), dNTPs, metal cations, protein protectants, and buffers.

9. The reagent kit according to claim 8, characterized in that, The recombinase is selected from the group consisting of: RecA, DrRecA, Rad51, RadA or UvsX recombinase or its homologs.

10. Use of the composition of any one of claims 1-6 or the kit of any one of claims 7-9 in the early diagnosis and / or typing of acute promyelocytic leukemia (APL).

11. A method for detecting the PML / RARA fusion gene in a sample, characterized in that, include: RPA-Nfo amplification was performed on the sample to be tested using the composition of any one of claims 1-6 or the kit of any one of claims 7-9. The amplification products were then measured by electrophoresis, fluorescent probe method or lateral flow chromatography. The presence and / or type of PML / RARA fusion gene in the sample were determined by the measurement results.

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