Primer pair for detecting TTMV-RARA fusion gene and kit thereof

Abstract

The invention provides a primer pair and a kit for detecting a TTMV-RARA fusion gene, the primer pair comprises a first upstream primer TTMV-RARA-F1, a second upstream primer TTMV-RARA-F2 and a downstream primer TTMV-RARA-R, the sequence of the first upstream primer TTMV-RARA-F1 is 5 '-GGGCGGGWGCYGAAG-3', and the sequence of the second upstream primer TTMV-RARA-F2 is 5 '-GGGCGGGWGCYGAAG-3'. The sequence of the first upstream primer TTMV-RARA-F2 is 5 '-CGAATGGCTGGAGGCGCYAGACGGAGA-3', the sequence of the second upstream primer TTMV-RARA-F2 is 5 '-CGAATGGCTGAGTTTATGCCGCYAGACGGAGA-3', and the sequence of the downstream primer TTMV-RARA-R is 5 '-GCTTGTAGATGCGGGGTAGA-3'. By designing the special primers, the problem of leak detection caused by large change of TTMV genomes can be solved, and more than 95% of known variation types can be detected.

Description

Technical Field

[0001] This invention belongs to the field of fusion gene detection technology, specifically relating to a primer pair and kit for detecting the TTMV-RARA fusion gene. Background Technology

[0002] Acute promyelocytic leukemia (APL) is a specific subtype of acute myeloid leukemia. Traditionally, approximately 95% of APL patients have a specific chromosomal translocation t(15;17)(q22;q21), forming the PML-RARA fusion gene. This fusion protein is a key driver of APL and can be treated with targeted drugs such as all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), achieving a complete remission rate of over 90%. However, some patients in clinical practice have bone marrow morphology consistent with APL, yet the PML-RARA fusion gene cannot be detected by routine karyotype analysis, FISH, or RT-PCR. These patients are classified as "atypical APL." Studies have shown that children with atypical APL have a significantly higher relapse rate and respond poorly to ATRA treatment, requiring conventional AML chemotherapy regimens.

[0003] In 2025, Professor Zhu Honghu's team at Beijing Chaoyang Hospital discovered a novel fusion gene—TTMV-RARA—in APL patients for the first time using whole transcriptome sequencing (WTS). This fusion is formed by the integration of the Torquetenominivirus (TTMV) genome into the RARA gene, replacing the traditional PML-RARA. TTMV is a non-enveloped circular single-stranded DNA virus that is widespread in the population, and seven cases of APL caused by the TTMV-RARA fusion have been reported to date.

[0004] Currently, TTMV-RARA fusion gene detection faces three major technical challenges: First, the TTMV genome is highly variable, with significant differences in integration fragment length among different patients (1164bp-2859bp), making it impossible for traditional single primers to cover all variants. Second, conventional PCR methods have low detection rates for low-abundance fusion transcripts, affecting the monitoring value of minimal residual disease. Third, existing methods rely on whole transcriptome sequencing (WTS), but this is costly and time-consuming, hindering clinical application. Summary of the Invention

[0005] In view of this, the present invention provides a primer pair and kit for detecting the TTMV-RARA fusion gene, which realizes efficient and accurate detection of novel viral-human fusion oncogenes, fills the technical gap in non-classical APL molecular diagnostics, and provides an important tool for precision medicine practice.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a primer pair for detecting the TTMV-RARA fusion gene, comprising a first upstream primer TTMV-RARA-F1, a second upstream primer TTMV-RARA-F2, and a downstream primer TTMV-RARA-R. The sequence of the first upstream primer TTMV-RARA-F1 is 5'-GGGCGGGWGCYGAAG-3'; the sequence of the second upstream primer TTMV-RARA-F2 is 5'-CGAATGGCTGAGTTTATGCCGCYAGACGGAGA-3'; and the sequence of the downstream primer TTMV-RARA-R is 5'-GCTTGTAGATGCGGGGTAGAG-3'.

[0007] Preferably, the primer pairs are determined using a degenerate primer design method.

[0008] Secondly, the present invention provides a probe for detecting the TTMV-RARA fusion gene, wherein the sequence of probe P is: 5'FAM-CTCTGGGTCTCAATGG-NFQ-MGB3'.

[0009] Preferably, the probe P has a reporter luminescent group FAM at its 5' end and a non-fluorescent quencher group NFQ at its 3' end, and the probe is also connected to an MGB modifying group.

[0010] Thirdly, the present invention provides a primer pair and a probe.

[0011] Preferably, it also includes RT-PCR premix, ROX correction dye, primer pairs for the internal reference ABL gene, probe for the internal reference ABL gene, negative control, positive control, and nuclease-free water.

[0012] Preferably, the primer pair for the internal reference ABL gene includes the sequence of ABL1-F: 5'-GCCGTGAAGACCTTGAAGGAG-3'; and the sequence of ABL1-R: 5'-ATGATATAGAACGGGGGCTC-3'.

[0013] Preferably, the probe for the internal reference ABL gene is 5'FAM-ACCTGGTGCAGCTCCTTGGG-NFQ-MG B3'.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) By designing special primers, this invention can solve the problem of missed detection caused by large TTMV genomic variations and can detect more than 95% of known variant types.

[0015] (2) This invention combines MGB probe technology and dynamic threshold algorithm, which can detect pathogens as low as 0.001%, and is suitable for monitoring minute lesions with high sensitivity.

[0016] (3) The present invention first performs preliminary screening by qPCR and then confirms the results by targeted sequencing, which effectively avoids false positives and has a high accuracy rate.

[0017] (4) The kit provided by this invention can be used on conventional molecular diagnostic laboratory equipment, reducing the high cost of comprehensive testing. Attached Figure Description

[0018] Figure 1 This is a sequence diagram of the TTMV-RARA fusion gene positive provided in Example 1 of the present invention; Figure 2 This is a sequence diagram of the TTMV-RARA fusion gene positive provided in Example 1 of the present invention. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0020] Example 1: Primer Pair and Probe Design This embodiment designs a set of efficient and highly compatible primers and probes for the precise detection of specific variant regions in the TTMV and RARA genes. Two primers are designed to target the transcriptional regulatory region (i.e., the upstream portion of ORF2) in the TTMV genome and the exon portion in the RARA gene, respectively. These two regions contain known variant hotspots, so the primers are designed specifically for these regions to ensure the detection of relevant variants. Degenerate bases are intentionally used at key positions in the primer design. For example, "W" represents A or T (i.e., the degenerate base of A / T), and "Y" represents C or T (i.e., the degenerate base of C / T). This is done to allow the primers to match different DNA sequence variants, thereby increasing primer compatibility and ensuring the capture of more possible gene variants. Short probes with a 3' end Minor GrooveBinder (MGB) modification are designed. This modification enhances the binding specificity of the probe to the target sequence, allowing the probe to more accurately identify the target DNA sequence during detection. The primer and probe sequences of this invention are shown in Table 1.

[0021] Table 1

[0022] The general steps for detecting viruses or pathogens using the primers and probes described above are as follows: Phase 1: Initial screening using real-time fluorescent PCR Use the primer pairs listed in Table 1 to perform multiplex qPCR (real-time quantitative PCR) tests.

[0023] The criteria for a positive result are: The Ct value (the threshold of fluorescence signal) is less than or equal to 35, and the amplification curve exhibits a typical S-shape.

[0024] The ΔCt value (the Ct value of the sample minus the Ct value of the negative control) is less than 3.

[0025] Phase 2: Transcriptome sequencing validation Further verification of positive samples from the initial screening involves the following steps: Construct strand-specific cDNA libraries.

[0026] The comparison was performed using reference sequences from the TTMV subtype database containing 326 strains.

[0027] The CLIP-seq algorithm was used to identify the fused DNA sequences and assemble them into a complete fused sequence.

[0028] Developing a bioinformatics analysis module using the primers and probes described above. Variant classification system: Based on the location of the fusion breakpoint, the TTMV-RARA virus is divided into 3 subtypes: Type I: Located upstream of the ORF2 gene.

[0029] Type II: Located inside the ORF2 gene.

[0030] Type III: This is a multi-segment fusion type.

[0031] Dynamic threshold setting: The positive judgment value is automatically adjusted based on the background load of TTMV in the sample (i.e. the basic amount of virus in the sample), thereby reducing false positive results.

[0032] Kit development using the primers and probes described above. The detection kit developed by combining the above steps includes the following core components: Multiplex qPCR module: includes degenerate primers, probes, and reaction buffer.

[0033] Library construction and validation module: includes reagents for constructing transcriptome libraries.

[0034] Comparison system: Positive control: Recombinant plasmid containing a conserved fusion sequence.

[0035] Negative control: cDNA from healthy human leukocytes.

[0036] Internal standard: Primer-probe system containing the ABL gene.

[0037] Example 2: Specific Detection Method 1. Sample collection and processing Sample type: Bone marrow fluid (preferably 3 mL EDTA anticoagulation) or peripheral blood (5 mL EDTA anticoagulation); Leukocyte separation: After diluting with an equal volume of physiological saline, the cells were centrifuged in lymphocyte separation solution (density 1.077 g / mL) at 2000 rpm for 15 minutes to collect the leukocyte layer; RNA extraction: A commercial RNA extraction kit was used. After extraction, the A260 / A280 ratio was measured (within acceptable range of 1.8-2.0), and the RNA was stored at -80℃.

[0038] 2. Real-time fluorescence PCR initial screening (1) Preparation of reaction system (20 μL system), see Table 2.

[0039] Table 2

[0040] Simultaneous detection using the internal control system: ABL gene primers (200nM each for F and R) + probe (100nM), upstream primer (ABL1-F) sequence: 5'-GCCGTGAAGACCTTGAAGGAG-3'; downstream primer (ABL1-R) sequence: 5'-ATGATATAGAACGGGGGCTC-3'; TaqMan-MGB probe (ABL1-P) sequence: 5'FAM-ACCTGGTGCAGCTCCTTGGG-NFQ-MG B3'.

[0041] (2) Amplification procedure, see Table 3 Table 3

[0042] (3) Result interpretation criteria Positive: FAM channel Ct value ≤ 35, and the amplification curve shows a typical S-shape; Figure 1-2 This is a sequence diagram of the TTMV-RARA fusion gene.

[0043] Suspicious: Ct value 35-38, repeated testing required; Negative: Ct value ≥ 38 or no amplification curve; Internal control validation: The Ct value of the ABL gene should be ≤32, otherwise the nucleic acid quality of the sample is unqualified.

[0044] 3. Validation by whole transcriptome sequencing (for initial positive samples) (1) cDNA library construction Use a strand-specific library preparation kit (such as the VAHTS Universal V10 RNA-seq Library Prep Kit for IIIumina). Fragmentation condition: 200-300bp; Connector connection: A personalized connector with a sample index sequence is used.

[0045] (2) Sequencing and data analysis Sequencing platform: SURFSeq 5000; Data volume: ≥50M reads / sample; Analysis process: 1. Raw data quality control (FastQC); 2. Compare with the reference genome (GRCh38 TTMV whole genome database); 3. Use the STAR-Fusion algorithm to identify fusion transcripts; 4. Breakpoint verification: IGV visualization is used to check fusion sites to support reads; Variant classification: Based on the location of the breakpoint, it is classified into type I / II / III fusion.

[0046] 4. Reagent Kit Implementation Case Reagent kit components (48 tests): qPCR module: Lyophilized primer / probe balls 2×RT-PCR premix ROX Correcting Dyes Database creation module: Fragmented enzymes Connector primers ligase mixture Reference standard: Positive control: Recombinant plasmid (containing the conserved fusion fragment 5'-CCGCCCGCGTTCCGGAGGTGACGGGGCGC-3') Negative control: HEK293 cell cDNA Nuclease-free water Operating procedures: 1. Sample RNA extraction; 2. First-strand cDNA synthesis (42℃ for 60 minutes); 3. Real-time fluorescence PCR detection; instrument compatible with ABI 7500 / QuantStudio, etc. 4. Positive samples from the initial screening are used to construct a transcription library and undergo sequencing; Data analysis report generated.

[0047] Comparative Example 1 This comparative example provides invention patent 202410580781.X, which discloses a primer pair and kit for real-time fluorescence PCR detection of the TTMV::RARA fusion gene. The primer pair includes an upstream primer (TTMV::RARA-F1), an upstream primer (TTMV::RARA-F2), and a downstream primer (TTMV::RARA-R). The probe for the primer pair is a TaqMan-MGB probe (TTMV::RARA-P). The sequence of the upstream primer (TTMV::RARA-F1) is: 5 The sequence of the upstream primer (TTMV::RARA-F2) is 5'-CGAATGGCTGAGTTTATGCCGCYAGACGGAGA-3'; the sequence of the downstream primer (TTMV::RARA-R) is 5'-GCTTGTAGATGCGGGGTAGAG-3'; and the sequence of the TaqMan-MGB probe (TTMV::RARA-P) is 5'FAM-CTCTGGGTCTCAATGG-NFQ-MGB 3'. Other reagents in the kit and specific usage instructions can be found in invention patent 202410580781.X.

[0048] The kit from Example 2 and the kit from Comparative Example 1 were used for detection and comparison. The results are shown in Table 4.

[0049] Table 4

[0050] As shown in Table 2, the kit containing the primer pairs and probes of this application can effectively address the issue of high TTMV genomic variation when detecting APL caused by TTMV-RARA fusion, thus ensuring that the detection covers more than 95% of known viral variants and avoiding missed detections. Employing MGB probe technology combined with a dynamic threshold algorithm, this detection method can detect extremely low concentrations of virus (as low as 0.001% viral load), making it highly suitable for monitoring trace amounts of residual virus. This invention first uses qPCR technology for rapid screening, followed by targeted sequencing for confirmation, which effectively reduces false positives and ensures the accuracy of the test results. The kit accompanying this technology can be used in conventional molecular diagnostic laboratory equipment, avoiding the high cost of comprehensive testing (such as WTS), making clinical diagnosis more economical and practical.

[0051] Effect verification Example 3: Clinical Sample Testing Sample source: APL Biobank, Beijing Chaoyang Hospital (n=120, including 32 morphologically consistent but PML-RARA negative samples). Comparative experiment: Traditional RT-PCR: Based on PML-RARA primers Comparative Example 1 Method of the present invention The results are shown in Table 5.

[0052] As can be seen from Table 5, this invention detected 7 positive TTMV-RARA cases, of which 2 cases were missed by Comparative Example 1 (sample APL-77 was missed because the fusion breakpoint was located in the new variant region of ORF2), showing that this invention has a wider coverage.

[0053] Example 4: Sensitivity Verification Sample construction: TTMV-RARA positive cell line RNA was diluted proportionally into healthy human leukocyte RNA (100% to 0.0001%). Test results: The method of this invention has a detection limit of 0.001% (three replicates all positive). Comparative Example 1 Method: Limit of Detection 0.1% Conclusion: Sensitivity is improved by 100 times, meeting the needs of minimal residual disease monitoring.

[0054] Example 5: Application of Treatment Monitoring Dynamic monitoring was performed on one TTMV-RARA-positive APL patient (male, 15 years old): At initial diagnosis: fusion gene load in bone marrow was 12.5%; After one course of ATRA+ATO treatment: the viral load decreased to 0.03% (partial remission); After 3 courses of treatment: viral load <0.001% (complete remission); At 6 months follow-up: the viral load rose to 0.1% (earlier than hematologic relapse).

[0055] Value proposition: This method can provide early warning of recurrence and guide the timing of intervention.

[0056] The TTMV-RARA fusion gene screening method and supporting system provided by this invention achieve efficient and accurate detection of novel virus-human fusion oncogenes, filling the technical gap in non-classical APL molecular diagnostics and providing an important tool for precision medicine practice.

[0057] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0058] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A primer pair for detecting TTMV-RARA fusion gene, characterized in that, Comprise: a first upstream primer TTMV-RARA-F1 with sequence of 5'-GGGCGGGWGCYGAAG-3'; a second upstream primer TTMV-RARA-F2 with sequence of 5'-CGAATGGCTGAGTTTATGCCGCYAGACGGAGA-3'; a downstream primer TTMV-RARA-R with sequence of 5'-GCTTGTAGATGCGGGGTAGAG-3'.

2. The primer pair according to claim 1, characterized in that, The primer pair is determined by a method of designing degenerate primers.

3. A probe for detecting a TTMV-RARA fusion gene, characterized in that, The sequence of the probe P is 5'FAM-CTCTGGGTCTCAATGG-NFQ-MGB 3'.

4. The probe of claim 3, wherein The 5' end of the probe P is labeled with a report light-emitting group FAM, and the 3' end is labeled with a non-fluorescent quenching group NFQ, and the probe is also connected with a MGB modification group.

5. A kit characterized in that, Comprise the primer pair of claim 1 or 2, and the probe of claim 3 or 4.

6. The kit of claim 5, wherein Also comprise RT-PCR premix, ROX correction dye, primer pair of internal reference ABL gene, probe of internal reference ABL gene, negative control product, positive control product and nuclease-free water.

7. The kit of claim 6, wherein The primer pair of the internal reference ABL gene comprises the sequence of ABL1-F: 5'-GCCGTGAAGACCTTGAAGGAG-3'; and the sequence of ABL1-R: 5'-ATGATATAGAACGGGGGCTC-3'.

8. The kit of claim 6, wherein The probe of the internal reference ABL gene is 5'FAM-ACCTGGTGCAGCTCCTTGGG-NFQ-MGB 3'.

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