Single-molecule detection kit for multiple non-coding RNAs in human mammary tissue by using programmable automatic cascade mechanism and application of single-molecule detection kit for multiple non-coding RNAs in human mammary tissue

Abstract

The invention discloses a single molecule detection kit for a plurality of non-coding RNAs (Ribonucleic Acid) in human breast tissues by using a programmable automatic cascade mechanism and application of the single molecule detection kit. The preparation method of the single molecule detection kit comprises the following steps: incubating piRNA-36026, lncRNA-DSCAM-AS1, a dumbbell probe-pi, a dumbbell probe-lnc, a primer-pi, a primer-lnc, a dNTP mixed solution, a ThermoPol reaction buffer solution and a reaction solution of Bst DNA polymerase (large fragment); and adding the reaction product into a reaction solution containing the signal probe 1, the signal probe 2, an NEBuffer 4 reaction buffer solution and T7 exonuclease, and incubating to obtain the single molecule detection kit. According to the present invention, the efficient automatic cascade mechanism and the high signal-to-noise ratio single molecule detection are adopted to achieve the sensitive simultaneous detection of a variety of ncRNAs, and the high sensitivity, the good selectivity and the multi-path analysis ability are provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological analysis, and particularly relates to a single-molecule detection kit for multiple non-coding RNAs in human breast tissue using a programmable automatic cascade mechanism, and a preparation method and use thereof. Background Art

[0002] As a major public health problem, cancer causes more than 10 million deaths worldwide each year, mainly due to late diagnosis and tumor metastasis. Noncoding RNAs (ncRNAs) are large amounts of transcribed "black matter" that cannot encode protein information, and they play crucial roles in complex biological processes. ncRNAs are considered to be tumor suppressors and oncogenic drivers in multiple cancer types. Based on their length, ncRNAs can be divided into two major categories: short noncoding RNAs (<200nt), including microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs); and long noncoding RNAs (lncRNAs, >200nt). Early studies confirmed that serum and plasma contain a large number of upregulated or downregulated miRNAs from various tissues / organs, making them very valuable biomarkers in liquid biopsy strategies. In recent years, lncRNAs and piRNAs have gradually attracted widespread attention as important biomarkers for cancer diagnosis, classification, and treatment evaluation. For example, lncRNAs can regulate gene expression and RNA splicing events by competitively binding to miRNA response elements (MREs), thereby promoting the proliferation and migration of tumor cells. lncRNAs are also involved in viral infection and subsequent antiviral immune responses. At the transcriptional level, piRNAs can guide Piwi proteins and cofactors to bind to primary transposon transcripts and induce the production of heterochromatin through DNA / histone methylation, thereby inhibiting transposon transcription. At the post-transcriptional level, piRNAs can guide Piwi proteins to cleave transposon mRNAs, thereby preventing transposon translation. In addition, the PIWI-piRNA pathway even regulates the expression of lncRNAs, leading to the degradation of lncRNAs. Increasing evidence has shown that abnormal expression levels of lncRNAs and piRNAs are associated with the pathogenesis of a variety of human cancers, such as gastric cancer, liver cancer, breast cancer, lung cancer, cervical cancer, prostate cancer, and colorectal cancer. It is worth noting that due to its RNase A resistance and high stability (half-life of 16 hours), lncRNA is stably present in blood, urine, saliva, cerebrospinal fluid and some cell-derived exosomes, and the 2'-O-methylation modification of piRNA 3' end can even protect piRNAs from degradation / oxidation in complex biological matrices. Therefore, accurate detection of multiple ncRNAs (such as lncRNAs and piRNAs) is of great significance for cancer biology and precision medicine.

[0003] Existing strategies for ncRNAs analysis include northern blotting, microarray, reverse transcription polymerase chain reaction (qRT-PCR), and RNA sequencing, but they are inevitably affected by hazardous radioactive labels, time-consuming and labor-intensive procedures, large sample consumption, poor sensitivity, and complex data analysis. To overcome these limitations, several nucleic acid-based amplification strategies, such as T7 RNA polymerase-mediated signal amplification, rolling circle amplification (RCA), target-induced interstrand ligation reaction, and hybridization chain reaction (HCR), have been incorporated into optical and electrochemical methods for ncRNAs detection. Although these strategies have improved sensitivity, they still have some inevitable disadvantages: (1) the multi-enzyme-assisted cascade amplification system relies on well-regulated incubation conditions (such as pH, reaction temperature, and ion concentration) of multiple enzymes, which inevitably increases the complexity of the experiment; (2) the multi-step experimental protocol (such as modification, separation, and washing steps) may lead to the risk of signal leakage and poor reproducibility. Therefore, it is very necessary to develop a sensitive and reliable method for the simultaneous detection of multiple ncRNAs.

[0004] Primer exchange reaction (PER) is a powerful amplification strategy in biosensing and bioimaging due to its simplicity, programmability, and ease of control. The PER strategy can achieve isothermal autonomous synthesis of arbitrary single-stranded DNA (ssDNA) using only a hairpin as a catalytic template, a short DNA primer (7-9 nt), and a strand-displacing polymerase (e.g., KF polymerase and Bst DNA polymerase) without the involvement of multiple enzymes and complex probe design. Summary of the invention

[0005] The purpose of the present invention is to provide a single molecule detection kit and use of a programmable automatic cascade mechanism for multiple non-coding RNAs in human breast tissue, by preparing a single molecule detection kit for single molecule detection of multiple non-coding RNAs in human breast tissue.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A method for preparing a single-molecule detection kit for multiple non-coding RNAs in human breast tissue using a programmable automatic cascade mechanism comprises the following steps:

[0008] Step 1, incubate a reaction solution of piRNA-36026 and lncRNA-DSCAM-AS1, dumbbell probe-pi, dumbbell probe-lnc, primer-pi, primer-lnc, dNTP mixture, ThermoPol reaction buffer, and Bst DNA polymerase (large fragment);

[0009] Among them, piRNA is a RNA that interacts with Piwi protein;

[0010] lncRNA is a long non-coding RNA;

[0011] Bst DNA polymerase (large fragment) is a part of the DNA polymerase protein of Bacillus stearothermophilus. The enzyme has 5′→3′ polymerase activity but lacks 5′→3′ exonuclease activity.

[0012] ThermoPol reaction buffer is the reaction buffer that comes with the Bst DNA polymerase (large fragment) product;

[0013] The dNTP mixture is an equimolar solution of ultrapure dATP, dCTP, and dTTP;

[0014] Step 2, adding the reaction product of step 1 to a reaction solution containing signal probe 1, signal probe 2, NEBuffer4 reaction buffer, and T7 exonuclease, and incubating to obtain a single molecule detection kit;

[0015] Among them, T7 exonuclease is a double-stranded DNA-specific exonuclease that catalyzes the removal of nucleotides from linear or nicked double-stranded DNA in the 5' to 3' direction;

[0016] NEBuffer4 reaction buffer is the reaction buffer that comes with the T7 exonuclease product.

[0017] In the step 1, 20 μL of a reaction solution of different concentrations of piRNA-36026 and lncRNA-DSCAM-AS1, 10 nM dumbbell probe-pi, 10 nM dumbbell probe-lnc, 100 nM primer-pi, 100 nM primer-lnc, 100 μM dNTP mixture, 1×ThermoPol reaction buffer, and 4 U Bst DNA polymerase (large fragment) was incubated at 37° C. for 60 minutes.

[0018] In the step 2, the reaction product was added to a reaction solution containing 300 nM signal probe 1, 300 nM signal probe 2, 1×NEBuffer 4 reaction buffer, and 10 U T7 exonuclease, and incubated at 37° C. for 30 min.

[0019] The ThermoPol reaction buffer solution includes 200mM Tris-HCl, 100mM (NH4)2SO4, 100mM KCl, 20mM MgSO4, 1% Triton X-100, pH 8.8. The NEBuffer4 reaction buffer solution includes 500mM Kac, 200mM Tris-Ac, 100mM Mg(Ac)2, 10mM DTT, pH 7.9.

[0020] The piRNA-36026 sequence is GGC CCCAUG GUG UAAUGG UCAGCACUC.

[0021] The lncRNA-DSCAM-AS1 sequence is: CUU UGG GAG GCU GAG GCA GG.

[0022] The dumbbell probe-pi sequence is: ACT AAA TTC AGG GCC TTT TGG CCC TGA ATT TAG TAATAA GAG AGG CCC GAG TGC TGA CCA TTA CAC CAT GGG GCC TCT CTT AT / Inverted dT / .

[0023] The probe dumbbell probe-lnc sequence is: ATTATTAAC AGG GCC TTT TGG CCC TGT TAATAATTAGTG TAA CTT TGG CCT GCC TCA GCC TCC CAAAGT TAC ACTA / Inverted dT / .

[0024] The probe primer-pi sequence is: TTT TTT TCT CTT ATT.

[0025] The probe primer-lnc sequence is: TTT TTT TTA CAC TA.

[0026] The probe signal probe 1 sequence is: FAM-TGA ATT TAG TTA TAA GAG A-BHQ1.

[0027] The sequence of the probe signal probe 2 is: Cy5-TGT TAA TAA TAA GTG TAA-BHQ2.

[0028] A programmable automated cascade mechanism for single-molecule detection of multiple noncoding RNAs in human breast tissue.

[0029] The programmable automatic cascade mechanism is used in the single molecule detection kit for multiple non-coding RNAs in human breast tissue in the preparation of a single molecule detection kit for detecting multiple ncRNAs in living cells and breast tissue.

[0030] The single-molecule detection kit can simultaneously quantify the expression of piR-36026 and DSCAM-AS1 in living cells and breast tissues, and can distinguish the expression levels of ncRNA in breast cancer patients and healthy people.

[0031] The single molecule detection kit is used to sensitively detect non-coding RNA, with a detection limit of 44.67 aM for piR-36026 and a detection limit of 45.71 aM for DSCAM-AS1.

[0032] Beneficial effects: The present invention provides a single-molecule detection kit and use of a programmable automatic cascade mechanism for multiple non-coding RNAs in human breast tissue, and develops a programmable automatic cascade mechanism for single-molecule detection of multiple ncRNAs (i.e., piR-36026 and DSCAM-AS1) in living cells and breast tissue. The analysis has several obvious advantages: (1) The input target ncRNAs can be autonomously and uniformly amplified into the functional ssDNA probe output by the PER cascade without the need for additional reverse transcription, effectively eliminating the contamination of genomic DNA; (2) The designed dumbbell probe integrates the target ncRNA recognition and the PER cascade in one probe, and due to the perfect folding of the dumbbell probe, the detection has extremely low background; (3) The integration of the programmable automatic cascade mechanism and single-molecule imaging makes the detection have higher sensitivity; (4) The entire amplification mechanism can be performed at a constant temperature, avoiding the necessity of tedious switching between multiple temperatures. Utilizing the high efficiency of the automatic cascade mechanism and the high signal-to-noise ratio of single-molecule imaging, the method has high sensitivity, good selectivity and multiplex analysis capabilities. This method can simultaneously quantify the expression of piR-36026 and DSCAM-AS1 in living cells and breast tissues, and can distinguish the expression levels of ncRNAs in breast cancer patients and healthy subjects. Importantly, by reprogramming the recognition sequence of the dumbbell probe, the proposed detection method can be used to detect other cancer-related ncRNAs, providing a new paradigm for multiplex detection and early clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Schematic diagram of combining a programmable automatic cascade mechanism with single-molecule detection to achieve simultaneous detection of multiple ncRNAs.

[0034] Figure 2: A is a 12% non-denaturing PAGE analysis of the PER cascade products initiated by piR-36026; B is a 12% non-denaturing PAGE analysis of the PER cascade products initiated by DSCAM-AS1; C is the FAM fluorescence emission spectrum in the presence (green curve) and absence (light green curve) of piR-36026; D is the Cy5 fluorescence emission spectrum in the presence (red curve) and absence (light red curve) of DSCAM-AS1.

[0035] Figure 3 : A is the FAM count determination induced by different concentrations of piR-36026; B is the linear relationship between the FAM count and the logarithm of the piR-36026 concentration; C is the Cy5 count determination induced by different concentrations of DSCAM-AS1; D is the linear relationship between the Cy5 count and the logarithm of the DSCAM-AS1 concentration.

[0036] Figure 4 : The FAM and Cy5 counts produced by piR-36026+DSCAM-AS1, piR-36026, DSCAM-AS1, miR-210, circMTO1, piR-36743, piR-823, HOTAIR, MALTAL and a control group without RNA were measured respectively.

[0037] Figure 5 : A is the FAM (green column) and Cy5 (red column) counts of HCT-116 cells, HepG-2 cells, MCF-7 cells, Hela cells, A549 cells and MCF-10A cells in response, respectively; B is the levels of piR-36026 (blue column) and DSCAM-AS1 (pink column) in HCT-116 cells, HepG-2 cells, MCF-7 cells, Hela cells, A549 cells and MCF-10A cells measured by qRT-PCR, respectively; C is the linear relationship between FAM counts and piR-36026 concentrations; D is the linear relationship between Cy5 counts and DSCAM-AS1 concentrations.

[0038] Figure 6 : A is the determination of ncRNA in clinical breast tissue; B and C are heat map analyses of piR-36026 (B) and DSCAM-AS1 (C) in breast cancer patient tissues and healthy human tissues, respectively; D and E are box plot analyses of piR-36026 (D) and DSCAM-AS1 (E) in breast cancer patient tissues and healthy human tissues, respectively; F is the ROC curve and AUC value showing the predictive ability of the test for breast cancer. DETAILED DESCRIPTION

[0039] The present invention will be further explained below in conjunction with the embodiments.

[0040] Reagents and Materials:

[0041] All oligonucleotides were synthesized and purified by HPLC by Sangon Biotechnology Co., Ltd. (Shanghai, China). Bst DNA polymerase (large fragment), 10×ThermoPol reaction buffer (200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, 1% Triton X-100, pH 8.8), T7 exonuclease, 10×NEBuffer4 (500 mM KAc, 200 mM Tris-Ac, 100 mM Mg(Ac)2, 10 mM DTT, pH 7.9), dATP, dCTP and dTTP were purchased from New England Biolabs (Ipswich, MA, USA). Diethylpyrocarbonate (DEPC)-treated water was purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). Human colon cancer cell line (HCT-116 cells), human liver cancer cell line (HepG-2 cells), human breast cancer cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), human lung adenocarcinoma cell line (A549 cells), and human normal breast cell line (MCF-10A cells) were purchased from the cell bank of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Paraffin-embedded breast tissue specimens from breast patients and healthy subjects were obtained from Nanjing Drum Tower Hospital (Nanjing, China), and the experiment was approved by the Ethics Committee of Nanjing Drum Tower Hospital.

[0042] Fluorescence spectrum detection and measurement of actual samples:

[0043] The fluorescence intensity of the reaction products was measured using an FLS-1000 fluorescence spectrometer (Livingston Instruments, Edinburgh, UK). The FAM fluorescence emission spectrum was measured at an excitation wavelength of 488 nm, and the fluorescence intensity at 520 nm was recorded for quantitative analysis of piR-36026. The Cy5 fluorescence emission spectrum was measured at an excitation wavelength of 635 nm, and the fluorescence intensity at 662 nm was recorded for quantitative analysis of DSCAM-AS1. Total RNA was extracted from human tissue samples using the miRNeasy FFPE kit according to the manufacturer's procedures. The resulting extract was transferred to a fresh tube and immediately used to detect the activity of piR-36026 / DSCAM-AS1.

[0044] Gel electrophoresis:

[0045] The target-induced polymerized transcripts were analyzed using 12% non-denaturing polyacrylamide gels in 1×TBE buffer (9mM Tris-HCl, 9mM boric acid, 0.2mM EDTA, pH 7.9) with 1×SYBR Gold as the fluorescent indicator at a constant voltage of 110V for 50min at room temperature. The gels were analyzed using the ChemiDocTM and ChemiDoc MP imaging systems.

[0046] The present invention develops a programmable auto-cascade mechanism for single-molecule detection of multiple ncRNAs (i.e., piR-36026 and DSCAM-AS1) in living cells and breast tissue. The analysis has several distinct advantages: (1) the input target ncRNAs can be autonomously and uniformly amplified into functional ssDNA probe outputs by PER cascade without the need for additional reverse transcription, effectively eliminating genomic DNA contamination; (2) the designed dumbbell probe integrates target ncRNA recognition with PER cascade in one probe, and the detection has extremely low background due to the perfect folding of the dumbbell probe; (3) the integration of the programmable auto-cascade mechanism with single-molecule imaging gives the detection higher sensitivity; (4) the entire amplification mechanism can be performed at a constant temperature, avoiding the necessity of tedious switching between multiple temperatures. Taking advantage of the high efficiency of the auto-cascade mechanism and the high signal-to-noise ratio of single-molecule imaging, the method has high sensitivity, good selectivity, and multiplex analysis capabilities. This method can simultaneously quantify the expression of piR-36026 and DSCAM-AS1 in living cells and breast tissues, and can distinguish the expression levels of ncRNAs in breast cancer patients and healthy subjects. Importantly, by reprogramming the recognition sequence of the dumbbell probe, the proposed detection method can be used to detect other cancer-related ncRNAs, providing a new paradigm for multiplex detection and early clinical diagnosis.

[0047] Embodiment 1:

[0048] A method for preparing a single-molecule detection kit for multiple non-coding RNAs in human breast tissue using a programmable automatic cascade mechanism comprises the following steps:

[0049] Step 1. Incubate 20 μL of a reaction solution containing different concentrations of piRNA-36026 and lncRNA-DSCAM-AS1, 10 nM dumbbell probe-pi, 10 nM dumbbell probe-lnc, 100 nM primer-pi, 100 nM primer-lnc, 100 μM dNTP mixture, 1× ThermoPol reaction buffer, and 4 U Bst DNA polymerase (large fragment) at 37 °C for 60 min.

[0050] Among them, piRNA is a RNA that interacts with Piwi protein;

[0051] lncRNA is a long non-coding RNA;

[0052] Bst DNA polymerase (large fragment) is a part of the DNA polymerase protein of Bacillus stearothermophilus. The enzyme has 5'→3' polymerase activity but lacks 5'→3' exonuclease activity.

[0053] ThermoPol reaction buffer is the reaction buffer that comes with the Bst DNA polymerase (large fragment) product;

[0054] dNTP mixture is an equimolar solution composed of ultrapure dATP, dCTP and dTTP.

[0055] Step 2, the reaction product was added to a reaction solution containing 300 nM signal probe 1, 300 nM signal probe 2, 1×NEBuffer 4 reaction buffer solution, and 10 U T7 exonuclease, and incubated at 37° C. for 30 min before subsequent measurement.

[0056] Among them, T7 exonuclease is a double-stranded DNA-specific exonuclease that catalyzes the removal of nucleotides from linear or nicked double-stranded DNA in the 5' to 3' direction;

[0057] NEBuffer4 reaction buffer is the reaction buffer that comes with the T7 exonuclease product.

[0058] ThermoPol reaction buffer solution includes 200mM Tris-HCl, 100mM (NH4)2SO4, 100mM KCl, 20mM MgSO4, 1% Triton X-100, pH 8.8.

[0059] NEBuffer4 reaction buffer solution includes 500mM KAc, 200mM Tris-Ac, 100mM Mg(Ac)2, 10mM DTT, pH 7.9.

[0060] The sequence of piRNA-36026 is GGC CCCAUG GUG UAAUGG UCAGCACUC.

[0061] The sequence of lncRNA-DSCAM-AS1 is: CUU UGG GAG GCU GAG GCAGG.

[0062] The sequence of the dumbbell probe-pi is: ACTAAATTCAGG GCC TTT TGG CCC TGAATT TAG TAA TAA GAGAGG CCC GAG TGC TGA CCA TTA CAC CAT GGG GCC TCT CTT AT / Inverted dT / .

[0063] The sequence of the probe dumbbell probe-lnc is: ATTATTAACAGG GCC TTT TGG CCC TGT TAATAA TTAGTGTAACTT TGG CCT GCC TCAGCC TCC CAAAGT TACACTA / Inverted dT / .

[0064] The sequence of probe primer-pi is: TTT TTT TCT CTTATT.

[0065] The sequence of probe primer-lnc is: TTT TTT TTACAC TA.

[0066] Probe signal probe 1 sequence is: FAM-TGAATTTAG TTATAAGAGA-BHQ1.

[0067] Probe signal probe 2 sequence is: Cy5-TGT TAATAATAAGTG TAA-BHQ2.

[0068] Embodiment 2: Figure 1 The mechanism of single-molecule detection of multiple ncRNAs is described. The present invention cleverly designs two dumbbell probes (dumbbell probe-pi and dumbbell probe-lnc), two linear primers (primer-pi and primer-lnc) and two linear signal probes (signal probe 1 and signal probe 2) for piR-36026 and DSCAM-AS1, respectively. The dumbbell probe of piR-36026 contains three functional domains, namely the complementary domain a of piR-36026, the binding domain b of primer-pi and the primer extension domain c. Three consecutive GC pairs) are located near the right loop domain of the dumbbell probe-pi, which serves as a stop codon to stop polymerization. The addition of dATP, dCTP and dTTP (without dGTP) can terminate the extension of the primer until the first guanine ribonucleotide is encountered during the polymerization process. Similarly, the dumbbell probe of DSCAM-AS1 contains three functional domains, namely the complementary domain a of DSCAM-AS1, the binding domain b of primer-lnc, the primer extension domain c and the stop codon domain with three consecutive GC pairs. Signal probe 1 ( Figure 1 , pink and orange) and signal probe 2 ( Figure 1, purple and blue) are labeled with a fluorophore (FAM or Cy5) at the 5' end and a quencher (BHQ1 or BHQ2) at the 3' end, respectively, and hybridize with functional probe 1 and functional probe 2, respectively, to initiate the cyclic liberation of FAM and Cy5 fluorophores assisted by T7 exonuclease (T7 Exo). The experiment consists of two consecutive steps: (1) primer exchange reaction (PER) induced by target ncRNA and (2) T7 Exo-assisted recycling cleavage of signal probes to release FAM and Cy5 fluorophores. In the presence of piR-36026, it can bind to the a domain of dumbbell probe-pi, exposing the b domain of primer-pi for binding. Subsequently, primer-pi hybridizes with domain b and is extended with the help of Bst DNA polymerase to append the nascent single-stranded sequence c* to the 3' end of primer-pi (the newly extended ssDNA is called functional probe 1), until the chain extension reaction stops at the stop codon of dumbbell probe-pi. Subsequently, the domain c* on the dumbbell probe-pi competes with the synthetic domain c* of the functional probe 1 through a random walk process of three-way branch migration, resulting in the spontaneous dissociation of the functional probe 1 from the dumbbell probe-pi. The free dumbbell probe-pi can bind to another primer-pi, inducing the next round of PER and producing abundant functional probe 1. Similarly, when DSCAM-AS1 is present, it can hybridize with the domain a of the dumbbell probe-lnc, exposing the binding domain b of the primer-lnc, and subsequently inducing the PER cascade to produce a large amount of functional probe 2. The resulting functional probes 1 and 2 can hybridize with the signal probes 1 and 2, respectively, to form two double-stranded DNA duplexes 1 and 2. The signal probes 1 and 2 in the dsDNA duplexes 1 and 2 can be digested stepwise by T7 Exo (a double-stranded DNA-specific exonuclease that catalyzes the removal of single nucleotides in the 5' to 3' direction), releasing FAM and Cy5 molecules as well as the functional probes 1 and 2. Notably, the released functional probes 1 and 2 can further hybridize with free signaling probes 1 and 2, respectively, initiating multiple rounds of digestion-release-hybridization, ultimately releasing a large number of FAM and Cy5 molecules. FAM and Cy5 signals can be simply monitored by TIRF-based single-molecule imaging for quantification of piR-36026 and DSCAM-AS1, respectively. However, in the absence of piR-36026 and DSCAM-AS1, the primer binding domain of the dumbbell probe is blocked and the auto-cascade mechanism cannot be initiated. As a result, neither FAM nor Cy5 signals are detected.

[0069] Example 3: Feasibility of verification experiment

[0070] The present invention carried out gel electrophoresis and fluorescence measurement to verify the feasibility of the method ( Figure 2 First, the products of the PER cascade were analyzed using 12% non-denaturing polyacrylamide gel electrophoresis (PAGE) with SYBR Gold as a fluorescent indicator. Figure 2 As shown in A, pi-36026 hybridizes with dumbbell probe-pi to form a double-stranded complex ( Figure 2 A, lane 3), whose migration rate is higher than that of the dumbbell probe-pi alone ( Figure 2 A, lane 5) is slow, which is a prerequisite for the subsequent PER cascade. Dumbbell probe-pi+primer-pi+Bst DNA polymerase did not detect the band of functional probe 1 ( Figure 2 A, lane 2), indicating that the PER cascade reaction did not occur. After adding piR-36026, the hybridization bands of piR-36026 and dumbbell probe-pi can be seen, and the functional probe-1 band also appears ( Figure 2 A, lane 1), indicating that piR-36026 can successfully initiate the PER cascade. In addition, in the absence of Bst DNA polymerase, the double-stranded complex cannot initiate the PER cascade ( Figure 2 Similarly, in the absence of DsCAM-AS1, no obvious functional probe 2 band was detected ( Figure 2 In contrast, in the presence of DSCAM-AS1, the characteristic band of functional probe 2 appeared ( Figure 2 B, lane 1), indicating that DSCAM-AS1 induced the PER cascade. Figure 2 B, lane 3) or Bst DNA polymerase ( Figure 2 In the case of (B, lane 4), no band of functional probe-2 was observed, indicating that these components played an important role in our experiment. The present invention further performed fluorescence spectroscopy measurements. When piR-36026 and DSCAM-AS1 were missing, the FAM fluorescence signal ( Figure 2 C, light green curve) and Cy5 fluorescence signal ( Figure 2 D, light red curve) were not detected. However, the FAM and Cy5 fluorescence signals generated by piR-36026 were significantly enhanced ( Figure 2 C, green curve) and DSCAM-AS1 ( Figure 2 D, red curve), indicating that piR-36026 and DSCAM-AS1 can activate the PER cascade to produce abundant functional probes and induce the cyclic liberation of FAM and Cy5 molecules assisted by T7 exonuclease. The above results demonstrate the feasibility of this method for simultaneous detection of piR-36026 and DSCAM-AS1.

[0071] Example 4: Sensitivity Detection

[0072] In order to investigate the detection sensitivity of the method, under the optimal reaction conditions, the present invention measured the responses of different concentrations of piR-36026 and DSCAM-AS1 to FAM and Cy5 fluorescent molecules. Figure 3 As shown in A, as the concentration of piR-36026 (C) increased from 0 to 1×10 -8 M, FAM counts (N) increased in a dose-dependent manner. -16 to 1×10 -9 There was a good linear relationship between FAM counts and the logarithm of piR-36026 concentration over a large dynamic range of 7 orders of magnitude ( Figure 3 The regression equation is N = 503.28 + 28.57lg C (R 2 =0.998), the detection limit is 44.67aM. Figure 4 As shown in C, as the concentration of DSCAM-AS1 (C) increased from 0 to 1×10 -8 M, Cy5 counts (N) increased in a concentration-dependent manner and increased from 1×10 -16 to 1×10 -9 A good linear correlation was obtained between the Cy5 counts and the logarithm of the DSCAM-AS1 concentration within a large dynamic range of 7 orders of magnitude ( Figure 3 The regression equation is N = 365.48 + 20.53lg C (R 2 =0.998), and the detection limit was calculated to be 45.71aM.

[0073] Example 5: Specificity detection

[0074] The specificity of this method was evaluated by introducing HOTAIR, MALTAL, piR-823, piR-36743, circMTO1, and miR-210 as negative controls. Figure 4As shown, neither FAM nor Cy5 fluorescence signals were observed in the presence of HOTAIR, MALTAL, piR-823, piR-36743, circMTO1, and miR-210, which was similar to the control group without RNA. In contrast, the addition of piR-36026 induced a significant enhancement of the FAM fluorescence signal, but not the Cy5 fluorescence signal, while the addition of DSCAM-AS1 induced a significant enhancement of the Cy5 fluorescence signal, but not the FAM fluorescence signal. When piR-36026 and DSCAM-AS1 coexisted, both FAM and Cy5 fluorescence signals were detected, indicating that only piR-36026 and DSCAM-AS1 specifically recognized and switched the structures of dumbbell probe-pi and dumbbell probe-lnc, respectively, initiating the subsequent auto-cascade mechanism to release Cy3 and Cy5 fluorophores. These results confirmed that the method has good selectivity for piR-36026 and DSCAM-AS1.

[0075] Example 6: Actual sample analysis

[0076] In order to explore the performance of this method in actual sample analysis, the present invention analyzed the expression levels of piR-36026 and DSCAM-AS1 in six cell lines, including HCT-116 cells, HepG-2 cells, MCF-7 cells, Hela cells, A549 cells and MCF-10A cells. Figure 5 As shown in A, the FAM counts of HCT-116 cells, HepG-2 cells, MCF-7 cells, Hela cells, and A549 cells were significantly higher than those of MCF-10A cells ( Figure 5 A, green column), indicating that piR-36026 is highly expressed in human cancer cells. Compared with the low Cy5 counts produced by MCF-10A cells, DSCAM-AS1 is expressed at higher levels in HCT-116 cells, HepG-2 cells, MCF-7 cells, Hela cells, and A549 cells ( Figure 5 A, red column), indicating that DSCAM-AS1 is upregulated in human cancer cells. We further compared our results with the standard qRT-PCR results of the same batch of extracted samples of HCT-116 cells, HepG-2 cells, MCF-7 cells, Hela cells, A549 cells, and MCF-10A cells. Figure 5 Figure B shows that the results of this example are consistent with the results of qRT-PCR. In addition, the present invention simultaneously detects piR-36026 and DSCAM-AS1 extracted from MCF-7 cells. Figure 5As shown in Figures 5C and 5D, the FAM and Cy5 counts (N) were linearly correlated with the logarithm of the MCF-7 cell number (X) in the range of 1 to 100,000 cells. The correlation equations for the piR-36026 method and the DSCAM-AS1 method were N = 47.79 + 36.75 lg X (R 2 =0.995) and N = 51.63 + 37.1lg X (R 2 =0.995). The detection limits of the piR-36026 method and the DSCAM-AS1 method were calculated as 1 cell. These results indicate that this method can accurately quantify multiple ncRNAs at the single-cell level and has great potential for application in clinical diagnosis.

[0077] Example 7: Clinical sample analysis

[0078] In order to evaluate the applicability of this method in clinical diagnosis, the expression levels of piR-36026 and DSCAM-AS1 in tissue samples of 8 breast cancer patients and 5 healthy subjects were analyzed in this example. Figure 6 (A). Figure 6 As shown in Figure B, the FAM count in breast cancer tissue is much higher than that in healthy human tissue. The average FAM count produced by breast cancer tissue was detected to be 219.03±20.06, which is 4.27 times higher than the average FAM count produced by healthy tissue (51.31±9.07). Figure 6 (B), indicating that piR-36026 is upregulated in breast cancer. In addition, the Cy5 counts of breast cancer tissues were significantly higher than those of healthy tissues. The average Cy5 counts produced by breast cancer tissues were detected to be 202.71±11.95, which is 3.36 times the average Cy5 counts produced by healthy tissues (60.41±7.57). Figure 6 B), indicating that DSCAM-AS1 is highly expressed in breast cancer. The experimental results (6 in FIG. 6) are consistent with the qRT-PCR results. The present embodiment generates a receiver operating characteristic (ROC) curve to further verify the diagnostic performance of the method. It is estimated that the area under the curve (AUC) values ​​of piR-36026 and DSCAM-AS1 are both 1 (>0.8), indicating that piR-36026 and lncRNA-DSCAM-AS1 can be used as tumor biomarkers to accurately predict breast cancer.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a single molecule detection kit for multiple non-coding RNAs in human breast tissue using a programmable automatic cascade mechanism, characterized in that: The following steps are involved: Step 1, incubate a reaction solution of piRNA-36026 and lncRNA-DSCAM-AS1, dumbbell probe-pi, dumbbell probe-lnc, primer-pi, primer-lnc, dNTP mixture, ThermoPol reaction buffer, and Bst DNA polymerase (large fragment); Among them, piRNA is a RNA that interacts with Piwi protein; lncRNA is a long noncoding RNA; Bst DNA polymerase (large fragment) is a part of the DNA polymerase protein of Bacillus stearothermophilus. The enzyme has 5′→3′ polymerase activity but lacks 5′→3′ exonuclease activity. ThermoPol reaction buffer is the reaction buffer that comes with the Bst DNA polymerase (large fragment) product; The dNTP mixture is an equimolar solution composed of ultrapure dATP, dCTP, and dTTP; Step 2, adding the reaction product of step 1 to a reaction solution containing signal probe 1, signal probe 2, NEBuffer4 reaction buffer, and T7 exonuclease, incubating to obtain a single molecule detection kit; Among them, T7 exonuclease is a double-stranded DNA-specific exonuclease that catalyzes the removal of nucleotides from linear or nicked double-stranded DNA in the 5' to 3' direction; NEBuffer4 reaction buffer is the reaction buffer that comes with the T7 exonuclease product.

2. A method for preparing a single molecule detection kit for multiple non-coding RNAs in human breast tissue using a programmable automatic cascade mechanism according to claim 1, characterized in that: In the step 1, 20 μL of a reaction solution of different concentrations of piRNA-36026 and lncRNA-DSCAM-AS1, 10 nM dumbbell probe-pi, 10 nM dumbbell probe-lnc, 100 nM primer-pi, 100 nM primer-lnc, 100 μM dNTP mixture, 1×ThermoPol reaction buffer, and 4 U Bst DNA polymerase (large fragment) was incubated at 37° C. for 60 minutes.

3. The method for preparing a single molecule detection kit for multiple non-coding RNAs in human breast tissue using a programmable automatic cascade mechanism according to claim 1, characterized in that: In the step 2, the reaction product was added to a reaction solution containing 300 nM signal probe 1, 300 nM signal probe 2, 1×NEBuffer 4 reaction buffer, and 10 U T7 exonuclease, and incubated at 37° C. for 30 min.

4. The method for preparing a single molecule detection kit for multiple non-coding RNAs in human breast tissue using a programmable automatic cascade mechanism according to claim 1, characterized in that: The ThermoPol reaction buffer solution includes 200mM Tris-HCl, 100mM (NH4)2SO4, 100mM KCl, 20mM MgSO4, 1% Triton X-100, pH 8.

8.

5. The method for preparing a single molecule detection kit for multiple non-coding RNAs in human breast tissue using a programmable automatic cascade mechanism according to claim 1, characterized in that: The NEBuffer4 reaction buffer solution includes 500mM KAc, 200mM Tris-Ac, 100mM Mg(Ac)2, 10mM DTT, pH 7.

9.

6. The method for preparing a single molecule detection kit for multiple non-coding RNAs in human breast tissue using a programmable automatic cascade mechanism according to claim 1, characterized in that: The piRNA-36026 sequence is GGC CCCAUG GUG UAAUGG UCAGCACUC; The lncRNA-DSCAM-AS1 sequence is: CUU UGG GAG GCU GAG GCA GG; The dumbbell probe-pi sequence is: ACT AAA TTC AGG GCC TTT TGG CCC TGA ATT TAG TAA TAAGAG AGG CCC GAG TGC TGA CCA TTA CAC CAT GGG GCC TCT CTT AT / Inverted dT / ; The probe dumbbell probe-lnc sequence is: ATTATTAAC AGG GCC TTT TGG CCC TGT TAATAATTAGTG TAA CTT TGG CCT GCC TCA GCC TCC CAAAGT TAC ACTA / Inverted dT / ; The probe primer-pi sequence is: TTT TTT TCT CTT ATT; The probe primer-lnc sequence is: TTT TTT TTA CAC TA; The probe signal probe 1 sequence is: FAM-TGA ATT TAG TTA TAA GAG A-BHQ1; The sequence of the probe signal probe 2 is: Cy5-TGT TAA TAA TAA GTG TAA-BHQ2.

7. A single-molecule detection kit for multiple non-coding RNAs in human breast tissue using a programmable automatic cascade mechanism obtained by the preparation method of claim 1.

8. Use of the programmable automatic cascade mechanism described in claim 7 for a single molecule detection kit for multiple non-coding RNAs in human breast tissue in the preparation of a single molecule detection kit for detecting multiple ncRNAs in living cells and breast tissue.

9. The use according to claim 8, characterized in that: The single-molecule detection kit can simultaneously quantify the expression of piR-36026 and DSCAM-AS1 in living cells and breast tissues, and can distinguish the expression levels of ncRNA in breast cancer patients and healthy people.

10. The use according to claim 8 or 9, characterized in that: The single molecule detection kit is used to sensitively detect non-coding RNA, with a detection limit of 44.67 aM for piR-36026 and a detection limit of 45.71 aM for DSCAM-AS1.

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