NL2034340B1

NL2034340B1 - MICRO RIBONUCLEIC ACID (miRNA) DETECTION KIT BASED ON PROBE-ANCHORED DUPLEX AND USE THEREOF

Info

Publication number: NL2034340B1
Application number: NL2034340A
Authority: NL
Inventors: Chen Ran; Xu Yili; Wang Kai
Original assignee: Univ Zhejiang
Priority date: 2022-11-08
Filing date: 2023-03-15
Publication date: 2024-05-24
Also published as: CN116200492B; CN116200492A; ZA202301656B

Abstract

The present disclosure provides a micro ribonucleic acid (miRNA) detection kit based on a probe-anchored duplex and use thereof. The miRNA detection kit based on a 5 probe-anchored duplex includes a main probe, an auxiliary probe, an anchor probe, and a fluorescent probe, as well as an amplification primer pair for target miRNAs, and a magnetic bead sample releasing agent, the main probe includes an auxiliary probe binding region, a target miRNA binding region, and an anchor probe binding region sequentially, the auxiliary probe binding region includes an upstream primer 10 complementary region and a fluorescent probe complementary region for amplifying the target miRNAs, the anchor probe binding region is complementary to a downstream primer for amplifying the target miRNAs, a 5'-end of the anchor probe or the main probe is labeled with biotin, and the magnetic bead sample releasing agent includes a streptaVidin-modif1ed magnetic bead.

Description

MICRO RIBONUCLEIC ACID (miRNA) DETECTION KIT BASED ON

PROBE-ANCHORED DUPLEX AND USE THEREOF

TECHNICAL FIELD

[0001] The present disclosure belongs to the technical field of nucleic acid detection, and in particular relates to a micro ribonucleic acid (miRNA) detection kit based on a probe-anchored duplex and use thereof.

BACKGROUND

[0002] Micro ribonucleic acid (miRNA) is a kind of single-stranded and non-coding small molecule RNA with a length of about 20 to 24 nucleotides (nt), widely existing in human tissues, cells, blood, and body fluids. The miRNA has an important gene regulatory function, and its abnormal expression is closely related to the occurrence, development, and metastasis of various diseases, especially tumors. Cell-free miRNA (cfmiRNA), also known as circulating miRNA (ctmiRNA), is a class of miRNA released into blood and other body fluids through active secretion by cells or passive leakage such as tissue damage and cell rupture. The cfmiRNA exists stably in the form of being encapsulated by exosomes or microvesicles or bound to proteins, and is suitable as a molecular marker for disease detection. Abnormally enriched tumor-derived cfmiRNAs in blood may occur in the early stages of tumors, originating from the expression and secretion of tumor cells in situ in tissues. Meanwhile, circulating tumor cells (CTCs) and cell-free DNAs (ctDNAs) are generally the result of direct metastases of tumor cells into the blood in advanced tumor stages. Therefore, cfmiRNA detection can achieve higher sensitivity than that of CTC and ctDNA detection, and is also superior to protein markers such as conventional tumor markers and autoantibodies in tumor detection.

[0003] The ctmiRNA amplification detection method can achieve better sensitivity and accuracy than those of other methods, and is the most commonly-used mainstream method. The method is generally based on real-time fluorescence quantitative PCR amplification, including: preparing a PCR template using a target miRNA, and then conducting the fluorescence quantitative PCR amplification. There is an inverse relationship between a Ct value of the amplification result and a copy number of the target miRNA: the smaller Ct value indicates more copies of the target miRNA; while if there is no Ct value, no target miRNA is detected. There are two main categories of methods for preparing PCR templates from target miRNAs. The first category is reverse transcription, including PolyA-tailed reverse transcription, tailed long primer reverse transcription, stem-loop primer reverse transcription (stem-loop method), and locked nucleic acid (LNA) primer reverse transcription. The stem-loop method is a mainstream method. In this type of method, the miRNA has an overall length of only about 22 nt, and parts that can be used for reverse transcription and parts used for the upstream primer are only about 11 nt separately. As a result, it is difficult for this type of method to exclude the interference of non-target nucleic acids present in large quantities in the sample, to hardly ensure the specificity of reverse transcription and

PCR amplification, resulting in high background and poor repeatability. Moreover, this type of method is not compatible with universal primer-based PCR, and is also difficult to conduct multiplex PCR, showing a low performance of the combination detection. The second category is a main probe method. In the early stage, our team invented a method for hybrid capture of a main probe using a target RNA to prepare a

PCR template and then conduct amplification detection, including: (1) "A PCR detection method for RNA with a washing-free main probe" (ZL201310205271.6) provides a PCR detection method for RNA. In this method, universal primers can be used; however, the detection of miRNAs directly by the universal primers is easily interfered by the hybridization of precursors and genomic DNA, which cannot avoid the high background and poor repeatability. (2) "A method and a kit for combined amplification detection of miRNAs" (202011041856.5) is a PCR detection method for miRNAs. Hybrid capture is conducted on multiple target miRNAs using single-stranded main probes, excess single-stranded main probes are removed by mung bean nuclease digestion, followed by conducting purification to obtain main probes that form duplexes with the target miRNAs; and PCR amplification detection is conducted to indicate the presence of the target miRNAs. This method is beneficial to eliminate interference and improve detection combination performances. However, during the expanded application, it is found that this method has two main shortcomings: (1) The method is not conducive to the detection of diseases with high genetic heterogeneity such as tumors. In this method, multiple target miRNAs need to exist in the sample simultaneously, but the high genetic heterogeneity leads to a low probability of simultaneous occurrence of multiple target miRNAs in different individual patients. (2) The method is not conducive to the detection of samples with high nucleic acid content. Mung bean nuclease has no selectivity for single-stranded nucleic acids. When there is a high nucleic acid content in the sample, the mung bean nuclease in the system is saturated by the large amount of single-stranded nucleic acids in the sample and cannot remove the single-stranded main probes in time, resulting in serious background amplification.

SUMMARY

[0004] In view of this, an objective of the present disclosure is to provide a miRNA detection kit based on a probe-anchored duplex and use thereof. The kit has simple operation, desirable repeatability, and excellent combination, and can suppress background amplification to improve tumor detection sensitivity.

[0005] The present disclosure provides a miRNA detection kit based on a probe-anchored duplex, including a probe set, an amplification primer pair for target miRNAs, and a magnetic bead sample releasing agent; where

[0006] the probe set includes a main probe, an auxiliary probe, an anchor probe, and a fluorescent probe; the main probe includes an auxiliary probe binding region, a target miRNA binding region, and an anchor probe binding region sequentially; and the main probe has a length consistent with a total length of the auxiliary probe, the target miRNAs, and the anchor probe;

[0007] the auxiliary probe binding region includes an upstream primer complementary region and a fluorescent probe complementary region for amplifying the target miRNAs; and the auxiliary probe binding region has a length consistent with a total length of an upstream primer for amplifying the target miRNAs and a fluorescent probe;

[0008] the anchor probe and the anchor probe binding region have a same length, and the anchor probe binding region is complementary to a downstream primer for amplifying the target miRNAs;

[0009] a 5'-end of the anchor probe or the main probe is labeled with biotin; and

[0010] the magnetic bead sample releasing agent includes 40 mM to 80 mM of zinc chloride, 40 mM to 80 mM of sodium acetate, 40 mM to 80 mM of cysteine, 10% to 18% of ethylene carbonate by mass percentage, 10% to 30% of formamide by volume percentage, 4% to 10% of sodium lauryl sulfate by mass percentage, and 90 pg/ml to

200 pg/ml of a streptavidin-modified magnetic bead.

[0011] Preferably, a base at a 3'-end of the main probe is adenine.

[0012] Preferably, the anchor probe binding region has a nucleotide sequence shown in SEQ ID NO: 3; and

[0013] the anchor probe has a nucleotide sequence shown in SEQ ID NO: 4.

[0014] Preferably, the downstream primer for amplifying the target miRNAs has a nucleotide sequence shown in SEQ ID NO: 5.

[0015] Preferably, the auxiliary probe binding region has a nucleotide sequence shown in SEQ ID NO: 1; and

[0016] the auxiliary probe has a nucleotide sequence shown in SEQ ID NO: 2.

[0017] Preferably, the upstream primer for amplifying the target miRNAs has a nucleotide sequence shown in SEQ ID NO: 6; and

[0018] the fluorescent probe has a nucleotide sequence shown in SEQ ID NO: 7.

[0019] Preferably, the kit further includes a washing solution, mung bean nuclease, a

PCR amplification buffer, and a 1x positive control; where

[0020] the washing solution is an aqueous solution including 5 mM to 20 mM of 3-morpholinopropanesulfonic acid, 40 mM to 80 mM of cysteine, 30 mM to 50 mM of sodium acetate, and 0.05% to 0.1% of Tween 20 by volume percentage.

[0021] The present disclosure further provides use of the miRNA detection kit based on a probe-anchored duplex in miRNA detection of a non-diagnostic purpose.

[0022] Preferably, the miRNA includes at least one miRNA selected from the group consisting of miR-191, miR-93, miR-16, miR-3662, and an miR-181 family; and

[0023] the miR-181 family includes at least one of miR-181a-Sp, miR-181b-5p, miR-181c-5p, and miR-181d-5p.

[0024] The present disclosure further provides a method for detecting miRNAs in a non-diagnostic purpose using the kit, including the following steps:

[0025] mixing a sample to be tested, the main probe, the auxiliary probe, the anchor probe, and the magnetic bead sample releasing agent, and conducting incubation; removing a supernatant under a magnetic field, washing, and conducting enzymolysis with the mung bean nuclease solution; removing a supernatant under the magnetic field, washing, and conducting fluorescence quantitative PCR amplification with the

PCR amplification buffer, the fluorescent probe, and the amplification primer pair for target miRNAs; where a detection result with a Ct value of less than or equal to 35 indicates that the sample to be tested has the target miRNAs, while the detection result with the Ct value of greater than 35 indicates that the sample to be tested does not have the target miRNAs.

[0026] The present disclosure provides a miRNA detection kit based on a 5 probe-anchored duplex, including a probe set, an amplification primer pair for target miRNAs, and a magnetic bead sample releasing agent. During detection, the main probe, the auxiliary probe, and the anchor probe are mixed with the sample and subjected to thermal insulation. The main probe can simultaneously hybridize with the auxiliary probe, the anchor probe, and the target miRNA to form a complete duplex, and bind to the streptavidin-modified magnetic bead through the biotin labeled to the end of the anchor probe or the main probe. After aspirating a supernatant and washing, the excess nucleic acid in the sample is washed away, and then the mung bean nuclease system is added for enzyme cleavage. The main probe that does not bind to the target miRNA is in a single-stranded or partially single-stranded state and then degraded, and the main probe that has captured the target miRNA remains intact in the duplex. After washing again, a fluorescence quantitative PCR buffer is added for PCR amplification.

A Ct value indicates that the target miRNA exists. Compared with the miRNA detection methods disclosed in the prior art, the kit uses universal auxiliary probe, anchor probe, and main probe to conduct hybridize capture on a single target miRNA to form a duplex. The kit does not rely on the simultaneous presence of multiple target miRNAs, and introduces a biotin-streptavidin magnetic bead to conduct extraction and washing before the mung bean nuclease digestion, thereby removing other nucleic acids that compete with mung bean nuclease in the sample. Accordingly, it is convenient for the single-stranded main probe to be degraded and removed to eliminate the background, thereby ensuring the accuracy and reliability of detection results.

[0027] The present disclosure further provides a method for detecting miRNAs in a non-diagnostic purpose using the kit. In the present disclosure, RNA is extracted by direct cleavage, and probe-specific hybridization with biotin-streptavidin magnetic beads is simultaneously conducted to capture the main probe and target miRNAs. The method can not only eliminate various mung bean nuclease inhibitors, but also eliminate a large number of non-target nucleic acids, thereby greatly reducing the mung bean nuclease load, giving full play to the enzymatic cleavage activity of mung bean nuclease, and clearing the probe background. Meanwhile, the main probe bound to the target miRNA forms a complete matching duplex with the auxiliary probe and the anchor probe, and the duplex is retained in the system to obtain a purified PCR template, which is conducive to improving the efficiency of PCR amplification. Thus, the method improves both the specificity and sensitivity of miRNA amplification detection. Experimental verification shows that the method of the present disclosure can eliminate the interference of excess nucleic acid, can identify mismatches with cleavage as low as 1 nt, and can identify similar miRNAs in the miRNA family.

Therefore, the method is not only superior to the traditional stem-loop method, but also superior to the method of patent 202011041856.5. The method has sensitivity as low as 100 copies/ml, which is 10 to 100 times higher than the method of patent 202011041856.5 or the stem-loop method. The method uses universal primers and universal fluorescent probes, and more than eight kinds of miRNAs can be detected in one reaction, which greatly improves the combined detection performances and is beneficial to the detection of genetic heterogeneity diseases, especially tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows a flow principle of a technical solution provided by the present disclosure; and

[0029] FIG. 2 shows an analysis result of sequence homology of a miR-181 family.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The present disclosure provides a miRNA detection kit based on a probe-anchored duplex, including a probe set, an amplification primer pair for target miRNAs, and a magnetic bead sample releasing agent.

[0031] In the present disclosure, the probe set includes a main probe, an auxiliary probe, an anchor probe, and a fluorescent probe. The main probe includes an auxiliary probe binding region, a target miRNA binding region, and an anchor probe binding region in sequence. The auxiliary probe binding region and the anchor probe binding region are common regions, while the target miRNA binding region is a variable region, which changes correspondingly according to target miRNA sequences. The main probe has a length consistent with a total length of the auxiliary probe, the target miRNA, and the anchor probe. The auxiliary probe is 100% complementary to a sequence of the auxiliary probe binding region. The target miRNA binding region is 100% complementary to a sequence of the target miRNA. The anchor probe binding region is 100% complementary to a sequence of the anchor probe.

[0032] In an example of the present disclosure, four sets of main probes are designed for mirRNAs of the miRNA: miR-191, miR-93, miR-16, and miR-3662. The miR-191, the miR-93, the miR-16, and the miR-3662 have nucleotide sequences shown in SEQ

ID NO: 8 to SEQ ID NO: II, respectively, and the corresponding main probes have nucleotide sequences shown in SEQ ID NO: 12 to SEQ ID NO: 15.

[0033] In the present disclosure, the auxiliary probe binding region includes an upstream primer complementary region and a fluorescent probe complementary region tor amplifying the target miRNAs; and the auxiliary probe binding region has a length consistent with a total length of an upstream primer for amplifying the target miRNAs and a fluorescent probe. The upstream primer complementary region for amplifying the target miRNAs is 100% complementary to a sequence of the upstream primer for amplifying the target miRNAs.

[0034] In the present disclosure, the anchor probe and the anchor probe binding region have a same length, and the anchor probe binding region is 100% complementary to a sequence of a downstream primer for amplifying the target miRNAs. A 5'-end of the anchor probe or the main probe is labeled with biotin.

[0035] In the present disclosure, when designing probes and primers, the design principles preferably include: a homologous part of the upstream primer, a homologous part of the fluorescent probe, and the anchor probe binding region have a length of 20 to 30 nt, and the sequence has no secondary structure and no more than 4 consecutive single nucleotides; the homology part of the upstream primer and the anchor probe binding region have a Tm value of 55°C to 60°C, and the homology part of the fluorescent probe has a Tm value of 65°C to 70°C.

[0036] In the present disclosure, a base at a 3'-end of the main probe is preferably adenine. The anchor probe binding region has a nucleotide sequence of preferably SEQ

ID NO: 3; and the anchor probe has a nucleotide sequence of preferably SEQ ID NO: 4.

The downstream primer for amplifying the target miRNAs has a nucleotide sequence of preferably SEQ ID NO: 5. The auxiliary probe binding region has a nucleotide sequence of preferably SEQ ID NO: 1; and the auxiliary probe has a nucleotide sequence of preferably SEQ ID NO: 2. The upstream primer for amplifying the target miRNAs has a nucleotide sequence of preferably SEQ ID NO: 6; and the fluorescent probe has a nucleotide sequence of preferably SEQ ID NO: 7.

[0037] In the present disclosure, the magnetic bead sample releasing agent includes 40 mM to 80 mM of zinc chloride, 40 mM to 80 mM of sodium acetate, 40 mM to 80 mM of cysteine, 10% to 18% of ethylene carbonate by mass percentage, 10% to 30% of formamide by volume percentage, 4% to 10% of sodium lauryl sulfate by mass percentage, and 90 pg/ml to 200 pg/ml of a streptavidin-modified magnetic bead. In the magnetic bead sample releasing agent, the sodium acetate, zinc chloride, cysteine, ethylene carbonate, and glycerol provide suitable ionic strength, pH value, and nucleic acid hybridization conditions. The sodium lauryl sulfate, zinc chloride, and cysteine provide sample lysis, protein denaturation, and RNase enzyme inhibition. The streptavidin-modified magnetic bead specifically adsorbs the anchor probe and the duplex formed with the main probe.

[0038] In the present disclosure, the kit further includes preferably a washing solution, mung bean nuclease, a PCR amplification buffer, and a 1x positive control. The washing solution is an aqueous solution including 5 mM to 20 mM of 3-morpholinopropanesulfonic acid, 40 mM to 80 mM of cysteine, 30 mM to 50 mM of sodium acetate, and 0.05% to 0.1% of Tween 20 by volume percentage. The PCR amplification buffer is an aqueous solution containing common components of fluorescence quantitative PCR, including hot-start DNA polymerase and dNTP. There is no special limitation on a source of the PCR amplification buffer, and PCR amplification buffers well known in the art can be used; in an example, the PCR amplification buffer is purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0039] The present disclosure further provides use of the miRNA detection kit based on a probe-anchored duplex in miRNA detection of a non-diagnostic purpose.

[0040] In an example of the present disclosure, the miRNA includes preferably at least one miRNA selected from the group consisting of miR-191, miR-93, miR-16, miR-3662, and an miR-181 family. The miR-191, the miR-93, the miR-16, and the miR-3662 have nucleotide sequences shown in SEQ ID NO: 8 to SEQ ID NO: 11; and the miR-181 family include at least one of miR-181a-5p, miR-181b-5p, miR-181c-5p, and miR-181d-5p. The miR-181a-5p, the miR-181b-5p, the miR-181c-5p, and the miR-181d-5p have nucleotide sequences shown in SEQ ID NO: 20 to SEQ ID NO: 23.

[0041] The present disclosure further provides a method for detecting miRNAs in a non-diagnostic purpose using the kit, including the following steps:

[0042] mixing a sample to be tested, the main probe, the auxiliary probe, the anchor probe, and the magnetic bead sample releasing agent, and conducting incubation; removing a supernatant under a magnetic field, washing, and conducting enzymolysis with the mung bean nuclease solution; removing a supernatant under the magnetic field, washing, and conducting fluorescence quantitative PCR amplification with the

[0043] In the present disclosure, the main probe has a working concentration of preferably 110 nM to 130 nM, more preferably 120 nM. The auxiliary probe has a working concentration of preferably 110 nM to 130 nM, more preferably 120 nM. The anchor probe has a working concentration of preferably 110 nM to 130 nM, more preferably 120 nM. The sample to be tested includes preferably a plasma sample or tissue fluid or cell culture fluid. The sample to be tested and the magnetic bead sample releasing agent have a volume ratio of (9-10):4, more preferably 9.5:4.

[0044] In the present disclosure, the incubation is conducted preferably at 28°C to 35°C for 8 min to 12 min, more preferably at 30°C for 10 min. The mung bean nuclease solution has a concentration of preferably 0.15 U/ul to 0.25 U/ul, more preferably 0.2 U/ul. A solvent of the mung bean nuclease solution is an aqueous solution containing 30 mM of sodium chloride and 1 mM of zinc chloride in 50 mM of a sodium acetate buffer at a pH value of 5.0. The mung bean nuclease solution is added at 1/2 volume of the sample to be tested.

[0045] In the present disclosure, a fluorescence quantitative PCR amplification program includes: 95°C for 10 min; 93°C for 10 sec and 65°C for 40 sec, conducting 40 cycles; and collecting fluorescence signals at 65°C. The sample to be tested and the

PCR amplification buffer have a volume ratio of preferably (9-10):3, more preferably 9.53.

[0046] In the present disclosure, the method has strong anti-interference ability and high detection sensitivity (with sample cell concentration of 0.1 cells/ul and miRNAs of 10 copies/ml), which is more advantageous than the method of patent

202011041856.5. The method can achieve high specificity in distinguishing different miRNAs in the same family, and has a blood positive rate in detecting lung cancer of 100%, as shown in Table 1.

[0047] Table 1 Comparison of results in detecting miRNAs by different methods

[0048]

Detection PADSA MCAP Stem-loop method method of miRNAs

Fluorescence = quantitative PCR

PCR 3 steps: (1) direct cleavage to | 3 steps: (1) direct cleavage to 6 steps: (1) enrichment and pretreatment extract RNA, and extract RNA, and isolation of exosomes; (2) steps simultaneous probe-specific simultancous probe-specific washing; (3) cleavage to hybridization and hybrid capture; (2) mung extract RNA: (4) DNase I biotin-streptavidin magnetic bean nuclease digestion; (3) digestion to remove DNA; bead capture; (2) mung bean | collection of purified nucleic (5) collection of purified nuclease digestion; (3) acid samples with high yield nucleic acid samples with collection of purified PCR low yield; (6) stem-loop template reverse transcription nuclease recognizes acid content of the sample is mismatches in miRNA mismatches that cut as low as high enough to saturate the families

I nt mung bean nuclease, the specificity may be severely reduced

Combination Desirable: by universal Acceptable: 4 miRNAs Poor: 1 miRNA canbe primers and universal simultaneously present can detected in one reaction fluorescent probes, more than be detected in one reaction 8 miRNAs can be detected in one reaction

Anti-interference Desirable: integrated General: the reaction is General: the reaction is hybridization, susceptible to interference susceptible to interference biotin-streptavidin magnetic | from excess nucleic acid and | from excess nucleic acid and bead capture, and digestion cleavage inhibitors reverse transcription can remove various inhibitors interfering substances

[0049] The miRNA detection kit based on a probe-anchored duplex and the use thereof provided by the present disclosure are described in detail below with reference to the examples, but these examples should not be understood as limiting the claimed scope of the present disclosure.

[0050] Table 2 is the sequence and corresponding number.

[0051] Table 2 Number and sequence information

[0032]

Sequenc Sequence (5'-3") Modi e name fication ee eeen] ee eeens

Anchor CCTCCACGTGACCCTGACGTA (SEQ ID NO: 3) probe binding region

GABO1 ACGTCAGGGTCAGCTGGAGG (SEQ ID NO: 4) 5’bio tin enn

ET

GPPO1 TCCGGATGCTGCAGTGATGGCA (SEQ ID NO: 7) 3'FAM.3’

BHQ!

TS0191 | GAGGCACAGCAGGTGCAGGTCCGGATGCTGCAGTGATGGCACAGCTGCTTTTG

GGATTCGTTGCCTCCACGTGACCCTGACGTA (SEQ ID NO: 12)

TS3662 | GAGGCACAGCAGGTGCAGGTCCGGATGCTGCAGTGATGGCACATCAGTCACTA

CTCATCATTTTCCCTCCACGTGACCCTGACGTA (SEQ ID NO: 15)

TS0016 | GAGGCACAGCAGGTGCAGGTCCGGATGCTGCAGTGATGGCACGCCAATATTTAC -

GTGCTGCTACCTCCACGTGACCCTGACGTA (SEQ ID NO: 14)

TS18la | GAGGCACAGCAGGTGCAGGTCCGGATGCTGCAGTGATGGCAACTCACCGACAG

CGTTGAATGTTCCTCCACGTGACCCTGACGTA (SEQ ID NO: 16)

TS181b | GAGGCACAGCAGGTGCAGGTCEGGATGETGCAGTGATGGCAACCCACCGACAG

CAATGAATGTTCCTCCACGTGACECTGACGTA (SEQ ID NO: 17)

TS181c | GAGGCACAGCAGGTGCAGGTCCGGATGCTGCAGTGATGGCAACTCACCGACAG -

GTTGAATGTTCCTCCACGTGACCCTGACGTA (SEQ ID NO: 18)

TS181d | GAGGCACAGCAGGTGCAGGTCCGGATGCTGCAGTGATGGCAACCCACCGACAA

CAATGAATGTTCCTCCACGTGACCCTGACGTA (SEQ ID NO: 19)

OL1009 GATGGACGTGCTTGTCGTGAAAC (SEQ ID NO: 27) 3 =| aen

TT

OLI366 GAAAATGATGAGTAGTGACTGATG (SEQ ID NO: 29) 2

OLI1001 TAGCAGCACGTAAATATTGGCG (SEQ ID NO: 30) 6

OL1181 AACATTCAACGCTGTCGGTGAGT (SEQ ID NO: 31) - a

OLI181 AACATTCATTGCTGTCGGTGGGT (SEQ ID NO: 32) b

OLII81 AACATTCAACCTGTCGGTGAGT (SEQ ID NO: 33) ©

OL1181 AACATTCATTGTTGTCGGTGGGT (SEQ ID NO: 34) - d

TS0411 | GAGGCACAGCAGGTGCAGGTCCGGATGCTGCAGTGATGGCAGGTTAGTGGACC

GTGTTACATACCTCCACGTGACCCTGACGTA (SEQ ID NO: 24)

SPO411 ATGGCAGGTTAGTGGACCGTGTTACATA (SEQ ID NO: 25) S'FAM.3°

BHQI ee 1 oy ee——— mee ee 62 1a-5p he eee 1b-5p

Ie-5p wel mmm OL 1d-5p

[0053] Example 1

[0054] Detection of miRNAs in cell medium supernatant by method of the present disclosure with universal fluorescent probes

[0055] A549 lung cancer cells and a lung cancer-associated miRNA miR-411 were taken as an example. A main probe for detection was designed and synthesized, namely the main probe TS0411 of miR-411, with a sequence of: 5'-GAGGCACAGCAGGTGCAGGTCCGGATGCTGCAGTGATGGCAGGTTAGT

GGACCGTGTTACATACCTCCACGTGACCCTGACGTA-3', where 5'-end and 3'-end (underlined) were universal sequences. The 5'-end was completely homologous to an upstream universal primer and an universal fluorescent probe, and the 3'-end was completely complementary to an universal anchor probe; the middle part (shown in bold) was a target miRNA binding region, which was completely complementary to a target miRNA miR-16. The auxiliary probe (SEQ ID NO: 2), anchor probe (SEQ ID

NO: 4), PCR primers (SEQ ID NO: 5 and SEQ ID NO: 6), and fluorescent probe (SEQ

ID NO: 7) each were universal. In the fluorescent probe, a 5'-end was labeled with a fluorophore FAM, and a 3'-end was labeled with a quencher BHQ1. The preparation of the system was as described above. 100 pl of an A549 cell medium supernatant or 100 ul of purified water (as a negative control) was centrifuged at 12,000 rpm for 1 min; 90 ul of a resulting supernatant was transferred to a tube containing 40 pl of a magnetic bead sample releasing agent, mixed well, and incubated at 30°C for 10 min; a product was adsorbed with a magnet to remove a supernatant, mixed well with 900 ul of a washing solution, then absorbed with a magnet to remove a supernatant, added with 50 ul of a mung bean nuclease solution, and incubated at 30°C for 1 min; a product was adsorbed with a magnet to remove a supernatant, mixed well with 900 u of the washing solution, then absorbed with a magnet to remove the supernatant, added with 30 ul of a PCR, mixed well by pipetting and transferred to a PCR tube; on an ABI7500

PCR thermal cycler, the fluorescence quantitative PCR was conducted by: 95°C for 10 min, followed by 40 cycles of 93°C for 10 sec and 65°C for 40 sec, and a fluorescent signal was collected at 65°C (FAM channel). The result of the A549 cell medium supernatant sample was positive (amplification with a Ct value of 35), indicating that the content of miR-411 in the sample reached a detectable level. The result of the negative control was negative (no amplification, that is, no Ct value), indicating that the operation process was normal without pollution.

[0056] Example 2

[0057] Detection of miRNAs in cell medium supernatant by method of the present disclosure with specific fluorescent probes

[0058] In the application scenario where synchronous amplification is required to detect and distinguish different target miRNAs, the method of the present disclosure can adopt specific fluorescent probes. A549 lung cancer cells and miR-411, and miR-16 were taken as an example. A main probe for detection was designed and synthesized, the main probe TS0411 of miR-411 was the same as that in Example 1, and a main probe TS0016 of miR-16 was shown in the sequence listing. The auxiliary probe, anchor probe, and PCR primer were universal and had sequences as described above. The fluorescent probes were specific to the target miRNAs. The miR-411-specific ~~ fluorescent probe (SP0411) had a sequence of: 5'-ATGGCAGGTTAGTGGACCGTGTTACATA-3', where a 5'-end was labeled with a fluorophore FAM, and a 3'-end was labeled with a quencher BHQI. The miR-16-specific fluorescent probe (SP0016) had a sequence of: 5'-ATGGCACGC

CAATATTTACGTGCTGCTA-3', where a 5'-end was labeled with a fluorophore HEX, and a 3'-end was labeled with a quencher BHQI. The part in bold was a part completely complementary to the target miRNA, and the part underlined upstream was a part of the universal sequence to increase a Tm value of the probe. The preparation of the system was as described above. 100 ul of an A549 cell medium supernatant or 100 ul of purified water (as a negative control) was centrifuged at 12,000 rpm for 1 min; 90 ul of a resulting supernatant was transferred to a tube containing 40 ul of a magnetic bead sample releasing agent, mixed well, and incubated at 30°C for 10 min; a product was adsorbed with a magnet to remove a supernatant, mixed well with 900 u of a washing solution, then absorbed with a magnet to remove a supernatant, added with 50 ul of a mung bean nuclease solution, and incubated at 30°C for 1 min; a product was adsorbed with a magnet to remove a supernatant, mixed well with 900 pl of the washing solution, then absorbed with a magnet to remove the supernatant, added with 30 ul of a PCR, mixed well by pipetting and transferred to a PCR tube; on an ABI7500

PCR thermal cycler, the fluorescence quantitative PCR was conducted by: 95°C for 10 min, followed by 40 cycles of 93°C for 10 sec and 65°C for 40 sec, and a fluorescent signal was collected at 65°C (FAM/HEX dual channel). The results of FAM channel and HEX channel of A549 cell medium supernatant samples each were positive (amplification with a Ct value), indicating that the contents of miR-411 and miR-16 in the sample each reached a detectable level. The results of FAM channel and HEX channel of the negative control each were negative (no amplification, that is, no Ct value), indicating that the operation process was normal without pollution.

[0059] Example 3

[0060] Detection of miRNAs in cell medium supernatant by method of the present disclosure with universal fluorescent dyes

[0061] Taking A549 lung cancer cells and miR-411 as an example: the main probe, auxiliary probe, anchor probe, and PCR primers were the same as those in Example 1, instead of using fluorescent probes, SYBR Green I was added to 0.4. The preparation of the remaining systems was as described above. 100 u of an A549 cell medium supernatant or 100 u of purified water (as a negative control) was centrifuged at 12,000 rpm for 1 min; 900 ul of a resulting supernatant was transferred to a tube containing 40 ul of a magnetic bead sample releasing agent, mixed well, and incubated at 30°C for 10 min; a product was adsorbed with a magnet to remove a supernatant, mixed well with 900 pl of a washing solution, then absorbed with a magnet to remove a supernatant, added with 50 ul of a mung bean nuclease solution, and incubated at

30°C for 1 min; a product was adsorbed with a magnet to remove a supernatant, mixed well with 900 ul of the washing solution, then absorbed with a magnet to remove the supernatant, added with 30 u of a PCR, mixed well by pipetting and transferred to a

PCR tube; on a Hongshi SLAN-96p PCR thermal cycler, the fluorescence quantitative

PCR was conducted by: 95°C for 10 min, followed by 40 cycles of 93°C for 10 sec, 65°C for 30 sec, and 74°C for 34 sec, and a fluorescent signal was collected at 74°C (SYBR channel). The result of the A549 cell medium supernatant sample was positive (amplification with a Ct value), indicating that the content of miR-411 in the sample reached a detectable level. The result of the negative control was negative (no amplification, that is, no Ct value), indicating that the operation process was normal without pollution.

[0062] Example 4

[0063] Components of the kit in the present disclosure

[0064] The production of 95 sets according to the conventional specification of the kit, 8 reactions/package, was taken as an example. According to a common 95% yield, 100 sets were planned. The components and required quantities were shown in Table 3, and the materials were prepared according to instructions of the kit (Table 4).

[0065] Table 3 Components of 100 sets of planned quantities

[0066]

No. Component Specificatio 100 sets of Remar n quantities ks

PADSACI 1.1x magnetic bead 300 ul 30 mi sample releasing agent

PADSAC3 1x mung bean nuclease 420 wd 42 mi solution me | | ee]

[0067] Table 4 Listing of materials

[0068]

No. Name Specification Property Quantity | Dilution Remarks factor

PADSALOMOO1 | Reagent cryovial 2 ml Raw material, 300 sterile

PADSALOMO02 | Reagent cryovial I ml Raw material, 200 sterile

PADSALOMO03 Tag 30 mm>20 Raw material, 500 mm printing

PADSALOMO0O4 Zinc chloride 600 mM Intermediate, 12 ml 10x PADSACI mother liquor sterilized 700 ud 600x PADSAC3

PADSALOMO05S Sodium acetate 600 mM Intermediate, 29 mi 10x mother liquor sterilized

PADSALOMO06 | Cysteine mother 600 mM Intermediate. 12 ml 10x PADSACI liquor sterilized 2.8 ml 60= PADSAC2

PADSALOM007 MOPS mother IM Intermediate. 1.7 mi 100x liquor sterilized

PADSALOMO0S Sodium acetate 2M Intermediate, I ml 40% {pH=5.0) stenlized

PADSALOMO09 Sodium chloride iM Intermediate, 420 ud | 106% mother liquor sterilized

PADSALOMO10 | Tris-HCl (pH=8.8) | IM Intermediate, 1.3 mi 20x sterilized

PADSALOMOII Potassium 2M Intermediate, 630 ud 40x chloride mother sterilized liquor

PADSALOMO12 | Glycerol mother 80% Intermediate, 1.6 ml 16x liquor sterilized

PADSALOMO13 Magnesium 1M Intermediate, 630 ul 400% chloride mother sterilized liquor

PADSALOMO14 Tween 20 10% Intermediate, 1.7 mi 106% sterilized

PADSALOMO13 Universal 12 uM Intermediate, 1.2 mi 100x auxiliary probe cryopreserved

GPCO1

PADSALOMO16 Universal biotin 12 uM Intermediate, 1.2 mi | 100x probe GABO1 crvopreserved

PADSALOMOL7 Universal 20 uM Intermediate, 250 ul 100x fluorescent probe cryopreserved

GPPO1

PADSALOMOI8 | Universal primer 1 20 pM Intermediate, 250 ul 100% cryopreserved

PADSALOMO19 | Universal primer 2 20 uM Intermediate, 250 ul 100x cryopreserved

PADSALOMO20 DTT mother IM Intermediate, 25 ud 1000x liquor cryopreserved

PADSALOMO21 Formamide Analytically Raw material, 87 mi 33x pure aliquoted

PADSALOMO22 Streptavidin 10 mg/ml Raw material, 1.2 mi 100x magnetic bead aliquoted

PADSALOMO23 dNTP Mix 10 mM Raw material, 5m 50x aliquoted

PADSALOMO24 Mung bean 10 U/ml Raw material, 840 ul 50x nuclease aliquoted

PADSALOMO25 Hot-start DNA SUM Raw material, 2.5 mi 100% polymerase aliquoted

PADSALOMO26 SDS Analytically Raw material, 728 pure aliquoted

PADSALOMO27 Ethylene Analytically Raw material, 216g carbonate pure aliquoted

PADSALOMO0O28 1x positive 2 uM Intermediate. 100 ud 1000= control probe cryopreserved

TS0016

PADSALOMO29 A549 medium 5 mg/ml Raw material, 100 mi supernatant aliquoted

[0069] Example 5

[0070] Anti-interference experiment

[0071] In the present disclosure, main probes for four human miRNAs were designed and synthesized, including miR-93, miR-191, miR-3662, and miR-16, which were marked as: TS0093, TS0191, TS3662, and TS0016, respectively. Meanwhile, oligonucleotides whose sequences were homologous to these four miRNAs were artificially synthesized as: OLI0093, OLIO191, OLI3662, OLI0O016, respectively. Table 1 showed specific sequence information.

[0072] With DHS5a E. coli medium and homologous oligonucleotides, an anti-interference comparison experiment was conducted between the method of the present disclosure and the method of patent 202011041856.5. Negative samples NLO1 and NHO1 were 20 pl and 200 pl of 10%/pl E. coli medium, respectively, and positive samples PLOT and PHOI were the 20 pl and 200 pl of 10%/ul E. coli medium added with 10° copies of the above four homologous oligonucleotides, each group was conducted in triplicate. When the method of the present disclosure was used for experiments, the sample was supplemented with a PBS, such that a sample volume reached 100 pl, and then the detection was conducted according to the above-mentioned steps. When the method of patent 202011041856.5 was used for experiments, 10 times the volume of a probe-containing treatment solution 1 was directly added to the sample according to this method, followed by conducting detection according to the steps of this method.

[0073] The results showed that when the E. coli content was 20x 105, the two methods could both obtain a negative result of "No Ct" without background amplification, and also gave positive results of desirable amplification with average Ct values (Cta) of 27.67 and 28.52, respectively. When the €. coli content was increased by 10 times to reach 200%10°, the method of the present disclosure could still obtain the negative result of "No Ct" without background amplification and the positive result of desirable amplification of 27.66. However, the method of patent 202011041856.5 could no longer obtain the negative result without background amplification; meanwhile, fluctuation of the amplification results increased, indicating that it was severely disturbed. The specific data were shown in Table 5:

[0074] Table 5 Experimental comparison between the method of the present disclosure (PADSA) and the method of patent 202011041856.5 (MCAP)

[0075]

Ctl C2 C3 Cta Ctl C12 Ci Cta oe Jo Jo Jo Joe ee

Jon fo Joo foe of oe

[0076] Note: Ctl to Ct3 were the results of three parallel experiments, and Cta was average.

[0077] The method of the present disclosure and the method of patent 202011041856.5 were further used for anti-interference comparison experiments with total RNA of £. coli and homologous oligonucleotides. The total RNA of £. coli was extracted and purified from DH5a £. coli by alkaline lysis, and adjusted to a required concentration with ultrapure water. Negative samples NL02 and NHO2 were 20 ul of 0.1 pg/ul and 1.0 ug/ul £. coli total RNAs, and positive samples PL02 and PHO2 were the 20 ul of 0.1 pg/ul and 1.0 ug/ul E. coli total RNAs added with 10° copies of the above four homologous oligonucleotides, each group was conducted in triplicate.

When the method of the present disclosure was used for experiments, the sample was supplemented with a PBS, such that a sample volume reached 100 ul, and then the detection was conducted according to the above-mentioned steps. When the method of patent 202011041856.5 was used for experiments, 10 times the volume of a probe-containing treatment solution 1 was directly added to the sample according to this method, followed by conducting detection according to the steps of this method.

The results showed that when the total RNA concentration of £. coli was 0.1 pg/pl, the two methods could both obtain a negative result of "No Ct" without background amplification, and also gave positive results of desirable amplification with average Ct values (Cta) of 27.61 and 28.20, respectively. When the total RNA concentration of F. coli was increased by 10 times to reach 1.0 ug/l, the method of the present disclosure could still obtain the negative result of "No Ct" without background amplification and the positive result of desirable amplification of 27.69. However, the method of patent 202011041856.5 could no longer obtain the negative result without background amplification; meanwhile, fluctuation of the amplification results increased, indicating that it was severely disturbed. The specific data were shown in Table 6:

[0078] Table 6 Experimental comparison between the method of the present disclosure (PADSA) and the method of patent 202011041856.5 (MCAP)

[0079]

Ctl Cl Ct3 Cta Ctl C12 Ci Cia

Me) waa wa wa ves ese

[0080] Note: Ctl to Ct3 were the results of three parallel experiments, and Cta was average.

[0081] Example 6

[0082] With lung cancer A549 cell comparison, the method of the present disclosure and the method of patent 202011041856.5 were detected to compare the sensitivity of cell detection.

[0083] Freshly harvested A549 cells were adjusted to a cell concentration of 100 cells/ul with PBS; 20 pl of a cell suspension was serially diluted by 10 times with PBS to obtain 100 ul for each of cell suspension samples at 10 cells/ul, 1 cell/ul, and 0.1 cells/ul, recorded as PAO10, PAOOL, and PAO. 1, respectively. 10 ul of each sample was collected, and each sample was replicated three times. The method of the present disclosure was used for combined detection of the four target miRNAs of miR-191, miR-93, miR-16, and miR-3662, and the method of patent 202011041856.5 was compared at the same time. When the method of patent 202011041856.5 was used for experiments, 10 times the volume of a probe-containing treatment solution 1 was directly added to the sample according to this method, followed by conducting detection according to the steps of this method. The results showed that when the sample cell concentrations were 10 cells/pl and 1 cell/ul, both methods could obtain positive results with amplification, and the method of the present disclosure obtained a smaller Ct value, indicating a better amplification effect. When the sample cell concentration was 0.1 cells/ul, the method of the present disclosure could still obtain a positive result of desirable amplification, but the comparison method could no longer obtain amplification. Therefore, the method of the present disclosure could achieve better cell detection sensitivity (100 cells/ml) than the method of patent 202011041856.5. The specific data were shown in Table 7:

[0084] Table 7 Experimental comparison between the method of the present disclosure (PADSA) and the method of patent 202011041856.5 (MCAP)

[0085]

Ctl C2 C3 Cta Ctl ct2 Ct3 Cla

PAO.1 0.1 28.35 28.36 29.18 28.63 NoCt | NoCt | NoCt | NoCt celis/ul

[0086] Note: NC was the negative control of purified water. Note: Ctl to Ct3 were the results of three parallel experiments, and Cta was average.

[0087] Example 7

[0088] The A549 cells were continuously cultured for 3 d under a growth density of close to 100%, and then a medium supernatant was collected and aliquoted at 10 pl/part to obtain a sample of the A549 medium supernatant, recorded as ECO10. By the method of the present disclosure and the method of patent 202011041856.5, the combined detection of four target miRNAs, miR-191, miR-93, miR-16, and miR-3662, was conducted in three replicates. When the method of the present disclosure was used for experiments, the sample was supplemented with a PBS, such that a sample volume reached 100 pl, and then the detection was conducted according to the above-mentioned steps. When the method of patent 202011041856.5 was used for experiments, 10 times the volume of a probe-containing treatment solution 1 was directly added to the sample according to this method, followed by conducting detection according to the steps of this method. The results showed that both methods could obtain positive results with amplification, and the method of the present disclosure obtained a smaller Ct value, indicating that better amplification was achieved, that is, a better detection sensitivity of cfmiRNA was achieved. The specific data were shown in Table 8:

[0089] Table 8 Experimental comparison between the method of the present disclosure (PADSA) and the method of patent 202011041856.5 (MCAP)

[0090]

Ctl C2 Ci Cta Ctl CQ Ct3 Cta eee foe foe foe eo

[0091] Note: NC was a negative control, and PAO10 was a positive control of A549 cells at 10 cells/ul. Note: Ctl to Ct3 were the results of three parallel experiments, and

Cta was average.

[0092] Example 8

[0093] The method of the present disclosure was tested for sensitivity using a homologous oligonucleotide OLI0016 of miR-16.

[0094] The 10° copies/ml OLIO016 sample was serially diluted by 10 times with a 0.1 ug/ul £. coli total RNA solution to obtain 1 ml for each of 100 copies/ml (LC0016100), 10 copies/ml (LCO016010), and 1 copy/ml (LC0016001) of the positive dilution samples; the detection was conducted according to the steps described above, and then repeated 3 times. The results showed that the positive dilution samples LCO016100 and

LC0016010 were significantly amplified, and LCO016001 might be amplified, but had large fluctuation that was close to a sensitivity limit. Accordingly, it was inferred that the sensitivity of the method of the present disclosure could reach 10 copies/ml stably.

The specific data were shown in Table 9:

[0095] Table 9 Sensitivity test of the method of the present disclosure (PADSA)

[0096]

Sample Property PADSA PADSA PADSA PADSA

Ctl cu CH Cta tomer teen | | ws | wa

NCRO1 Negative No Ct No Ct No Ct 27.69 control

[0097] Note: NCRO1 was a negative control for E. coli. Ctl to Ct3 were the results of 3 parallel experiments, and Cta was average; if there were both Ct values and No Ct values in parallel experiments, the average was not calculated.

[0098] Example 9

[0099] Further, the specificity of the method of the present disclosure was investigated by detecting homologous oligonucleotides of a human miRNA family miR-181.

[0100] The human miR-181 family has mainly four different 5p mature forms: miR-181a-5p, miR-181b-5p, miR-181c-5p, and miR-181d-5p. These forms have very similar sequences, with only 1 to 5 nt differences (FIG. 2), but have different expression and regulatory functions in different disease pathologies. For example: more and more evidence shows that miR-181a and miR-181b are up-regulated in gastric cancer, which may promote the development, invasion, and metastasis of gastric cancer; and a low expression level of miR-181c in gastric cancer tissue may have the effect of inhibiting gastric cancer. The expression and role of miR-181d in gastric cancer are still unclear. Therefore, the amplification detection method of miRNA should not only achieve high sensitivity, but also high specificity capable of distinguishing different miRNAs in the same family. 4 kinds of oligonucleotides completely homologous to 4 kinds of miR-181, marked as: OLI181a, OLI181b,

OLI181c, and OLI181d, respectively, as well as corresponding main probes, marked as:

TS181a, TS181b, TS181c, and TS181d, respectively, were artificially synthesized (specific sequence information was shown in the sequence listing); an oligonucleotide sample concentration was adjusted to 10° copies/ml. The method operation of the present disclosure was as described above. The results showed that the combination of the oligonucleotide sample and the corresponding main probe could be significantly amplified, while the cross combination had no amplification, showing that the method of the present disclosure could achieve the high specificity for distinguishing different miRNAs in the same family. The specific data were shown in Table 10:

[0101] Table 10 Detection of homologous oligonucleotides of human miRNA family miR-181 by the method of the present disclosure (PADSA)

[0102]

Samp | TSIS | TSI8 | TSI8 | TS18 | TSI8 | TSI8 | TSI8 | TSI8 | TSI8 | TSI8 | TSI8 | TSIS le la la la Ib 1b 1b Ic lc lc 1d Id 1d

Ctl Cn Ct3 Ctl C2 CG Ctl Ct2 C3 Ctl C2 Ct3

[0103] Note: Ctl to Ct3 were the results of three parallel experiments.

[0104] Example 10

[0105] Combined detection of miRNAs, that is, simultaneous detection of the expression of one or more of multiple target miRNAs in a sample, is the key to achieving high performance in the detection of diseases with high genetic heterogeneity such as tumors. The 8 oligonucleotides (OLI0093, OLI0191, OLI3662,

OLI0016, OLII81a, OLII81b, OLII8lc, and OLI181d) were further used to investigate the combined performance of the method of the present disclosure.

[0106] 8 main probes (TS0093, TS0191, TS3662, TS0016, TS181a, TS181b, TS181c, and TSI181d) were added to the magnetic bead sample releasing agent at a concentration of 15 nM. The composition of the remaining reagents was the same as before, and the detection operation was the same as before. The concentration of each oligonucleotide sample was adjusted to 10* copies/ml, and the concentration of the 8-oligonucleotide mixed sample (OL1TO8) was 8x10* copies/ml. The results showed that each oligonucleotide sample and oligonucleotide mixed samples could be significantly amplified, showing that the method of the present disclosure could achieve the high specificity for distinguishing different miRNAs in the same family.

The specific data were shown in Table 11:

[0107] Table 11 Combined detection of 8 kinds of oligonucleotides by the method of the present disclosure

[0108]

Ctl 2589 | 2573 | 2592 | 2552 | 2584 | 2540 | 2579 | 2604 | 2209 | No

Ct

CO | 2538 | 2551 | 2526 | 2554 | 2531 | 2511 | 2591 | 25.53 | 2268 | No

Ct ci 2565 | 2530 | 2558 | 2577 | 2601 | 2575 | 2543 | 2546 | 2284 | No

Ct

LLL LL Le

[0109] Note: NC was a negative control, Ctl to Ct3 were the results of three parallel experiments, and Cta was average.

[0110] Example 11

[0111] The miR-885-5p, miR-34b, and miR-3662 are three miRNAs that are highly expressed in lung cancer tissues but not in paracancerous tissues, as verified by multiple literature reports. The main probes of these three target miRNAs were synthesized (referring to sequence listing for specific information on the sequence), which were denoted as: TS0885, TS034b, and TS3662, respectively.

[0112] 2 ml of heparin sodium anticoagulated whole blood samples were separately collected from 10 confirmed lung cancer patients and 10 healthy volunteers, while purified water negative control and A549 cell positive control were set up; after centrifugation at 5,000 rpm for 3 min, about 95 ul of a supernatant was transferred to a tube containing 40 pl of the magnetic bead sample releasing agent, mixed well, and incubate at 30°C for 10 min; after being adsorbed by a magnet to remove a supernatant, 950 ul of a washing solution was added to mix well, then absorbed with a magnet to remove a supernatant, 50 u of a mung bean nuclease solution was added, and incubated at 30°C for 1 min; a product was adsorbed with a magnet to remove a supernatant, 950 pl of the washing solution was added to mix well, then absorbed with a magnet to remove a supernatant, 30 ul of a PCR solution was added, incubated at 95°C for 10 min, centrifuged briefly at 12,000 rpm, and 30 u of a supernatant was transferred to a PCR tube, and the fluorescence quantitative PCR was conducted with the aforementioned PCR procedure. Test results were: the negative control had no amplification, and the positive control had a Ct value of 22.21, indicating that the quality control of the experiment was qualified; the Ct values of blood samples from 10 patients with lung cancer each were 24.29 to 30.43, all of which were positive, showing that the combined detection of lung cancer blood positive rate of these 3 target miRNAs by the method of the present disclosure 1s 100%; meanwhile, none of the blood samples of the 10 healthy volunteers was amplified, showing that the combined detection of the three target miRNAs by the method of the present disclosure had a blood-negative coincidence rate of 100%.

[0113] Example 12

[0114] Detection of 3 lung cancer-specific miRNAs in human blood by using the kit of the present disclosure

[0115] 3 lung cancer-specific and highly-expressed miRNAs, including miR-885-5p, miR-34b, and miR-3662, were amplified and detected using the kit produced. The main probes of the 3 target miRNAs were synthesized (referring to sequence listing for specific sequence information): TS0885, TS034b, and TS3662, mixed in equal proportions such that a total concentration was 1.2 uM (each 0.4 uM); 40 pl of the main probe was pipetted to 300 ul of a 1.1x magnetic bead sample releasing agent provided in the kit. After mixing, a mixture was divided into 2 ml centrifuge tubes at 40 pl/tube, and 15.3 ml of purified water was added to a 10x washing solution; specific composition and detection operation of the remaining components were the same as above. 2 ml of heparin sodium anticoagulated whole blood samples were separately collected from 10 confirmed lung cancer patients and 10 healthy volunteers, and purified water negative control and A549 cell positive control were set up; after centrifugation at 5,000 rpm for 3 min, about 100 pl of a supernatant was transferred to a tube containing 40 ul of the magnetic bead sample releasing agent, mixed well, and incubated at 30°C for 10 min; after being adsorbed by a magnet to remove a supernatant, 950 pl of a washing solution was added to mix well, then absorbed with a magnet to remove a supernatant, 50 ul of a mung bean nuclease solution was added, and incubated at 30°C for | min; a product was adsorbed with a magnet to remove a supernatant, 950 pl of the washing solution was added to mix evenly, then absorbed with a magnet to remove a supernatant, 30 ul of a PCR solution was added, pipetted to mix well and transferred to a PCR tube; on an ABI7500 PCR instrument, fluorescence quantitative PCR was conducted by: 95°C for 10 min, followed by 40 cycles of 93°C for 10 sec and 65°C for 40 sec, and a fluorescence signal was collected at 65°C (FAM channel). The results were analyzed and determined by an automatic threshold value of the computer: if the sample had a Ct value, it was determined to have amplification; if the Ct value was less than 35, it was determined to have significant amplification, showing positive; if there was no Ct value, it was determined as no amplification, showing negative; if the Ct value was 35 to 40, which was a gray area, it was determined as weakly positive when it could be repeated. The test results were: the negative control had no amplification, and the positive control had a Ct value of 23.03, indicating that the quality control of the experiment was qualified; the Ct values of blood samples from 10 patients with lung cancer were 25.13 to 33.19, all of which were positive, showing that the combined detection of these three target miRNAs by the kit of the present disclosure had a blood positive rate of 100% for lung cancer; meanwhile, none of the blood samples of the 10 healthy volunteers was amplified, showing that the combined detection of the three target miRNAs with the kit of the present disclosure had a blood-negative coincidence rate of 100%.

[0116] The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.

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OR CINEDQUalifiler name>mol type“ /INSDgualifier named

LES <“INSDOQualifier value>other DNA</INSDOuallfier value> ied </INSDOQualijiier>

AE <INSDOualifier ld=swgign> iá8 <INSDQualifiler namednote</IN3DCualifier name» ag <INSDQualifler valuevGPFOl</TINSDOuallfier value» </INSDQuali fier!»

LL <INSDguelifier id="gli">

LE v“INSDQualifier namerorganism</IN3DQualifier name>

Lis <INSDQualifier value>synthetic construct </INSDQualifier value» 174d </INSDQualifier> ii </INSDFeature cuals> ij </INSDFealurex> 1H </INSDSey feahure-table>

Lis <INSDSeq sequemcergaggcacagcaggtgcagg/INSDSeq seguenced

EG </INSUE aa»

Lai </EegquencebData>

LSL “SequenceData seguencellsumben=N"js>

RE <INSDSeq> ie: <INSDSegq lengtb>b22</INSDSeq length>

Ld <INSDSeq molbype>DNA</INSDSeqg moltype> ian <INEDZeq division>PAT</INSDEsqg division»

LEE <INSDSeq festure-table>

LEY <INSD¥eature» law <INSDFeature key>source</INSDFeature key»

LED <INSDFeature location»l1..22/INSDFeature location» 134 <INSDFesture duels»

REEN CINSDQuUalifiso> 182 <INSDQualifier namedmol type</INSDOualifisr name> an <INSDQualifier valuerother DNA /INSDgualifier valuer

ERE </INSDOualifier:>

Lan CINSUQualifler in=NgVTn>

LE <INSDOualijier namernote</INSDQualifisr name> 107 CINSDQualifier valued>GPPOl</INangualifier value» 138 </INSDQualifier> u ins “INSDoualifier id=sgidnx 200 <INSDGualifier namerorganism</INSDQualifisr name> 200 <INSDQualifier valuersynthetic construct </INSDQuali fier values

EDE <SINSDOualifier>

LS </INSDFeature duels» did </INSDFeature> 205 </INSDSeg feature-tabler 23% <INSDSeq zequence>tececggatgetgcagtgatggea</IN3DE=ag sequenced 203 “/INSDSeoa> 208 </Segquaencebata>

EASE <Segquencebalta seguantailiicghar=ngns

ZL <INSDSeq>

LLL “INSDSeq length>23</INSkSeq Length> did <INSDSeq moltype>RNA</INSDSeq moliype> zi <INSDSeq division>PAT</INSDSeg division: zld <INSLSeq Ieature-labier

Zil <INSDFeabure> zieë <INSDFsature key>source“/INSDFeature key> a <INSDFearure location>l..23</INSDFeature location ale “IiNSDFeanure gqualsh

Ein <INSDQOualifier»

ZED <INSDQualifier nams>mol type“ /INSDOualifier name

ZEE <INSDQualifiler wvaluerother RNA</INSDO0uelifier value 222 <ATNaSDQuali fis»

Da <INSDQualifier 1d=V"glosvs 224 <INSDQualifier namernote</INZDOualifier name>

Zal <INSDOualifier valuermiR-191</INSDQualifier value»

ZEE <SINSDOualifier> ra <INSDQualifier aa=ngligy>

ZEE <INSDQualifier name>organism</INSDQualifier names

ZEB <INSDQualifier value>synthetic construct </INSDQualifier value»

D220 </INSDQualifisan» 23% </INSDFeature quals>

Zan </INSDFeature>

SRE </INSDSeg faature-table> aE “INSDSeq sequencecaacggaatcccaaaagcagetg</INSDSeq sequence» 255 << INIDSeg>

E38 </SeguenceDatar

Zj <SequenceData samencelósuben=NSN>

ZI <INSDSeq> 239 <INSDSeq lengih>23</INSDSeg length»

ZA <INSDSeq moliype>RNA</INSDSeq moltype>

AAL “INSDSeq division»PAT</ INDE division

LE <INSDSeq feature-itable> di <INSDFeature»> zäë <INSDFesture keyrsource“/INSDFealure key» 245 <INSDFeature location»l..23</INSDFeaturs location 246 <INSDFeature guals> 247 <INSDQualifier>

SAS CINEIDOualifier namermol type“ /INSDUualifier name> dès JINSDOQualifier valuerother RNA INIDOualifier value»

ZE </INSDOualifier> u gl <INSDQualifier id="glijnn 257 <INSDQualifier name>note</INSDQualifier name> 253 <INSDQualifler valuermiR-93</INaDOualiflier value» 254 </INSDQvalifier: 255 <INSDQuaiifler id="glijr2> and CINMEDOualifier namerorganism</INSDQualifier name on <INZDQualiflsr value>synthetie construct </INSDQualifier value> 258 </INSDOuaiifiers 253 </INSDFeature guals> 250 x“/INSDFeacturex Ĳ

Zo </INSDSeg feature-tabled> 282 <INSDSeq sequence>gatggacgtgcttgtegtgaaac</INSDSey sequence’

Zos </INSDSeg>

Sh <j Gaemquencebata>

PEG <Gequencelala seguantellManbhao=2187>

LEE <IiNSsDseqd> 257 <INSDSeq iength>22</INSDSeq length> 268 <IN3DSeq moliype>RNA</INSDSeq moltyper 268 <INSDSeq division>PAT</INSDSeg division» 2730 <INSDieq festure-table>

ZL <INSDFeature> aE “INSDFearture keyrsourced/INIDFeaturs Key»

Zi <“INSDFeature locabtion>l..22</IN3DFeature location» zijd <INSDFeature gualis>

ZD SINSDOualifier> 26 <INSDQualifler name>mol type“ /INSDgvalifier name>

DE <INSDOualifier valuerother RNA</INSHGualifier value» 28 </INSDQuali fier!» ain CINSDQualifiler id="glijv>

SED <INZDOualifisr namernote“/INSDQualifier name> aa CINSDQualifiler value >miR-16</INsDgualifier value»

LEE </INSDOualiiier>

FER CINSDOualifier id="gil2r> ahd <INSDQualifier name>organism</INSDQualifisr name>

DEH <INSDQualifier valuebsynthetic construct </INSDOualijier value»

RE </INSDOualifier:> an “{INSDFeature quals>

ZEE </INSDFealure»>

EEA </INSDSeg feature-tabler za <INSDSeq sequence>tagcagcacgtaaatattggeg“/INSDSeq zeguence> 23: </INSDSegs 282 </SeguenceDatas 283 <Sequencebata zaogquencaildiunbhay=viivs pa <iNSDSeg>

PACES “INSDSeq lengih>24</INSDSeg lengths

Lel <INSDSeq moltype>RNA</INSDSeg moltype> at <INSDSeq divislon»PAT</INIDSeg division

ZR <INSDSeq Ieatureriabier 233 “INSDFeaturex

Zij <INSDFeature key>source“/INSDFealure key>

BL <INSDFearure location>l..24</INSDFeature Lccation»

SOA <INSDFearure guals:»

Ho <CINSDQualifiers

Aird <SINSDQuUalifier name>mol type /INSDQualifier name»

HOR <INSDQualifier value>other RNAC/INSDOualifier value»

SE </INSDQualifier> -

Bee] JINSDoualifier id="gil5ns 338 CINADGualifler name>note</IN3Doualifier name>»

TOO <INSDQualifier valuermiR-3662</INSD@ualifier value»

ILO </INSDOualifier:>

LL CINSDQualifler ao=vglidys

SLE <INSDOualijilen namerorganism“/INSDQualifier name»

ERC <INSDQuaiifler value>synthetic construct </INSDQualifier valued 214 </INSDQuali jier

TG </INSDFaature cualss»

ILE </INSDPeature>

SLT </INSDSeg feature-teblex> sle “INSDSed sequsncergaaaatgatgagtagtgactgatg/INSDSeg sequence 31% <SINBDE eg

SED </SeguenceData>

JE <Sequencalata sopeancalliumber="313%> 282 CINEDSeqr za <INSDSeq Length>84</INSDSeq length»

FA <IMEDZeq moliype>DNA</INSDSeqg moltyper

IEE <INSDSeq division»PAT</INEDIag division>

IEG CINEDSeq feature-table>

Sd <INSDFeature»

SEE <INSDFeature key>source</INSDFeature key»

IED <INSDFesture locationrl..84</INSDFeature Location»

S28 CINSDFeature quals>

IEA <INSDQualifier> 332 <INSDQualifier name>mol type</INSDQualifier name>

IIB <INSDOualifier valuerother DNA4/INSDQualilfier value» 334 </INSUQualifiers>

S&D <INSDQualifler i0=NgS81%> dae <INSDQualifier name>note</INSDQualifier name>

Ra <INSDQualifier valuerTs019L</INSDQualifier value» 228 </INSDQuali jier

TE <INSDQualifier id="g28Nx>

Zan <INSDQualifier namevorganism“/INSDguali fier name>

RE <INSDOualifier valuersynthetic construct

</INSDQuali jier valus>

Sad </INSDOualilfier>

REIN </INSDFreaturs guals> 44 </INSDFeaturer u </INSDEeg Zearture-tabier

Tie <INSISeq sequence> gaggcacagcaggtgcaggtcecggatgctgcagtgatggcacagetgettttgggattegttgcctccacgtga ccctgacgta“/INSDSeq sequence»

GET </INSUE aa»

Sd </EegquencebData>

SAD “SequenceData seguencellsumben="i3®>

S50 <INSDSeq> 251 <INSDSeg length»85</INIDIeqg length»

IH <INSDSey moliype>DNA</INSDSeg moltvper 353 <INSDSeqg divisiom>PAT:/INSDSeg divisions

Ig <INSDSeq festure-table>

ILL <INSDFeature>

SLE <INSDFeature key>source</INSDFeature key»

Sn <INSDFeature Llocation>l..85/INSDFeature location» 358 <INSDFealure guals> 258 SINEZDOualifiso> za <INSDgualifier namexmol type“/INSDgualifier name>

IAL <INSDQualifier valuerother DNA</INSDQualifier value»

En </INSDOualifier:>

HES CINSUQualifler ia=vqRIY> sed <INSDOualijier namernote</INSDQualifisr name>

SES CINSDQualifier value>»Ts0093-/INSDOQualifier value»

RARE </INSDQualifier> - -

SE CINZDQuallifier 1d=9gRen»

Tan <INSDGualifier namerorganism</INSDQualifisr name> zee <INSDQualifier value»>synthetic construct </INSDQualifier valuer

SID </INSLQualifiers

STL </INSDFeature duels»

STE </INSDFeature>

Si </INSD8eq featura-tabler nig <INSDSeq zeguence> gaggcacagcaggtgcaggtcecggatgctgcagtgatggcagtttcacgacaagcacgtccatccctccacgtg accctgacgta</INSDSeq sequence»

GEE </INSDSeg> u

STE <j Gaemquencebata>

Ra <Gequencelala segquenceliNuubec=Nidr>

STE <IiNSsDseqd> 373 <INSDSeq iength>84</INSDSeq length> 280 <INSDSeq moliype>DNAC/ INSDSeq moliyper

TE <INSDSeg division>PAT</INSDSeq division»

BEL <INSDSeq feature-table>

BES <INSDFeature>

SEG CINEDFeature keyvsource</INSDFeature key» san “INSDFeabture locabtion>l..B84</INSDFeature location»

SES <INSDIesture guals>

SEF SINSDOualifier> aus <INSDQualifler named>mol type</INSDRualifizp names

IN <INSDQualifier valuerother DNA //INSDGualifler value»

Zan </INSDQuali fier!»

ES <INSDguelifier id="g8S3"> wan <INSDOualifier namernotes/INSDQuali fier name>

Ss <INSDQualifier value>»TS0016/INSDQualijier valued

Ld </INSDOQualijier> 335 <INSDOualifier id="qg390>

RACES <INSDQualifler namedorganism</INSDQualifisr name> 8 <INSDQualifier valuersynthetic construct </INSDOualifier value» zes <fINSDUualifler»>

Si x“/INSDFeature gquals> ie </INSDFeature»

B «/INSDSeg featurse-tabier> 302 <INSDSeq sequence> gaggcacagcaggtgcaggtcecggatgctgcagtgatggcacgccaatatttacgtgetgectacctccacgtga ccctgacgta</INSDSeq sequence» 403 </INSDSeg> u 404 <SBequencelata>

4050 <HGegquencelbata seguanselilMonhar=niSY > 405 LINSDSeq> dT <INSDSeq length>86</INSDSeg length> 4038 <INSDSeq molityperDNAC/TNSDSeg moliype> 4098 <INSDSeg division>PAT</INSDSeq division»

SLD <INSDSeg feabture-tablel

ALL <INSDFesture>

AL <INSDFearure key>sources/INSDFeature key> 413 “INSDFearure location>l..86</INSD¥Featiure location 414 <“INSDPFeacure gqualis> 415 <INSDOQualifier> 41 <INSDQualifier name>mol type</INsSDQualifier nama» 317 CINBDQualifiler valuerother DNA</INSD{ualijier value» dis </INSDQualifisan» ale <INSDQuelifier id='gS84ns

A450 <INSDOualifier namernote</INSDOualifisr name>

AEL <INSDOualifier valuerTs3662/INSDGualifler value» dd </INSDOualilfier> u - tE <INSDQualifier id="qg33V> jie <INSDgualifier namedorganism</INSDQualifier name> 495 <INBDQualifier valuersynthetic construct </INSDOualifier value» 426 </INSDQualifiern> u

ZT </INSDFearture qualss </INSLFeature» ds </INSDSeg feature-itablex 450 <INSDSeq sequence» gaggcacagcaggtgcaggtcecggatgctgcagtgatggcacatcagtcactactcatcattttccctccacgt gaccctgacgta/INSDSeqg sequencer

EE </INSDSeg> Ĳ

A32 </SegusncsDara>

A433 LSequencebalta seqguancailiioghar=i8% > ds <INSDSeq> 4505 <INSDSeq length>»85</INSDSeg length»

HEE <INSDSeq moltype>DNA/INSDSeg moliype> 437 <INSDSeq divislon»>PATC/INSDSeg divisions 428 <INSDSeq feature-takbled 48 <INSDFealure> 440 <INSDFsature key>source</INZDFsature key>

AA <INSDFearure location>»1..85</INSDFeerure location 44 CINEDFeature gualse ds <INSDQOualifier» dd <INSDQuelifier name>mol type</INIDQuaiifier name) 44h <INSDQualifiler wvaluerother DNA<C/INSDJualifier value 448 <ATNaSDQuali fis» 447% <INSDQualifier id="g985n»> 448 <INSDQualifier namernote</INZDOualifier name>

A440 <INSDOualifier valuerTs18la</!NSDovelifler value» 450 “/INSDOualifier> Ĳ 45% LINSDGualiifler ia=wgl3dy> ane <INSDgualifier nemssorganism“/TINSDOQualifier name 352 <INSDguelifier valievsynthetic construct “/ENSDOualifier valuer 454 </INSDQualifisan» - 455 </INSDFeature quals>

ALE </INSDFeature> 407 </INSDSeg faature-table> ds <INSDSeq sequencer gaggcacagcaggtgcaggtcecggatgctgcagtgatggcaactcaccgacagcgttgaatgttcctccacgtg accctgacgta/INSDSeq sequence 453 </INSDSea: jon </SecuenceDala»> dn <Sequencebata zegvencaiDNumber=nNijsD

AGZ <INSDSeq> 403 CINEDSeq lengthi»85</INsSDSeqg length» did <INSDSeq moltype>DNA</INSDSeg moliype> deb <INSDSeq division»>PAT</INSDSeg divisions 455 <INSDSeq Ieatureriabier da CINEDFeaturae» 458 <IN3LFeature key>source“/INSDFealure key» 450 <INSDFeature iocation»>l..85</INSDPeature location>

ATG <INSDFeature quals> u

AFL <CINSDQualifiers dE <INSDOualijilen name>mol type“/INSDQualifier ame» dE <INSDQualifier value>other DNA</INSDO0uelifier value» 474 </INSDQualifier> - - 475 CINZDQuallifier id="gn8nx> 478 CINADGualifler name>note</IN3Doualifier name>» 477% <INSDQualifier value>TS181b</INSDoualifier value 475 <fINSDUualifler»> 4 CINSUDQualifler in="g3ën> dg <INSDQUalifiler naemerorganism/INSDOualifier name dal <INSDQualifier value>synthetic construct </INSDQualifier valued 482 </INSDQuali jier 383 </INSDFeature gualss» 484 </INSDFeature>

An </INSDSeg feature-teblex>

ABE <INEDSeq sequencer gaggcacagcaggtgcaggtcecggatgctgcagtgatggcaacccaccgacagcaatgaatgttcctccacgtg accctgacgta“/INSDSeqd sequence 487 </INSDSegr 35E </SecuenceDatas 38 <SequenceData saguencaliunbaer="318"> 280 <iNSDSed>

AL <INSDSeq liengith>84</INSDSeg length

ESE CINEDSeqg moliyperDNA</INSDSeg moltypex 49% <INSDSeq division»PATL/INIDSeg division» ind <INSDSeq feature-itableX 435 <INSDFeaturer> 33e <INSDFeature keyssource“/INSDFealure hay» 357 <INSDFeature locationsl..84</INSDFeature Location»

A488 {INEDFeature gualis>

An <INSDQualifiers

S00 SINEDOualifiler name>mol type</IN3DQualifier name»

Li <“INSDOQualifier value>other DNA</INSDOuallfier value>

Sip </INSDOQualifier> u 52 <INSDOualifier id="qg87n> 5304 <INSDQualifiler namednote</IN3DCualifier name»

BH <INSDOualifier valuer»T8l8le</INSDOualifiser value»

SDE </INSDQuali fier!»

SOY <INSDguelifier id="g38">

DOG “INSDOualifier namevorganism /INSDQualifier named

Li <INSDQualifier value>synthetic construct </INSDQualifier value 510 </INSDOualifisr>

Sh <SINSDFaature guals>

B12 </INSDFeatune> -

SLS </INSDSeg Íesalure-teble>

Vid <INSDSeqg sequencer gaggcacagcaggtgcaggtcecggatgctgcagtgatggcaactcaccgacaggttgaatgttcctccacgtga ccctgacgta:/INSDSeq zeqvence> 515 </INSDSeq> u

Sid </SeguenceDatar

DLT <SequenceData samencelósubern=NLS8nx>

GLE <INSDSeq> sle <INSDSeq length>85</INSDSeg lengths

Lal <INSDSeq moltype>DNAC/ INSDEaeqg moltyper

LE CINEDSeq division»PAT</ INDE division

La <INSDSeq feature-itable>

L233 <INSDFearture:»

SEE <INSDFeature keyrsource</INSDFeature key> 325 <INSDFeature locations>l..85</INSDFeature Locations

SRG <INGDFeature quals> u 527 <INSDQualifier>

REE CIMNEDOuAalifieyr namermol type</iNsDoualifier name >

LE <INSDOualifier valuerother DNA</INIDOuzalifier value»

EE </INSDOualilfier> 3k <INSDQualifier id="qgS88">

S357 <INSDQualifier name>note</INSDQualifier name> 323 <INSDQualifler valuerTs181d</INSDoualifier value»

SDE </INSDQvalifier: - u

Gal <INSDQuelifier id='ga0ns

SRE CINMEDOualifier namerorganism</INSDQualifier name

LRT CINZDQualifiler value>synthetic construct </INSDQvalifier valus> 548 </INSDOualifiers 533 </INSDFeature guals> x“/INSDFeacturex Ĳ

Sak </INEDEeg feature-tabled>

SAL CINSDSeq sequence gaggcacagcaggtgcaggtcecggatgctgcagtgatggcaacccaccgacaacaatgaatgttcctccacgtg accctgacgta/INSDS3eqg sequence

LAG <SINEDE E> nad </SeguenceData> 545 <Sequencalata sopeancalliumber="208> 5d8 VINSDSeq»

Say <INSDSey lenghh>23</IN3DSeq length»

SAR <INEDSeq moelitype>RNAC/ INSDIeg moltype:r

Law <INSDSeq division>PAT4/INSDSeg division

DLO CINEDSeq feature-table>

SIG <INSDFeaturer

LIE <INSDFeature keyirsource</INSDFeature key: 352 <INSDFeature location»1..23</INSDFeature location» 554 CINSDFeature quals> u iii <INSDQualifier>

SRG <INSDQualifier name>mol type“ /INSDQualifier name>

SEE <INSDOualifier valuerother RNA4/INSDQualilfier value»

LG “/INSDOualifier> u

Die LINSDGualifiler in=Ngilj*>

LEG <INSDQualifier name>note</INSDQualifier name>

DE: <CINSDQualifier value>miR-181a-5p</1NSDQualifier valued

BAZ <ATNaSDQuali fis»

BAY <INSDQualifier ìd="glist2> vod <INSDQualifier namerorganism“/INSDQualifier name>

Ges <INSDOQualifier valuersynthetiec construct <{/INSDQuali jier valuex>

LEE SINEDQualifiers>

DET </INSDPearure guals>

Len </INSDFeaturer u

Da </INSDEeg Zearture-tabier

BO <INSDSey sequencs>aacattcaacgetgteggtgagt</INIDSeg sequence

B </INSDSeg> u

VIE </SemtenceDala>

LES <HGegquencelbata seguanselilMonhar=n2ivs 44 <INZDIeq>

Ss <INSDSeq length»>23</INSDSeg length>

Sid <INSDSeq moltype>RNA{/INSDSeg moliype>

DET <INSDSeq division»PAT</INSDSeg divisions

Sie <INSDSeg feabture-tablel

Sia <INSDFesture>

LEO <INSDFearure key>source</INIDFeature key»

LHL “INSDFearure location»1..23</INSDFeature location

Lo <“INSDPFeacure gqualis>

GEE <INSDQualifier»>

Sid “INSDgvelifier named>mol type</INSDQualifier name>

S385 CINBDQualifiler valuerother RNA</IN3DGualifler value» 386 </INSDQvalifier:

SR <INSDQuaiifler id="gliS"2>

EARS <INSDOualifier namernote</INSDOualifisr nama»

LER <INSDQualifier value >miR-181b-5p</INSDOuali fier valueX

Len </INSDOualifier> Ĳ

Gel <INSDQualifier id="glig> 5332 <INSDgualifier namedorganism</INSDQualifier name> 533 <INBDQualifier valuersynthetic construct </INSDoualifier valuer

Sd </INSDQuali fier!» u

DL </INSDFearture qualss

Lu </INSDFaaturel

Lu </INSDSeg feature-itablex

RCE <INSDSeg seguenceraacattcaacgetgteggtgggt</IN3DEsqg sequencer 539 </INSDSeq> u

ADD </SecuenceDatas

AOL <SequenceData ssqvueanoslDNumber=NgaN)»>

SOD <iNSDSed>

Ss <INSDSeq liengih>22</INSDSeg lengths

Lid CINZDSeq moltype»RNAC/INSDSag moltype> £05 <INSDSeq division»PATL/INIDSeg division» sO <INSDSeq feature-itableX

S07 CINEDEeatures

ADE <INSDFeeture keyrsource“/INSDFeature key>

Hi3G <INSDFeature locationsl1..22</INSDFeature Location»

S10 {INEDFeature gualis>

SE <INSDGualifier>

Sie “INSDQualifier name>mol type“ /INSDgualifier named

ELS <“INSDOQualifier valuesother RNA</INSDOuallfier values» gie </INSDOualiiier>

S15 CINSDOualifier 1d=svglais»

SLE <INSDQualifiler namednote</IN3DCualifier name» ai <INSDQualifier valuermiR-18le-5p</INa3Doualifier value»

S18 </INSDQuali fier!»

Sie CINSDQualifiler id="glgi0r>

RVR CINZDQuallfier namerorganism</IN3DQualifier name>

Sl <INSDQualifier value>synthetic construct </INSDQualifier value»

S37 </INSDQualifier>

Aa </INSDFeature cuals>

Ald </INSDFealurex> - 82% </INSDSey feahure-table>

SIT <INEDSeq sequenceraacattcaacctgteggtgagt</iNSDSey sequencer

GET </INSDE ag»

LG </EegquencebData>

LD “SequenceData seguencellNumben="g3®> £30 <INEDSedq>

Aal <INSDSegq lengih>23</INSDSeq length>

Ala <INSDSeq molbype>RNA</INSDSeqg moltype> 433 <INEDSeq divisiom>PAT</INSDSeg division»

G34 <INSDSeq festure-table>

SSL <INSD¥eature»

L348 <INSDFeature key>source</INSDFeature key» £57 <INSDFeature location>l..23</IN3DFeature location»

Gif <INSDFesture duels» 423 CINSDQuUalifiso>

SD <INSDQualifier name»mol type“ /INSDQuali fier name>

SAL <INSDQualifier valuerother RNA /INSDgualifier valuer

San </INSDOualifier:>

Sis CINSDQualifler io=vglalys gid <INSDOualijier namernote</INSDQualifisr name> 4h <INSDQualifier value>miR-181d-5p</INSDQualifier values

S48 </INSDQualifier> aa CINSZDOualifiler 1d="qRRave

Aj <INSDQualifier namerorganism“/INSDQuali fier name>

S48 <INSDQualifier valuersynthetic construct </INSDQuali fier values

GLO <SINSDOualifier>

SOL </INSDFeature duels»

En <JINSDFeature» £52 </INSD8eq featura-tabler aid <IN3D3eq zequencs>aacattecattgttgteggtgagt</INsUS=qg sequenced en </INSDSeg:> 858 </Segquaencebata> sij <SSequencebala segsenceinNvumbsc=n2an>

Ds <INSDSeq>

GLD “INSDSeq length>84</INSkSeq Length>

EE <INSDSeq moltype>DNA</INSDSeq moliype>

Gel <INSDSeq division>PAT/INSDSeg divisions

Aa: <INSDSeq fearvurertabier» and <INSDFeabture> 594 <INEDFeature key>source:/INSDFeature kKey>

SoL <INSDFeature Iscation>l..84</INSDFealure location [ES CINEDFeature gualse

SET CINBDOualifierns

LER <INSDQualifier name>mol type“ /INSDQualifier name» 553 <INSDQualifiler wvaluerother DNA</INSDO0uaelifier value

AEG <ATNaSDQuali fis»

SL <INSDQualifier Ld=UvgRann

Sid <INSDQualifier name>note</INSDQualijier name>

Sis <SINSDOualifier valuerTs041l1</!NSDovelifler value»

Ga <SINSDOualifier>

SUD <INSDQualifler in='gB97>

Ee <INSDgualifier nemssorganism“/TINSDOQualifier name

SU? <INSDQualifier value>synthetic construct <SINSDOualifier value» aia </INSDQualifisan» -

Sis </INSDFeature quals>

SSD </INSDEFeature> u

SRE </INSDSeg faature-table>

Lo <INSDSeq sequence gaggcacagcaggtgcaggtcecggatgctgcagtgatggcaggttagtggaccgtgttacatacctccacgtga ccctgacgta“/INSDSeq sequenced

A872 </TNSDS eq

Hid </SeguenceDatas

SEG <Sequencebata zaogquencaildiunbhay="28%:

SRE <INSDSeq>

Ly CINEDSeq length>»28</INSDSeqg length»

Len <INSDSeq moltype>DNA</INSDSeg moliype> (ee <INSDSeq diviglon»>PAT</INSDSeg divisions £34 <INSDSeq Ieatureriabier 43: “INSDFeaturex

ABE <INSDFeature key>source“/INSDFealure key>

S93 <INSDFearure location>l..28</INSDFeature Lccation»

Sud <INSDFearure guals:» den CINSDOualifiers [SCR <INSDOualifisr name>mol type</IN3DQualifier ame»

Es <INSDQualifier value>other DNA</INSDO0uelifier value» 538 </INSDQualifier> - - 438 “INSDoualifier id="gBaänx

ZD CINADGualifler name»note/INSDOualifier name>»

GO <INSDQualifier valuer»8P0411</INSDOualifier value

TOE <fINSDUualifler»>

Tk CINSUQualifler ia=nq8Rv>

REE <INSDQUalifiler naemerorganism/INSDOualifier name

TOR <INSDQualifier value>synthetic construct </INSDQualifier valued

TOE </INSDQuali fier»

TDT </INSDFeature gualss»

TOE </INSDFeature>

Tow </INSDSeg feature-teblex>

VG “INSDSeqg sequmcevatggcaggttagtggaccgtgttacata“/INSDSeq sequence»

LL </INSDSedg>

Tie </SeguenceData>

Jil <SedquenceData semuencelnNumbLer=Ng&r>

Tid “INSDSeg»

TAH <INSDSey lenghh>28</IN3DSeq length»

Tie <INEDSeq moltype>DNA</INSDSeg moltype:>

TE <INSDSeq divisicon>PATA/INSDSeag division» ls “INSDSeq feature-tabled>

LD <INSDFeature»

TE <INSDFeature kevy>source</INSDPFeature key:

GEL <INSDFesture locaticon»l1..28</INSDFeature Location»

TEE CINSDFeature covals> u

Tan <INSDQualifier>

Tad <INSDQualifier name>mol type“ /INSDQualifier name>

EE <INSDOualifier valuerother DNA4/INSDQualilfier value»

TEE </INSUQualifiers> u

Lj <INSDQualifler in=\gS5r>

EE <INSDQualifier name>note</INSDQualifier name>

TES CINSDQualifier wvalue>8P0016</iNsnnualifier values

TEN </INSDQuali fier»

Ta <INSDQualifier id="gBa4nx»>

Tal <INSDQualifier namerorganism“/INSDQualifier name>

ES <INSDOQualifier valuersynthetiec construct <{/INSDQuali jier valuex>

Tad </INEDOualifiers>

TES </INSDPeature guals>

Ta8 </INSDFeaturer -

Ta </INSDEeg Zearture-tabier iis <INSDZeg sequenceratggcacgccaatatttacgtgetgeta:/INSDSeq zeqvence> 72e </INSDSeg> u

TAD <SBequencelata>

TAL <Sequencebata seguanceiiNunbeo=NS3y>

AE LINSDSeq>

TAR <INSDSeq length>23</INSDSeq length>

Fad <INSDSeq moltype>DNA</INSDSeq moliypex

Fan <INSDSeq division>PAT/THSDSeq divisions

TAG <INBDZeq feabture-tablel

TAT <INSDFesture>

TAB <“INSDFearure keyrsource</INSDieature key

Ta “INSDFearure location»1..23</INSDFeature location

FL <“INSDPFeacure gqualis>

EN <INSDQualifier»> 352 <INSDgualifier name>mol type:/INSDOQuali fier name>

THI CINBDQualifiler valuerother DNAC/IN3DGualifler value»

Tag </INSDQualifisan» an <INSDQuelifier id='gS&n>

The CINSDOualifier namernotex/INSDQualifier name>

TE <INZDOualifisr valuerOLI0093«/INshoualifier value»

EE </INEDQualifier> -

TIED <INSDQualifier id="gBöv>

EE <INSDQualifier name>organism</INSDQualifisr names

FE <INBDQualifier valuersynthetic construct </INSDOualifier value

Taz </INSDQuali fier!» u

Tos </INSDFearture qualss

JEE </INSDFaaturel

TED </INSDSeg feature-itablex

TEE <INSDSeq ssqguencer>gatggacgtgettgtegtgaaac</INSDhEeg seguencel 757 </INSDSeq> u

Tan <fSeguenceDalar

TH <SequencaData ouencalDiiumbar="280

TO <INSDSeq>

TEL <INSDSeq lengih>23</INSDSeq length

VE CINZDSeq moltype»DNA</INSDSag moltype>

TIL <INSDSeq division»PAT</INSDSeg division»

Te <INSDSeq feature-itableX

EE LIMIDFeaturas

Tie <INSDFeature keyssource“/INSDFealure hay»

TTT <INSDFeature location»l1..23</INSDFeature location

Fis <INSDFeature guals>

Ti <INSDguelifiler»

Tn CINEDQUalifiler name>mol type“ /INSDgualifier named gl <“INSDOQualifier valuse>other DNA</TINSDOualifier value>

TE </INSDOQualijiier>

JEL <INSDOualifier id="qgg87n>

Tad <INSUQualifier named>note</IN3DQualifier name>

TEE <INSDQualifier value>oLI0191</INSDouali fier valiue>

TRE </INSDQuali fier!»

FES <INSDguelifier id="g5g">

TEE CINZDQuallfier namerorganism</IN3DQualifier name>

Tan <INSDQualifier value>synthetic construct </INSDQualifier value

TE </INSDQualifier>

TH </INSDFaature quals>

Fa </INSDFeature> -

TAS </INSDSeg feature-tabhlad

Tod <“INSDSeg sequmcercaacggaatcccaaaagcagetg/INSDSeg seqmience»>

Fan </INSUE aa»

Tal </EegquencebData>

ERE <SequsnceData seguenceIiNumbber="g8N> 3E <INSDSeq>

TE <INSDSeg length» r24</INIDIeqg length»

Zan <INSDSeq molbype>DNA</INSDSeqg moltype>

SOL <INSDSeq division>PAT</INSDSeqg division»

BOA <INSDSeq festure-table>

SDS <INSDFeature»

Gd <INSDFeature key>source</INSDFeature key»

HOE <INSDPeature location>l..24</IN3DFeature location» 2a <INSDFesture duels»

DT SINSDOualifie>

S48 <INSDQualifier namedmol type</INSDOualifisr name>

Ze <INSDQualifier valuerother DNA /INSDgualifier valuer

BLD <fINSDUualifler»> si CINSUQualifler ia=vgqbRY> fe <INSDOualifisr name>noted/INSDQualifisr name> 415 <INSDQualifier value>OLI3662</INSDOQualifier values sia </INSDQualifier>

Sis “INSDoualifier Ld=0geQn» oie <INSDGualifier namerorganism</INSDQualifisr name>

SLT <INSDQualifier value»>synthetic construct en </INSDQuali fier values

BLE <SINSDOualifier>

Hi </INSDFeature duels» on <JINSDFeature»

HEL </INSDSeg feature-tabler

BE <INSDSeq sequence>gaaaatgatgagtagtgactgatg</INiDieq sequencer 22% </INSDSeqg»

S2á </SegusncsDara>

Bs <SSequencebala seqgsenceinNvumbsc=n3dN>

SEE <INSDSeq>

Gad <INSDSeq length>22</INSkSeq length>

EER <INSDSeq moltype>DNA</INSDSeq moliype>

LED <INSDSeq division>PAT</INSDSeg division: 220 <INSLSeq feature-itabled

Sa CINSDFeabure>

S32 <IiNSDFearure key>source“/INSDFeature key> en CINEDFeature location>»l..22</INSDFeature location»

SS CINEDFeature guals>

S&D <INSDQOualifier» pa <INSDQualifier name>mol type“ /INSDQualifier name» 537 <INSDQualifier valuerother DNAC/INSDQualifier valued cae <ATNaSDQuali fis» 239 <INSDQualifier id="g88Nx> <INSDQualifier namernote</INIDQualifier names

Ba <INSDOualifier value>oLI0016</INSDQualifier valued

Sa <SINSDOualifier>

Fan

Gis <INSDQualifler in=\gS2r> fad <INSDQualifier name>organism</INSDQualifier names

IS <INSDQualifier value>synthetic construct =_ <SINSDOualifier value» saa </INSDQualifisan» 847 </INSDFeature quals>

RE </INSDEFeature>

Fah </INSDSecg feature-table>

Fa ; i

Gli <INSDSeq sequenceltagecagecacgtaaatattggeg</ INSDSeq sequences

SDL << INIDSeg> 252 </SeguenceDatar 253 <Seduencepata samencoalóNuber=s2Ln»>

Dd <INSDSeq>

BEG <INSDSeq lengih>23</INSDSeg length»

BEE <INSDSeq moliype>»DNA</INSDSeq moltypas

SLT CINZDSeq divizion»PAT</INSDS=q division»

LE <“INSDSeq feature-itable>

LEG CINZDFeature> zen <INSDFesture keyrsource“/INSDFealure key»

NY <INSDFeature location>l..23</INSDFeature locations 282 “INSDFeature guals>

BED <INSDQualifier> u en CL

NEG CIMNEDOuAalifieyr namermol type“ /INSDQualifier name> sor <INSDQualifier value>other DNA</INSDQualifier value» ws

SEE </INSDOualifier>

ET <INSDQualifier id="gl90> sae <INSDQualifier name>note</INSDQualifier name> 249 <INSDQualifier value»OLI1B8la</INSDQualifisr value» oan </INSDQvalifier: 8 <INSDQuelifier id='gsdnn

BE <INSDOQualifier namerorganism“/INSDQualifier name fr “INSDoualifier value>synthetic construct rR Le </INSDQvali fien valued 474 </INSDOualiiier> 2S </INSDFeature guals> 276 </INSDFealure»

SET </INSDSeg feature-tabled>

Sis CINSDSed sequenceraacattcaacgctgteggtgagt/INSDSeu sequenced

BEG </INSDSear

BRU <j Gaemquencebata>

Gol <Gequencelala segquanceliNuubsc=NSgN>

NEY <IiNSsDseqd>

HED <INSDSeq iength>23</INSDSeq length>

SE <IN3DSeq moliype>DNA</INSDSeq moltyper

SS <INSDSeq division>PAT</INSDSeg division»

BEG <INSDSeq feature-table>

ERY <INSDFeature>

She “INSDFsature keyrsourece</INIDFeaturs key»

SED <“INSDFeature Llocation»l..23/INSDFeature location»

Sn <INSDFeature gualis>

ROL <INSDOualifier>

S52 <INSDuualifier name>mol type</INSDOualifisr name>

S47 <INSDQualifler valuerother DNA«/INSIGualiflier value»

Bag </INSDQuali fier!»

Zes CINSDQualifiler id="glolv>

Fug <INSDOualifier namernotes/INSDQuali fier name>

Da <INSDQualifiler value>OLI1l8lb</INaloualifier value»

HR </INSDOQualijier> 559 CINSDOualifisr id="qg8&r>

S00 <INSDQualifler namedorganism</INSDQualifisr name>

Gia <INSDQualifier valuersynthetic construct </INSDOualijier value»

SOE <fINSDUualifler»>

S03 </INSDFeaturs gquals>

Gd </INSDFealure»>

G05 </INSDSeg feature-tabledr 30a <INSDSeq sequencsraacattcattgetgteggtggst /INSDSeg seguence> 307 </INSDSegs u

ZOE </SecuenceDala»>

Gan <Sequencebata zegvenaoaiDNumber=n33"D

SLD <INSDSeq>

SL “INSDSeq lengih>22</INSDSeg Length>

GLE <INSDSeq moltype>DNA</INSDSeg moltype> #13 <INSDSeq divislon»PAT</INIDSeg division

Bld <INSDSeq Ieatureriabier

Zij “INSDFeaturex

ERE <INSDFeature key>source“/INSDFealure key>

SLT <INSDFearure location>l..22</INSDFeature Lccation»

SLS <INSDFearure guals:»

Sl <CINSDQualifiers

Gi <INSDQuUalifier namebmol type /INSDQualifier name>

DEL <INSDQualifier value>other DNA</INSDO0uelifier value»

BED </INSDQualifier> 323 CINSZDOualifiler 1d="qRQane

G24 CINADGualifler name>note</IN3Doualifier name>»

SEE <INSDQualifier value>OLIl8le/INSDogualifier value»

GE </INSDOualifier:>

Gnd CINSUQualifler ia=vQq8R9>

GG <INSDQuUalifier namelorganism</INIDQuallifier name>

LE <INSDQualifier value>synthetic construct </INSDQualifier valued 3240 </INSDQuali jier

BEL </INSDFaature cualss»

Sa </INSDFeature>

GRE </INSDSeg feature-teblex> sd “INSDSeqg sequmceraacattcaacctgteggtgagt:/INSDSeq sequencer <SINBDE eg

GEE </SeguenceData> 337 <Sequencalata sopeancallilumber="34%>

Bee VINSDSeq» 533 <INSDSey lenghh>23</IN3DSeq length»

BAG <INSDSedq moliype>DNA</INSDSeg moltype»>

SAL <INSDSeq division>PAT4/INSDSeg division

Ga CINEDSeq feature-table>

SiS <INSDFeature»

Gb <INSDFeature keyirsource</INSDFeature key: 345 <INSDFesture locaticon»l1..23</INSDFeature locations

Sada <INSDFesture gualz> u

S47 <INSDQualifier>

G48 <INSDQualifier namermol type</INSDQualifier name>

EE <INSDOualifier valuerother DNA4/INSDQualilfier value»

GE <SINSDOualifier>

EN LINSDGualifiler ia=ngdQ3v> mie <INSDQualifier name>note</INSDQualifier name>

EN <INSDQuelifier valiuevOLIl8ld</INSDQualifier valua> 354 <ATNaSDQuali fis»

Gi <INSDQualifier id="g70nx>

SE <INSDQualifier namerorganism“/INSDQualifier name>

Gn <INSDOQualifier valuersynthetic construct <{/INSDQuali jier valuex>

GEE </INEDOualifiers>

ENE </INSDPeature guals>

FEN </INSDFeaturer -

Zó: </INSDEeg Zearture-tabier

Za: <INSDSeq sequence>aacattcattgttgteggtgggt/INSDSeq sequence 353 </INSDSeg> vod </SemtenceDala>

SOL </STi6Zeguencelisiing>