CN106636317B

CN106636317B - Lung cancer related microRNA detection kit

Info

Publication number: CN106636317B
Application number: CN201510737078.6A
Authority: CN
Inventors: 刘苏燕; 吴诗扬; 董艳
Original assignee: Surexam Bio Tech Co Ltd
Current assignee: Guangzhou Surexam Medical Laboratory Co ltd
Priority date: 2015-10-30
Filing date: 2015-10-30
Publication date: 2020-06-02
Anticipated expiration: 2035-10-30
Also published as: CN106636317A

Abstract

The invention relates to a lung cancer related microRNA detection kit, which comprises: the method is characterized in that each lung cancer related microRNA to be detected comprises at least one primary signal amplification probe, at least one secondary signal amplification probe, at least one tertiary signal amplification probe and a capture probe, wherein the P1 sequence of the capture probe of the lung cancer related microRNA is selected from SEQ ID NO. 1-SEQ ID NO. 21. The capture probe and the signal amplification probe selected by the invention can perform hybridization reaction under uniform reaction conditions, and non-specific binding does not exist among various probes; the designed probe has good specificity and high signal-to-noise ratio in detection. Meanwhile, the combined use of a plurality of probes enables the identification kit and the detection method to form a system with good detection effect.

Description

Lung cancer related microRNA detection kit

Technical Field

The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and particularly relates to a lung cancer related microRNA detection kit.

Background

MicroRNAs (miRNAs) are endogenous non-coding RNAs with a regulation function, and the size of the RNAs is about 20-25 nucleotides. Mature miRNAs are produced from a long primary transcript by a series of nuclease cleavage processes, then assembled into an RNA-induced silencing complex (RISC), recognize a target miRNA by way of base-complementary pairing, and direct the silencing complex to degrade the target miRNA or repress translation of the target miRNA according to the difference in degree of complementarity. Recent studies have shown that mirnas are involved in a wide variety of regulatory pathways including development, viral defense, hematopoietic processes, organogenesis, cell proliferation and apoptosis, fat metabolism, and the like.

The occurrence and development of lung cancer are complex polygenic events, and microRNAs participate in the polygenic events and play an important role. For example, research results show that hsa-miR-145, hsa-miR-155, hsa-miR-let-7a-2, hsa-miR-let-7b and the like are related to lung cancer prognosis, wherein hsa-miR-155 is an independent prognosis factor of lung cancer; in addition, miRNAs with increased expression levels in lung cancer tissues include hsa-miR-128b, hsa-miR-152, hsa-miR-125b, hsa-miR-27a, hsa-miR-146a, hsa-miR-222, hsa-miR-23a, hsa-miR-24, hsa-miR-150 and the like. In addition, various miRNAs related to the occurrence and development of lung cancer are disclosed in the prior art, such as hsa-miR-10b-5p, hsa-miR-34a, hsa-miR-141-3p, hsa-miR-155-5p, hsa-miR-17-3p, hsa-miR-21-5p, hsa-miR-106a-5p, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-miR-191-5p, hsa-miR-192-5p, hsa-miR-203a-3p, hsa-miR-205-5p and the like.

At present, the methods for detecting miRNA mainly include Northern Blot, gene chip, fluorescent quantitative probe method, and microsphere-based flow cytometry. However, northern blot method has low sensitivity, long time consumption and large RNA dosage, and is not suitable for high-throughput analysis; the gene chip technology can realize high-throughput analysis of miRNA, i.e. a plurality of miRNA are detected on one chip simultaneously, but the defects are low result accuracy, poor repeatability and expensive experiment price; the fluorescence quantitative probe method has high detection sensitivity, but the detection cost is expensive; the flow cytometry technology based on the microspheres fixes the probe on the microspheres and places the probe in a liquid phase, which is more beneficial to capturing miRNA sequences, so that the accuracy is improved.

The in situ hybridization technique is a method for positioning and morphologically detecting specific miRNA sequences in preserved tissue sections or cell preparations. However, the prior art has many difficulties in multiplex and parallel detection of miRNA. Firstly, preparing a labeled probe of each target miRNA respectively; secondly, it is difficult to detect the expression of multiple target mirnas simultaneously in situ. Therefore, the detection of multiple mirnas reported at present can only be performed by different labeling methods, however, the possible cross-hybridization of the probe to non-specific sequences in the cell cannot be well controlled by different labeling methods.

Disclosure of Invention

The invention aims to provide a lung cancer related microRNA detection kit with strong specificity and high sensitivity.

The technical scheme for achieving the purpose is as follows.

A lung cancer related microRNA detection kit comprises: aiming at each lung cancer related microRNA to be detected, the method comprises at least one primary signal amplification probe, at least one secondary signal amplification probe, at least one tertiary signal amplification probe and a capture probe, wherein the lung cancer related microRNA is selected from hsa-miR-486-5p, hsa-miR-29a-5p, hsa-miR-542-5p, hsa-miR-502-3p, hsa-miR-376a-3p, hsa-miR-500a-5p, hsa-miR-424-5p, hsa-miR-10b-5p, hsa-miR-34a, hsa-miR-141-3p, hsa-miR-155-5p, hsa-miR-17-3p, hsa-miR-21-5p, At least one of hsa-miR-106a-5p, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-miR-191-5p, hsa-miR-192-5p, hsa-miR-203a-3p, hsa-miR-205-5p, hsa-miR-212-3p and hsa-miR-214-3p, wherein:

the capture probes are used for connecting target nucleic acid and primary signal amplification probes, each capture probe is provided with a specificity P1 sequence, a spacer arm sequence and a P2 sequence from the 5 'end to the 3' end, and the P2 sequences aiming at different target genes are different from each other;

the primary signal amplification probe sequentially comprises from the 5 'end to the 3' end: a P4 sequence, a spacer arm sequence, a P3 sequence, a P3 sequence and a P2 sequence are complementary and matched;

the secondary signal amplification probe sequentially comprises from the 5 'end to the 3' end: p5 sequence, spacer arm sequence, P6 sequence;

the P4 sequence contains at least one base fragment which is complementarily paired with the P5 sequence, and a spacer arm sequence is arranged between each base fragment which is complementarily paired with the P5 sequence;

the three-stage signal amplification probe sequentially comprises from the 5 'end to the 3' end: the sequence of P8, the sequence of a spacer arm and the sequence of P7, wherein the 5' end of the sequence of P8 is also modified with fluorophores, and the fluorophores aiming at different target nucleic acids are different from each other in color or different from each other in emission wavelength;

the P6 sequence contains at least one base fragment which is complementarily paired with the P7 sequence, and a spacer arm sequence is arranged between each base fragment which is complementarily paired with the P7 sequence;

the P2 sequence, the P3 sequence, the P4 sequence, the P5 sequence, the P6 sequence, the P7 sequence and the P8 sequence are all sequences without hairpin structures, no dimer is formed inside each probe and between the probes, no mismatch exists, and no specific binding exists between the probes and other nucleic acids in the whole detection system;

the P1 sequence of the capture probe of the lung cancer related miRNA to be detected is selected from at least one of the following sequences: SEQ ID NO.1 for hsa-miR-486-5p, SEQ ID NO.2 for hsa-miR-29a-5p, SEQ ID NO.3 for hsa-miR-542-5p, SEQ ID NO.4 for hsa-miR-502-3p, SEQ ID NO.5 for hsa-miR-376a-3p, SEQ ID NO.6 for hsa-miR-500a-5p, SEQ ID NO.7 for hsa-miR-424-5p, SEQ ID NO.8 for hsa-miR-10b-5p, SEQ ID NO.9 for hsa-miR-34a, SEQ ID NO.10 for hsa-miR-141-3p, SEQ ID NO.11 for hsa-miR-155-5p, SEQ ID NO.12 for hsa-miR-17-3p, SEQ ID NO.13 for hsa-miR-21-5p, SEQ ID NO.14 for hsa-miR-106a-5p, SEQ ID NO.15 for hsa-miR-146a-5p, SEQ ID NO.16 for hsa-miR-191-5p, SEQ ID NO.17 for hsa-miR-192-5p, SEQ ID NO.18 for hsa-miR-203a-3p, SEQ ID NO.19 for hsa-miR-205-5p, SEQ ID NO.20 for hsa-miR-212-3p, and SEQ ID NO.21 for hsa-miR-214-3 p.

In one embodiment, the P2 sequence is selected from: SEQ ID NO.22 to SEQ ID NO. 43; the P5 sequence is selected from: SEQ ID NO. 66-SEQ ID NO.87, wherein the sequence of P7 is selected from SEQ ID NO. 110-SEQ ID NO. 131; the spacer sequence in the P4 sequence is 3-10T; the spacer sequence of the P6 sequence is 2-10T; the P8 sequence is polyT.

In one embodiment, the polyT is 3-10 Ts.

In one embodiment, the P4 sequence is selected from: SEQ ID No. 44-65, wherein the P6 sequence is selected from: SEQ ID NO.88 to SEQ ID NO. 109.

In one embodiment, the spacer arm sequence between the P4 sequence and the P3 sequence in the primary signal amplification probe is selected from 5-20T; the spacer arm sequence between the P5 sequence and the P6 sequence in the secondary signal amplification probe is selected from 5-10T; the spacer arm sequence between the P7 sequence and the P8 sequence in the three-stage signal amplification probe is selected from 3-10T.

In one embodiment, the fluorophore is selected from the group consisting of: FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705 and Alexa Fluor 488, and the fluorophores for different target nucleic acids are different from each other.

The main advantages of the invention are:

(1) the lung cancer related microRNA detection capture probe and the signal amplification probe selected by the invention are obtained by comprehensive evaluation, statistical analysis and optimized combination of various parameters through a large number of tests. The hybridization reaction can be carried out under uniform reaction conditions, and non-specific binding does not exist among various probes; the designed probe has good specificity and high signal-to-noise ratio in detection. Meanwhile, the combined use of a plurality of probes enables the identification kit and the detection method to form a system with good detection effect.

(2) The in situ hybridization method has the defect of low fluorescence signal sensitivity, but the invention adopts a novel in situ hybridization method and improves the fluorescence signal intensity through a signal amplification system. The detection process can be completed within 8h, and the miRNA hybridization probe with single copy is combined with the corresponding fluorescent probe through a signal amplification system, so that the detection sensitivity of miRNA in-situ hybridization is obviously improved.

(3) The invention uses the mode of multi-site specific pairing and cascade amplification of the probe to realize the amplification of the signal, but not the method of PCR amplification, thereby improving the detection signal, realizing the specificity of detection and avoiding the false positive of the reverse transcription PCR and real-time fluorescence quantitative PCR technology.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1 detection kit for lung cancer-related miRNA

The embodiment provides a lung cancer related miRNA detection kit, which comprises a capture probe and a signal amplification probe, wherein the signal amplification probe comprises a first-level signal amplification probe, a second-level signal amplification probe and a third-level signal amplification probe. The probe has the characteristics of strong specificity and high sensitivity.

1. Capture probe

The capture probes are used for connecting the target nucleic acid and the primary signal amplification probes, the base sequence of each capture probe sequentially comprises a specific sequence P1 combined with the target nucleic acid to be detected, a spacer arm sequence and a P2 sequence which can be complementarily paired with the P3 combined with the primary signal amplification probes from the 5 'end to the 3' end, and the P2 sequences aiming at different target genes are different from each other.

The spacer is used for spacing the capture probe P2 sequence from the specificity sequence P1, and the spacer with proper length is arranged in the probe, so that the steric hindrance can be reduced, and the efficiency of the hybridization reaction and the specificity of the hybridization reaction can be improved. The spacer arm of the capture probe of the invention is preferably 5-10T, preferably 5T in this embodiment.

This example is directed to hsa-miR-486-5p, hsa-miR-29a-5p, hsa-miR-542-5p, hsa-miR-502-3p, hsa-miR-376a-3p, hsa-miR-500a-5p, hsa-miR-424-5p, hsa-miR-10b-5p, hsa-miR-34a, hsa-miR-141-3p, hsa-miR-155-5p, hsa-miR-17-3p, hsa-miR-21-5p, hsa-miR-106a-5p, hsa-miR-146a-5p, hsa-miR-191-5p, hsa-miR-192-5p, hsa-miR-542 a-5p, Designing capture probes for hsa-miR-203a-3p, hsa-miR-205-5p, hsa-miR-212-3p and hsa-miR-214-3p, and specifically designing the capture probes according to the following steps in tables 1 and 2:

TABLE 1P 1 sequence of capture probes

TABLE 2P 2 sequence of capture probes

SEQ ID NO.	P2 sequence (5 '→ 3')	SEQ ID NO.	P2 sequence (5 '→ 3')
				22	TGACATGATG	33	AGTACTTGTG
23	GATTGTATTG	34	AGTCTTGAAG
				24	TGAAGACTGA	35	TGATGAATTG
25	ATTGATTGTG	36	ATGACGATAG

26	GTCTATAGTG	37	TTGACGTGAA
				27	GATTCAGTGA	38	TGTGACATGA
28	TTGAGTAATG	39	TGTAATTGAG
				29	TGTAATGAGT	40	TGATGCATGT
30	GATTAGTGAT	41	TGAAGTGCAT
				31	GTAGATTAGT	42	TGAAGTGATT
32	GATGACAGTA	43	TGTTGCAGTG

2. Signal amplification probe

1) First order signal amplification probe

The signal amplification component comprises one or more primary signal amplification probes, and each primary signal amplification probe sequentially comprises from the 5 'end to the 3' end: a P3 sequence, a spacer arm sequence and a P4 sequence which are combined with the reverse complementary pairing of the P2 sequence, wherein the P3 sequence realizes the cascade amplification of target signals through the combination with the capture probe P2 sequence.

In this example, the sequence P3 was a base sequence reverse-complementary to the sequence P2.

The spacer arm sequence between the P3 and P4 sequences of the invention can be selected from 5-20T, and the spacer arm used in the embodiment is 10T.

The P4 sequence contains one or more base segments which are reversely complementary with the P5 sequence of the secondary signal amplification probe, preferably 2-5 base segments which are complementarily paired with the P2 sequence, in the embodiment, the P4 contains 3 base segments which are reversely complementary with the P5, and spacer arm sequences are arranged among the same base segments, the spacer arm sequences are preferably 3-10T, and 3T are arranged in the embodiment.

TABLE 3P 4 sequences of first-order Signal amplification probes

SEQ ID NO.	P4 sequence (5 '→ 3')
		44	TCTCAT TTT TCTCAT TTT TCTCAT
45	CTACGA TTT CTACGA TTT CTACGA
		46	TACTAC TTT TACTAC TTT TACTAC
47	TGATAC TTT TGATAC TTT TGATAC
		48	GATCTC TTT GATCTC TTT GATCTC
49	ATATCA TTT ATATCA TTT ATATCA
		50	TATCTC TTT TATCTC TTT TATCTC
51	CACATC TTT CACATC TTT CACATC
		52	TCACAT TTT TCACAT TTT TCACAT
53	ACATCA TTT ACATCA TTT ACATCA
		54	CATCGA TTT CATCGA TTT CATCGA

55	TCGATC TTT TCGATC TTT TCGATC
		56	ACTCTC TTT ACTCTC TTT ACTCTC
57	ACTATC TTT ACTATC TTT ACTATC
		58	ACATCC TTT ACATCC TTT ACATCC
59	GCTCTA TTT GCTCTA TTT GCTCTA
		60	AGATGC TTT AGATGC TTT AGATGC
61	CTCAGA TTT CTCAGA TTT CTCAGA
		62	TCTATC TTT TCTATC TTT TCTATC
63	CATATC TTT CATATC TTT CATATC
		64	TGCTCA TTT TGCTCA TTT TGCTCA
65	CTGCTA TTT CTGCTA TTT CTGCTA

2) Two-stage signal amplification probe

The invention contains one or more than one secondary signal amplification probes, and the secondary signal amplification probes sequentially comprise from the 5 'end to the 3' end: a P5 sequence, a spacer arm sequence and a P6 sequence which are reversely and complementarily combined with the P4 sequence, wherein the 3' end of the P6 sequence is also modified with a fluorescent group; the P4 contains one or more P5 sequence reverse complementary base sequences;

the spacer arm sequence between the P5 and P6 sequences of the invention can be selected from 5-10T, and the spacer arm used in the embodiment is 6T.

TABLE 4P 5 sequences of Secondary Signal amplification probes

SEQ ID NO.	P5 sequence (5 '→ 3')	SEQ ID NO.	P5 sequence (5 '→ 3')
				66	ATGAGA	77	GATCGA
67	TCGTAG	78	GAGAGT
				68	GTAGTA	79	GATAGT
69	GTATCA	80	GGATGT
				70	GAGATC	81	TAGAGC
71	TGATAT	82	GCATCT
				72	GAGATA	83	TCTGAG
73	GATGTG	84	GATAGA
				74	ATGTGA	85	GATATG
75	TGATGT	86	TGAGCA
				76	TCGATG	87	TAGCAG

The P6 sequence contains one or more base sequences which are complementary with the P7 sequence of the three-level signal amplification probe in the reverse direction, preferably contains 2-5 base fragments which are complementary and matched with the P7 sequence, in the embodiment, the P6 sequence contains three base fragments which are complementary with the P7 sequence in the reverse direction, and spacer arm sequences are arranged among the same base sequences, the spacer arm sequences are preferably 2-10T, and in the embodiment, the spacer arm sequences are 2T.

TABLE 5P 6 sequences of Secondary Signal amplification probes

SEQ ID NO.	P6 sequence (5 '→ 3')
		88	CTACA TT CTACA TT CTACA
89	CATAC TT CATAC TT CATAC
		90	CACTC TT CACTC TT CACTC
91	CATGA TT CATGA TT CATGA
		92	ACGAC TT ACGAC TT ACGAC
93	GACTC TT GACTC TT GACTC
		94	CAGTT TT CAGTT TT CAGTT
95	ACACG TT ACACG TT ACACG
		96	AGCAT TT AGCAT TT AGCAT
97	CTCGA TT CTCGA TT CTCGA
		98	ACGTG TT ACGTG TT ACGTG
99	CTCAG TT CTCAG TT CTCAG
		100	GACGA TT GACGA TT GACGA
101	GCTGA TT GCTGA TT GCTGA
		102	TCGAG TT TCGAGTT TCGAG
103	GTGAC TT GTGAC TT GTGAC
		104	TCGAC TT TCGAC TT TCGAC
105	GTCGA TT GTCGA TT GTCGA
		106	CTCGT TT CTCGT TT CTCGT
107	TCTAG TT TCTAG TT TCTAG
		108	CGTAC TT CGTAC TT CGTAC
109	CTGGA TT CTGGATT CTGGA

3) Three-stage signal amplification probe

In this embodiment, the three-level signal amplification probe sequentially includes, from the 5 'end to the 3' end: a P8 sequence, a spacer arm sequence, a P7 sequence, wherein the P6 sequence contains 3 or more sequences which are reverse complementary to the P7 sequence; the 5' end of the P8 sequence is also modified with a fluorescent group;

the spacer arm sequence between the P7 and P8 sequences of the invention can be selected from 3-10T, and the spacer arm used in this embodiment is 5T.

TABLE 6P 7 sequences of three-stage Signal amplification probes

SEQ ID NO.	P7 sequence (5 '→ 3')	SEQ ID NO.	P7 sequence (5 '→ 3')
				110	TGTAG	121	CTGAG
111	GTATG	122	TCGTC
				112	GAGTG	123	TCAGC
113	TCATG	124	CTCGA
				114	GTCGT	125	GTCAC
115	GAGTC	126	GTCGA
				116	AACTG	127	TCGAC
117	CGTGT	128	ACGAG
				118	ATGCT	129	CTAGA
119	TCGAG	130	GTACG
				120	CACGT	131	TCCAG

In this embodiment, the P8 sequence is a polyT sequence of 5 bases, and its 5' end is labeled with a fluorophore selected from: FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705 and Alexa Fluor 488, wherein the fluorophores for different target nucleic acids are different from each other, i.e., the selected fluorophores are different from each other in color or different from each other in emission wavelength, so as to distinguish the different types of target nucleic acids.

Except for the situation that the specific probes defined under the conditions are reversely complementary and completely matched, the sequences of the P3, the P4, the P5, the P6, the P7 and the P8 are sequences without hairpin structures, no dimer is formed between the probe interior and the probe, no mismatch exists, and no specific binding exists between the probe interior and other nucleic acids of the whole detection system.

Example 2 detection kit for detecting miRNA related to lung cancer

The invention provides a lung cancer related miRNA detection kit, which can be used for independently or simultaneously detecting the expression level of one or more of the following target miRNAs: hsa-miR-486-5p, hsa-miR-29a-5p, hsa-miR-542-5p, hsa-miR-502-3p, hsa-miR-376a-3p, hsa-miR-500a-5p, hsa-miR-424-5p, hsa-miR-10b-5p, hsa-miR-34a, hsa-miR-141-3p, hsa-miR-155-5p, hsa-miR-17-3p, hsa-miR-21-5p, hsa-miR-106a-5p, hsa-miR-146a-5p, hsa-miR-155-5 hsp, hsa-miR-191-5p, In actual detection, hsa-miR-192-5P, hsa-miR-203a-3P, hsa-miR-205-5P, hsa-miR-212-3P and hsa-miR-214-3P can form a detection kit by using corresponding P1-P8 sequences according to specific requirements, and detection can be realized.

In this example, hsa-miR-486-5p, hsa-miR-29a-5p, hsa-miR-542-5p, hsa-miR-502-3p, hsa-miR-376a-3p, hsa-miR-500a-5p, hsa-miR-424-5p, hsa-miR-10b-5p, hsa-miR-34a, hsa-miR-141-3p, hsa-miR-155-5p, hsa-miR-17-3p, hsa-miR-21-5p, hsa-miR-106a-5p, hsa-miR-146a-5p, hsa-miR-191-5p, hsa-miR-192-5p, hsa-miR-192-5p, 21 miRNAs such as hsa-miR-203a-3p, hsa-miR-205-5p, hsa-miR-212-3p and hsa-miR-214-3p are randomly divided into 5 groups so as to detect kit components aiming at different miRNAs, and the kit components have reliability and repeatability.

The components of the detection kit of the embodiment comprise: the components of the capture probe, the signal amplification probe and the fluorescent group are shown in Table 7. In the detection kit of this embodiment, the detection is performed by randomly dividing the detection kit into 5 groups.

TABLE 7 detection kit for specific target genes (SEQ ID NO. in the form of Table.)

Example 3 detection of samples Using the kit of example 2

This example will use the kit of example 2 to detect lung cancer cells.

The sources of the lung cancer cells are as follows: the lung cancer cell strain NCI-H1975 can be obtained from the existing products by the technicians in the field according to the names of the cell strains.

The formulations of the various solutions are as follows:

the signal amplification probe mixtures used in this example all were as listed in the corresponding list of example 2.

Firstly, sample pretreatment, namely filtering CTCs (biological chemical centers) to a filter membrane

1. The blood sample was stored in a sample storage tube using a storage solution, centrifuged horizontally at 600 Xg for 5min, and the supernatant was discarded.

2. 4mL of PBS and 1mL of fixative were added, vortexed, mixed, and allowed to stand at room temperature for 8 min.

3. And (3) filtering a sample: transferring the liquid in the sample storage tube into a filter, and opening a vacuum pump to pump out the liquid; 4mL of PBS was added to the storage tube, and the tube wall was washed and the liquid was filtered off with suction.

4. The filters were transferred to a 24-well plate, 400. mu.L of 4% formaldehyde solution was added, and the mixture was fixed at room temperature for 1 hour.

5. The liquid was removed and washed three times with 1mL PBS per well for 2min each time.

Second, permeabilization treatment

1. Adding 50 mu L of permeabilizing agent into each new 24-pore plate, taking the filter membrane out of the PBS, contacting the edge of the filter membrane piece with absorbent paper, removing redundant liquid, and reversely buckling the filter membrane on the permeabilizing agent, namely, the side with the code engraved on the iron circle of the filter membrane is downward close to the liquid. Incubate at room temperature for 5 min.

2. The liquid was removed and washed twice with 1ml PBS per well for 2min each time. The filters were kept in PBS for further experimental work.

Digesting the cell, exposing miRNA, and hybridizing the miRNA with the probe

1. Preparing digestive enzyme working solution with corresponding concentration:

reagent composition	Dosage per sample
		Digestive enzymes	1.25μL

PBS	48.75μL
		Total volume	50μL

2. The digestive enzyme working solution is evenly mixed by vortex and is subpackaged into 24-hole plates, and each hole is 50 mu l.

3. And taking out the filter membrane, and reversely buckling the filter membrane onto digestive enzyme working solution in a 24-pore plate to ensure that the downward surface of the filter membrane is fully contacted with the liquid and no bubbles exist. Standing at room temperature for 1 h.

4. The liquid was removed and washed three times with 1ml PBS per well, 2min each time. The filters were kept in PBS buffer for further experimental work.

Fourthly, probe hybridization, the specific sequence of the probe is combined with the target miRNA sequence

1. The capture probe mixture and the probe buffer solution are preheated for 20min in a water bath at 40 ℃ before use.

2. Preparing a capture probe working solution:

reagent composition	Dosage per sample
		Capture probe mixture	8μL
Probe buffer (40 ℃ pre-heating)	42μL
		Total volume	50.0μL

Vortex and mix well and dispense into 24 well plates, 50. mu.l per well.

3. And taking out the filter membrane, and reversely buckling the filter membrane onto the capture probe working solution in the 24-pore plate to ensure that the downward surface of the filter membrane is fully contacted with the liquid and no bubbles exist.

4. Cover with 24-well plate and incubate at 40. + -. 1 ℃ for 3 hours.

5. The liquid was removed and washed three times by adding 1ml RI washing solution per well, each time for 2 min. And keeping the filter membrane in the washing liquid until the next experimental operation, wherein the soaking time of the sample in the washing liquid cannot exceed 30 min.

Fifthly, amplifying target mRNA sequence signals

1. The probe buffer solution is preheated for 20min in a water bath at 40 ℃ before use.

2. Preparing a probe working solution:

reagent composition	Dosage per sample
		Signal amplification probe mixed liquid	8μL
Probe buffer (40 ℃ pre-heating)	42μL
		Total volume	50.0μL

Vortex and mix well and dispense into 24 well plates, 50. mu.l per well.

3. And taking out the filter membrane, and reversely buckling the filter membrane onto the probe working solution in the 24-pore plate to ensure that the downward surface of the filter membrane is fully contacted with the liquid and no bubbles exist.

4. Cover with 24-well plate and incubate at 40. + -. 1 ℃ for 3 hours.

5. Removing liquid, adding 1ml washing solution into each hole, washing for three times, and soaking for 2min each time. And keeping the filter membrane in the washing liquid until the next experimental operation, wherein the soaking time of the sample in the washing liquid cannot exceed 30 min.

Sixthly, developing color and marking target signal by fluorescence

1. The chromogenic buffer (preheated at 40 ℃) was vortexed and mixed in the dark, and the mixture was dispensed into 24-well plates at 50. mu.l/well.

2. And taking out the filter membrane, and reversely buckling the filter membrane onto the chromogenic buffer solution in the 24-pore plate to ensure that the downward surface of the filter membrane is fully contacted with liquid and no air bubbles exist.

3. Cover with 24-well plate cover, incubate at 40 + -1 deg.C for 30 min.

4. Removing liquid, adding 1ml washing solution into each hole, washing for three times, and soaking for 2min each time. And keeping the filter membrane in the washing liquid until the next experimental operation, wherein the soaking time of the sample in the washing liquid cannot exceed 30 min.

Seventhly, observing CTCs by fluorescence microscope

The control of the present invention uses DAPI as the nuclear fluorophore, which emits a blue fluorescent signal.

1. Placing the filter membrane with cell surface facing upwards on a glass slide, cutting the filter membrane along the inner ring of the iron ring, adding 10 μ L of anti-quenching agent, covering with 18mm × 18mm cover glass, and directly performing microscopic examination or storing at-20 deg.C.

2. The number of CTC heterokaryons was counted through a 20-fold objective lens.

3. And (4) positioning the position of the heteronuclear according to the 10-time objective lens, dripping oil, observing an experimental result by using an oil scope, and photographing and recording the result.

4. And then positioning the next heterogenic nucleus position according to the 10-time objective lens, dripping oil, observing an experimental result by using an oil lens, and photographing in a visual field to record the result.

5. Repeating the operation until all the heterokaryons are photographed, wherein the number of the heterokaryons is consistent with the result of counting 20 times of the objective lens.

The microscope uses channels as follows:

TABLE 8 excitation and emission wavelengths of fluorophores

Eighthly, judging and analyzing detection results

1. Positive lung cancer cell identification standard

Lung cancer cells are enriched on a filter membrane, and the positive judgment standard of the lung cancer cells is as follows:

1) has a corresponding target miRNA specific marker, and shows a fluorescent signal point under a corresponding fluorescent channel in the kit.

2) Nuclear DAPI staining positive.

3) The lung cancer cells are irregular in shape, the diameter of the lung cancer cells is larger than 10 mu m, the lung cancer cells are obviously larger than the aperture of the filter membrane, and the aperture of the filter membrane is 7 mu m. The size of the white blood cells is similar to the size of the filter membrane pores.

2. Using the above detection method, each sample was detected and observed, wherein "-" or "+" was used to indicate whether fluorescence was detected for DAPI staining of cell nuclei; aiming at the fluorescence signal intensity of the target detection miRNA, respectively reading the number of miRNA fluorescence points of corresponding colors of 10 lung cancer cells in each sample, and calculating the average point number, wherein the specific result is as follows:

TABLE 9 sample test results (number of fluorescence signal points)

EXAMPLE 4 stability of the kit

1. Kit stability detection

The invention provides a miRNA detection kit, which selects different numbers of capture probes to form corresponding probe mixed liquor aiming at different target detection miRNAs, thereby realizing the parallel detection of different numbers of miRNAs.

In this example, the stability of the kit of the present invention was evaluated by detecting the expression of hsa-miR-486-5p, hsa-miR-29a-5p, hsa-miR-542-5p and hsa-miR-502-3p in 15 samples derived from three different cell lines (5 samples for each cell line) using the kit consisting of the probe set Group1 of example 2.

2. Relating to the detection of sample origin

In this embodiment, lung cancer cells derived from 3 different cell lines of lung cancer are used as detection targets, so as to verify the validity and stability (repeatability), specific cell lines and samples are shown in table 10, and those skilled in the art can obtain related cell lines from existing products according to the names of the cell lines. The experimental procedure is referred to example 2.

TABLE 10 cell lines and test specimens

Sample number	Lung cell strain	Experimental group
			Samples 26 to 30	NCI-H1975	Group6
Samples 31 to 35	HCC827	Group7
			Samples 36 to 40	PC9	Group8

3. The result of the detection

Detecting and observing each sample by using the kit, wherein the result of DAPI staining of cell nucleus indicates whether fluorescence is detected by using "-" or "+"; aiming at the fluorescence signal intensity of the target detection miRNA marker, respectively reading the number of miRNA fluorescence points of corresponding colors of 10 lung cancer cells in each sample, and calculating the average point number, wherein the specific result is as follows:

TABLE 11 sample test results (number of fluorescence signal points)

As can be seen from the above detection results, on one hand, the detection results of samples from different cell lines are different, and therefore, the present invention is capable of detecting different miRNA expression levels, and is effective; on the other hand, the fluorescence point number detection results of miRNA in 4 such as hsa-miR-486-5p, hsa-miR-29a-5p, hsa-miR-542-5p, hsa-miR-502-3p and the like of 5 samples from the same cell strain are similar (+ -3), and the samples are specifically shown in Group6 (samples 26-30), Group7 (samples 31-35) or Group8 (samples 36-40), so that the kit has good repeatability; therefore, the kit is effective and reliable in stability, and other kits aiming at different miRNA types still have stable and reliable results, and specific data are omitted.

Example 5 target miRNA number selection

1. Design of kit preparation (selection of number of Capture probes)

The invention provides a kit for detecting a target miRNA, which selects different numbers of capture probes to form corresponding probe mixed liquor aiming at different target detection miRNAs, thereby realizing the parallel detection of different numbers of miRNAs.

In this embodiment, a capture probe is selected for 1, 3, 5, and 7 mirnas, a first-order signal method probe, a second-order amplification probe, and a third-order signal amplification probe are selected as the signal amplification probes to form a detection kit, a sample from the same cell line NCI-H1975 is detected, and the detection effect is compared, where the specific compositions are shown in table 12, the probes are selected from embodiments 1 to 2, and the experimental steps refer to embodiment 3.

TABLE 12 selection of capture probes for different numbers of target miRNAs (Table number SEQ ID NO.)

2. Detecting and observing each sample by using the kit, wherein the result of DAPI staining of cell nucleus indicates whether fluorescence is detected by using "-" or "+"; aiming at the fluorescence signal intensity of the target detection miRNA marker, respectively reading the number of miRNA fluorescence points of corresponding colors of 10 lung cancer cells in each sample, and calculating the average point number, wherein the specific result is as follows:

TABLE 13 sample test results (number of fluorescence signal points)

As can be seen from the comparison of the 4 groups of experiments, the kit can detect target miRNAs with different quantities, can complete detection by using 1, 3, 5 and 7 miRNA capture probes aiming at different miRNA capture probes, and has good specificity and stability.

Other kits aiming at the marker gene and using different quantities of capture probes still have stable and reliable results, and specific data are omitted.

EXAMPLE 6 selection of labeled probes

1. Design of kit preparation (selection of Signal amplification Probe combination)

The invention provides a kit for detecting miRNA related to lung cancer, which selects different signal probe combinations to form corresponding probe mixed liquor aiming at different target detection miRNAs, thereby realizing the parallel detection of the miRNAs.

In this example, the kit composed of the signal amplification probes at different levels provided in example 1 was used to detect the expression of hsa-miR-502-3P, hsa-miR-141-3P, hsa-miR-146a-5ph and hsa-miR-212-3P in 5 samples from the same cell strain (NCI-H1975), so as to evaluate the applicability of the signal amplification probes P2, P3, P4, P5, P6 and P7 provided by the present invention. The specific kit comprises the following components:

TABLE 14 different signal amplification probe combinations (SEQ ID NO. number in table)

2. Using the above-described kit, each sample was subjected to detection (using the detection method described in example 3) and observation, wherein with respect to DAPI staining of cell nuclei, "-" or "+" was used to indicate whether fluorescence was detected; aiming at the fluorescence signal intensity of the target detection miRNA marker, respectively reading the number of miRNA fluorescence points of corresponding colors of 10 lung cancer cells in each sample, and calculating the average point number, wherein the specific result is as follows:

TABLE 15 detection results of different combinations of signal amplification probes

It can be known from the comparison of the 3 groups of experiments that the detection results of the miRNA detected by the kit composed of different signal probe combinations are consistent, except for the miRNA and the amplification probe combination thereof detected in this embodiment, the combination of other miRNA and other signal amplification probes provided by the present invention can also achieve the detection effect of this embodiment, and the specific test result is omitted. Therefore, the signal amplification probe provided by the invention can be matched randomly according to actual operation requirements, and can realize miRNA detection of the invention, and the detection results are consistent, which shows that the signal amplification probe provided by the invention has good applicability to miRNA detection related to lung cancer.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A lung cancer related microRNA detection kit is characterized by comprising: aiming at each lung cancer related microRNA to be detected, the method comprises a primary signal amplification probe, a secondary signal amplification probe, a tertiary signal amplification probe and a capture probe, wherein the lung cancer related microRNA comprises hsa-miR-486-5p, hsa-miR-29a-5p, hsa-miR-542-5p, hsa-miR-502-3p, hsa-miR-376a-3p, hsa-miR-500a-5p, hsa-miR-424-5p, hsa-miR-10b-5p, hsa-miR-34a, hsa-miR-141-3p, hsa-miR-155-5p, hsa-miR-17-3p, hsa-miR-21-5p, hsa-miR-106a-5p, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-miR-191-5p, hsa-miR-192-5p, hsa-miR-203a-3p, hsa-miR-205-5p, hsa-miR-212-3p and hsa-miR-214-3p, wherein:

the P1 sequence of the capture probe of the lung cancer related miRNA to be detected comprises: SEQ ID NO.1 for hsa-miR-486-5p, SEQ ID NO.2 for hsa-miR-29a-5p, SEQ ID NO.3 for hsa-miR-542-5p, SEQ ID NO.4 for hsa-miR-502-3p, SEQ ID NO.5 for hsa-miR-376a-3p, SEQ ID NO.6 for hsa-miR-500a-5p, SEQ ID NO.7 for hsa-miR-424-5p, SEQ ID NO.8 for hsa-miR-10b-5p, SEQ ID NO.9 for hsa-miR-34a, SEQ ID NO.10 for hsa-miR-141-3p, SEQ ID NO.11 for hsa-miR-155-5p, SEQ ID NO.12 for hsa-miR-17-3p, SEQ ID NO.13 for hsa-miR-21-5p, SEQ ID NO.14 for hsa-miR-106a-5p, SEQ ID NO.15 for hsa-miR-146a-5p, SEQ ID NO.16 for hsa-miR-191-5p, SEQ ID NO.17 for hsa-miR-192-5p, SEQ ID NO.18 for hsa-miR-203a-3p, SEQ ID NO.19 for hsa-miR-205-5p, SEQ ID NO.20 for hsa-miR-212-3p, and SEQ ID NO.21 for hsa-miR-214-3 p;

the P2 sequence includes: SEQ ID NO.22 to SEQ ID NO. 43; the P5 sequence includes: SEQ ID NO. 66-SEQ ID NO.87, wherein the P7 sequence comprises SEQ ID NO. 110-SEQ ID NO. 131; the spacer sequence in the P4 sequence is 3-10T; the spacer sequence of the P6 sequence is 2-10T, and the P8 sequence is polyT;

the P4 sequence includes: SEQ ID No. 44-65, and the P6 sequence comprises: SEQ ID NO.88 to SEQ ID NO. 109.

2. The lung cancer-related microRNA detection kit according to claim 1, wherein the polyT is 3-10 Ts.

3. The lung cancer-associated microRNA detection kit according to any one of claims 1-2, wherein the spacer arm sequence between the P4 sequence and the P3 sequence in the primary signal amplification probe is selected from 5-20T; the spacer arm sequence between the P5 sequence and the P6 sequence in the secondary signal amplification probe is selected from 5-10T; the spacer arm sequence between the P7 sequence and the P8 sequence in the three-stage signal amplification probe is selected from 3-10T.

4. The lung cancer-related microRNA detection kit according to any one of claims 1-2, wherein the fluorescent group is selected from the group consisting of: FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705, and Alexa Fluor 488.

5. The lung cancer-associated microRNA detection kit according to any one of claims 1 to 4, wherein the P2 sequence is: SEQ ID NO.22 for hsa-miR-486-5p, SEQ ID NO.23 for hsa-miR-29a-5p, SEQ ID NO.24 for hsa-miR-542-5p, SEQ ID NO.25 for hsa-miR-502-3p, SEQ ID NO.26 for hsa-miR-376a-3p, SEQ ID NO.27 for hsa-miR-500a-5p, SEQ ID NO.28 for hsa-miR-424-5p, SEQ ID NO.29 for hsa-miR-10b-5p, SEQ ID NO.30 for hsa-miR-34a, SEQ ID NO.31 for hsa-miR-141-3p, SEQ ID NO.32 for hsa-miR-155-5p, SEQ ID NO.33 for hsa-miR-17-3p, SEQ ID NO.34 for hsa-miR-21-5p, SEQ ID NO.35 for hsa-miR-106a-5p, SEQ ID NO.36 for hsa-miR-146a-5p, SEQ ID NO.37 for hsa-miR-191-5p, SEQ ID NO.38 for hsa-miR-192-5p, SEQ ID NO.39 for hsa-miR-203a-3p, SEQ ID NO.40 for hsa-miR-205-5p, SEQ ID NO.41 for hsa-miR-212-3p, and SEQ ID NO.42 for hsa-miR-214-3 p.

6. The lung cancer-associated microRNA detection kit according to any one of claims 1 to 4, wherein the P5 sequence is: SEQ ID NO.66 for hsa-miR-486-5p, SEQ ID NO.67 for hsa-miR-29a-5p, SEQ ID NO.68 for hsa-miR-542-5p, SEQ ID NO.69 for hsa-miR-502-3p, SEQ ID NO.70 for hsa-miR-376a-3p, SEQ ID NO.71 for hsa-miR-500a-5p, SEQ ID NO.72 for hsa-miR-424-5p, SEQ ID NO.73 for hsa-miR-10b-5p, SEQ ID NO.74 for hsa-miR-34a, SEQ ID NO.75 for hsa-miR-141-3p, SEQ ID NO.76 for hsa-miR-155-5p, SEQ ID NO.77 for hsa-miR-17-3p, SEQ ID NO.78 for hsa-miR-21-5p, SEQ ID NO.79 for hsa-miR-106a-5p, SEQ ID NO.80 for hsa-miR-146a-5p, SEQ ID NO.81 for hsa-miR-191-5p, SEQ ID NO.82 for hsa-miR-192-5p, SEQ ID NO.83 for hsa-miR-203a-3p, SEQ ID NO.84 for hsa-miR-205-5p, SEQ ID NO.85 for hsa-miR-212-3p, and SEQ ID NO.86 for hsa-miR-214-3 p.

7. The lung cancer-associated microRNA detection kit according to any one of claims 1 to 4, wherein the P7 sequence is: SEQ ID NO.110 for hsa-miR-486-5p, SEQ ID NO.111 for hsa-miR-29a-5p, SEQ ID NO.112 for hsa-miR-542-5p, SEQ ID NO.113 for hsa-miR-502-3p, SEQ ID NO.114 for hsa-miR-376a-3p, SEQ ID NO.115 for hsa-miR-500a-5p, SEQ ID NO.116 for hsa-miR-424-5p, SEQ ID NO.117 for hsa-miR-10b-5p, SEQ ID NO.118 for hsa-miR-34a, SEQ ID NO.119 for hsa-miR-141-3p, SEQ ID NO.120 for hsa-miR-155-5p, SEQ ID NO.121 for hsa-miR-17-3p, SEQ ID NO.122 for hsa-miR-21-5p, SEQ ID NO.123 for hsa-miR-106a-5p, SEQ ID NO.124 for hsa-miR-146a-5p, SEQ ID NO.125 for hsa-miR-191-5p, SEQ ID NO.126 for hsa-miR-192-5p, SEQ ID NO.127 for hsa-miR-203a-3p, SEQ ID NO.128 for hsa-miR-205-5p, SEQ ID NO.129 for hsa-miR-212-3p, and SEQ ID NO.130 for hsa-miR-214-3 p.