CN106636318B

CN106636318B - Nucleic acid signal amplification detection kit

Info

Publication number: CN106636318B
Application number: CN201510737340.7A
Authority: CN
Inventors: 吴诗扬; 许嘉森; 刘苏燕
Original assignee: Surexam Bio Tech Co Ltd
Current assignee: Surexam Bio Tech Co Ltd
Priority date: 2015-10-30
Filing date: 2015-10-30
Publication date: 2020-06-09
Anticipated expiration: 2035-10-30
Also published as: CN106636318A

Abstract

The invention relates to a nucleic acid signal amplification detection kit, which mainly comprises: aiming at each target gene, at least one primary signal amplification probe and at least one secondary signal amplification probe or a third signal amplification probe are provided, or the method mainly comprises the following steps: at least one primary signal amplification probe and at least one secondary signal amplification probe or a tertiary signal amplification probe and a capture probe are provided for each target gene. The invention adopts a detection kit designed by a novel in-situ hybridization method, and improves the intensity of a fluorescence signal through a signal amplification system. The detection process can be completed within 8h, and the single copy nucleic acid hybridization probe is combined with the corresponding fluorescent probe through a signal amplification system, so that the detection sensitivity of RNA in-situ hybridization is obviously improved.

Description

Nucleic acid signal amplification detection kit

Technical Field

The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and particularly relates to a nucleic acid signal amplification detection kit.

Background

In Situ Hybridization (ISH) is a technique that uses complementary base sequences between single strands of nucleic acid molecules, and matches radioactive or nonradioactive exogenous nucleic acid (i.e., probe) with DNA or RNA to be detected on tissue, cell or chromosome to form specific nucleic acid hybrid molecules, and displays the position of the nucleic acid to be detected on the tissue, cell or chromosome by a certain detection means, wherein the DNA or RNA to be detected can be endogenous DNA, messenger RNA (mRNA), micro RNA (miRNA), viral sequences or bacterial sequences. The sensitivity of this technique, i.e., the threshold level of detection, can be up to 10-20 copies of mRNA per cell.

The fluorescein labeling of the in situ hybridization probes can be carried out by direct and indirect labeling methods. Currently, the direct labeling method is to covalently bind fluorescein directly to the probe nucleotide or pentose phosphate backbone, or to incorporate fluorescein nucleoside triphosphate when the probe is labeled by the nick translation method. The direct labeling method has simple steps in detection, but has poor sensitivity because signal amplification cannot be performed. The indirect labeling is to label a DNA probe by adopting biotin, detect the probe by coupling fluorescein avidin or streptavidin after hybridization, and amplify a fluorescence signal by utilizing an avidin-biotin-fluorescein complex so as to detect a fragment of 500 bp. The sensitivity is higher than that of the direct labeling method.

However, the sensitivity and specificity of existing ISH-based detection techniques are often poorly characterized for the detection of low copy number DNA or RNA targets in cells, which limits their application. To overcome the limitations of ISH, a novel ISH signal amplification method called RNAscope was developed by Advanced CellDiagnostics, inc. This assay includes a uniquely designed oligomeric capture probe and a signal amplification system consisting of a preamplifier, an amplicon (amplifier) and a label probe, enabling significant signal amplification without amplification of background signal and single RNA molecule detection of almost any gene.

However, the use of RNAscope alone has not reliably detected some low copy genes in past formalin fixed, paraffin embedded (FFPE) tissue sections where RNA is significantly degraded, and in addition, the current RNAscope ○ R technology has not been able to allow single RNA molecules to be visualized at 40X magnification.

In view of the above, there is a need for a method for amplifying nucleic acids with high sensitivity and high specificity for the detection of low copy number DNA or RNA targets in cells.

Disclosure of Invention

The invention aims to provide a nucleic acid signal amplification detection kit with strong specificity and high sensitivity.

The technical scheme for achieving the purpose is as follows.

A nucleic acid signal amplification detection kit mainly comprises: at least one primary signal amplification probe and at least one secondary signal amplification probe are provided for each target gene, or the method mainly comprises the following steps: at least one primary signal amplification probe and at least one secondary signal amplification probe for each target gene, and a capture probe, wherein

The primary signal amplification probe sequentially comprises from the 5 'end to the 3' end: p4 sequence, spacer arm sequence, P3 sequence;

the secondary signal amplification probe sequentially comprises from the 5 'end to the 3' end: p5 sequence, spacer arm sequence, P6 sequence; the 3' end of the P6 sequence is also modified with a fluorescent group, the colors or emission wavelengths of the fluorescent groups aiming at different target nucleic acids are different, and the P5 sequence is 5 bp-15 bp;

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 capture probes can be used for connecting target nucleic acid and primary signal amplification probes, and each capture probe comprises the following components from the 5 'end to the 3' end: the specific P1 sequence, the spacer arm sequence and the P2 sequence which can be complementarily matched and combined with the nucleic acid of the target gene to be detected are different from each other aiming at the P2 sequences of different target genes, wherein the P1 sequence is 15 bp-30 bp, and the P2 sequence is 8 bp-25 bp;

when the capture probe is contained in the kit, the P3 sequence contains at least one base fragment which is complementarily paired with the P2 sequence, and a spacer arm sequence is arranged between each base fragment which is complementarily paired with the P2 sequence; when the kit does not contain a capture probe, the P3 sequence is a specific base fragment which can be combined with the complementary pairing of the nucleic acid of the target gene to be detected;

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

Or, the nucleic acid signal amplification detection kit mainly comprises: at least one first-level signal amplification probe, at least one second-level signal amplification probe and at least one third-level signal amplification probe for each target gene, or mainly comprises: for each target gene, there are at least one primary signal amplification probe, at least one secondary signal amplification probe, and at least one tertiary signal amplification probe, and a capture probe,

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 sequence of the P5 is 5 bp-15 bp;

the capture probes are used for connecting target nucleic acid and primary signal amplification probes, each capture probe is provided with a specific sequence P1, a spacer arm sequence and a P2 sequence which can be complementarily matched and combined with nucleic acid of a target gene to be detected from a 5 'end to a 3' end, and the P2 sequences aiming at different target genes are different; the sequence of the P1 is 15 bp-30 bp, and the sequence of the P2 is 8 bp-25 bp; when the capture probe is contained in the kit, the P3 sequence contains at least one base fragment which is complementarily paired with the P2 sequence, and a spacer arm sequence is arranged between each base fragment which is complementarily paired with the P2 sequence; when the kit does not contain a capture probe, the P3 sequence is a specific base fragment which can be combined with the complementary pairing of the nucleic acid of the target gene to be detected;

the three-stage signal amplification probe sequentially comprises from the 5 'end to the 3' end: the fluorescent probe comprises a P8 sequence, a spacer arm sequence and a P7 sequence, wherein the 5' end of the P8 sequence is also modified with fluorescent groups, the fluorescent groups aiming at different target nucleic acids are different, the colors of the corresponding fluorescent groups are different, or the emission wavelengths of the corresponding fluorescent groups are different, and the P7 sequence is 4 bp-10 bp;

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 probes, no mismatch exists, and no specific binding exists between the probes and other nucleic acids in the whole detection system.

In one embodiment, the P1 sequence of the capture probe is selected from SEQ ID NO.121 to SEQ ID NO.135, and the P2 sequence of the capture probe is selected from: SEQ ID NO.101 to SEQ ID NO. 120; the sequence of the primary signal amplification probe P3 is selected from SEQ ID NO. 1-SEQ ID NO.20, and the sequence of P4 is selected from SEQ ID NO. 21-SEQ ID NO. 40; the secondary signal amplification probe is selected from a P5 sequence SEQ ID NO. 41-SEQ ID NO.60, and the P6 sequence is selected from a SEQ ID NO. 61-SEQ ID NO. 80; the sequence of the third-level signal amplification probe P7 is selected from SEQ ID NO. 81-SEQ ID NO.100, and the sequence of P8 is polyT.

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

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 2-10T.

In one embodiment, when the capture probe is contained in the kit, the P3 sequence contains 2-5 base fragments complementarily paired with the P2 sequence, and a spacer arm sequence is arranged between each base fragment complementarily paired with the P2 sequence, and the spacer arm sequence is 4-10T.

In one embodiment, the P4 sequence contains 2-4 base fragments complementary paired with the P5 sequence, and a spacer arm sequence is arranged between each base fragment complementary paired with the P5 sequence, and the spacer arm sequence is 3-10T.

In one embodiment, the P6 sequence contains 2-4 base segments that are complementary paired with the P7 sequence, and a spacer sequence is provided between each base segment that is complementary paired with the P7 sequence, and the spacer sequence can be 2-10T.

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.

In one embodiment, the target nucleic acid is mRNA, comprising: the EGFR gene, the KIT gene and/or the B2M gene, wherein the P1 sequence aiming at the EGFR gene is selected from SEQ ID NO.121 to SEQ ID NO.125, and the P2 sequence is: SEQ ID NO.103, the P3 sequence is: SEQ ID NO.3, and the sequence of P4 is: the sequence of the P5 is SEQ ID NO.45, the sequence of the P6 is SEQ ID NO.67, and the sequence of the P7 is SEQ ID NO. 87; the P1 sequence aiming at the KIT gene is selected from SEQ ID NO. 126-SEQ ID NO.130, and the P2 sequence is: SEQ ID NO.109, the P3 sequence is: SEQ ID NO.9, wherein the sequence of P4 is: 30, 50 as the sequence of P5, 74 as the sequence of P6 and 94 as the sequence of P7; the P1 sequence aiming at the B2M gene is selected from SEQ ID NO. 131-SEQ ID NO.135, and the P2 sequence is: SEQ ID NO.115, the P3 sequence is: SEQ ID NO.15, the P4 sequence is: the sequence of the P5 is SEQ ID NO.55, the sequence of the P6 is SEQ ID NO.79, and the sequence of the P7 is SEQ ID NO. 99.

If there is more than one capture probe for each target nucleic acid, the P1 sequences are different and the P2 sequences are the same or different.

The main advantages of the invention are:

(1) the in situ hybridization method has the defect of low fluorescence signal sensitivity, but the invention adopts the detection kit designed by the novel in situ hybridization method to improve the fluorescence signal intensity through a signal amplification system. The detection process can be completed within 8h, the single copy nucleic acid hybridization probe is combined with the corresponding fluorescent probe through a signal amplification system, the detection sensitivity of the RNA in-situ hybridization is obviously improved, in addition, the amplification of the signal is realized in a multi-site specific pairing and cascade amplification mode instead of a PCR amplification method, the detection signal is improved, the detection specificity is realized, and the false positive of the reverse transcription PCR and real-time fluorescent quantitative PCR technology is avoided.

(2) The signal amplification probe selected by the invention is obtained by comprehensive evaluation, statistical analysis and optimized combination of various parameters through a large number of tests. The method adopts multiple signal amplification probes, the selection of the probes can realize secondary probe or tertiary probe use, signal amplification detection can be realized, and when three probes are used together, the detection sensitivity is greatly improved.

(3) The various specific probes designed by the invention aiming at the mRNA of the EGFR gene, the KIT gene and the B2M gene can perform hybridization reaction under uniform reaction conditions, and nonspecific combination basically does not exist among the 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.

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. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. 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

In this embodiment, a set of signal amplification detection system with high sensitivity is designed, and signal amplification components of the first-level signal amplification probe, the second-level signal amplification probe, the third-level signal amplification probe, and the capture probe will be described in detail.

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 P4 sequence, a spacer arm sequence, a P3 sequence combined with a target nucleic acid to be detected, and a P3 sequence realizes cascade amplification of a target signal through combination with a capture probe.

Except for the case of reverse complementary perfect matching of specific probes defined below, the sequences of P3, P4, P5, P6, P7 and P8 are sequences without hairpin structures, no dimer and no mismatch are formed between the probe and the probe, and no specific binding exists between the probe and other nucleic acids in the whole detection system.

The P3 sequence of the invention contains at least one base fragment reverse-complementary to the P2 sequence of the capture probe, preferably 5-15 identical base fragments, in this example, P3 contains 2 base fragments reverse-complementary to the P2 sequence of the capture probe, and spacer arm sequences are arranged among the identical base fragments, the spacer arm sequences can be selected from 4-10T, in this example, 6T are used, the P4 sequence can contain at least one base fragment reverse-complementary to P5, in this example, 3 base fragments reverse-complementary to P5.

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.

TABLE 1P 3 sequence of Primary Signal amplification probes

SEQ ID NO.	P3 sequence (5 '→ 3')
		1	CATCATGTCA TTTTTT CATCATGTCA
2	CAATACAATC TTTTTT CAATACAATC
		3	TCAGTCTTCA TTTTTT TCAGTCTTCA
4	CACAATCAAT TTTTTT CACAATCAAT
		5	CACTATAGAC TTTTTT CACTATAGAC
6	TCACTGAATC TTTTTT TCACTGAATC
		7	CATTACTCAA TTTTTT CATTACTCAA
8	ACTCATTACA TTTTTT ACTCATTACA
		9	ATCACTAATC TTTTTT ATCACTAATC
10	ACTAATCTAC TTTTTT ACTAATCTAC
		11	TACTGTCATC TTTTTT TACTGTCATC
12	CACAAGTACT TTTTTT CACAAGTACT
		13	CTTCAAGACT TTTTTT CTTCAAGACT
14	CAATTCATCA TTTTTT CAATTCATCA
		15	CTATCGTCAT TTTTTT CTATCGTCAT
16	TTCACGTCAA TTTTTT TTCACGTCAA
		17	TCATGTCACA TTTTTT TCATGTCACA
18	CTCAATTACA TTTTTT CTCAATTACA
		19	ACATGCATCA TTTTTT ACATGCATCA
20	ATGCACTTCA TTTTTT ATGCACTTCA

The P4 sequence contains one or more base segments which are reversely complementary with the P5 sequence of the secondary signal amplification probe; preferably 2-4 bases, in this example, the P4 sequence contains 3 bases complementary to the reverse of P5, and there is a spacer sequence between the same bases, which can be selected from 3-10T, in this example, 5T are used.

TABLE 2P 4 sequences of first-order Signal amplification probes

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: the probe comprises a P5 sequence, a spacer arm sequence and a P6 sequence which are reversely and complementarily combined with a P4 sequence, wherein when a three-stage signal amplification probe is not included in the technical scheme, the 3' end of the P6 sequence is also modified with fluorescent groups, and the fluorescent groups aiming at different target nucleic acids are different from each other, namely the colors of the corresponding fluorescent groups are different from each other or the emission wavelengths are different from each other; the sequence of the P5 is 5 bp-15 bp.

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 3P 5 sequences of Secondary Signal amplification probes

The P6 sequence contains one or more base sequences reverse complementary to the P7 sequence of the tertiary signal amplification probe, preferably 2-4 base fragments, in this embodiment, the P6 sequence contains three base fragments reverse complementary to the P7 sequence, and spacer arm sequences are arranged between the same base fragment, the spacer arm sequences can be selected from 2-10T, in this embodiment, 3T are used.

TABLE 4P 6 sequences of Secondary Signal amplification probes

SEQ ID NO.	P6 sequence (5 '→ 3')
		61	TACGA TTT TACGA TTT TACGA
62	CATAG TTT CATAG TTT CATAG
		63	TAGCA TTT TAGCA TTT TAGCA
64	CTAAG TTT CTAAG TTT CTAAG
		65	TGAAC TTT TGAAC TTT TGAAC
66	CCTAG TTT CCTAG TTT CCTAG
		67	CGATC TTT CGATC TTT CGATC
68	CTACG TTT CTACG TTT CTACG
		69	ATCAG TTT ATCAG TTT ATCAG
70	ACGCT TTT ACGCT TTT ACGCT
		71	TCTAG TTT TCTAG TTT TCTAG
72	CTCGA TTT CTCGA TTT CTCGA
		73	GTCGA TTT GTCGA TTT GTCGA
74	GTGTC TTT GTGTC TTT GTGTC
		75	TAGTC TTT TAGTC TTT TAGTC
76	GAGTC TTT GAGTC TTT GAGTC
		77	ACTAG TTT ACTAG TTT ACTAG
78	CATCT TTT CATCT TTT CATCT
		79	GCTAG TTT GCTAG TTT GCTAG
80	GCTCA TTT GCTCA TTT GCTCA

3) Three-stage signal amplification probe

The three-level signal amplification probe sequentially comprises the following components from a 5 'end to a 3' end: the fluorescent probe comprises a P8 sequence, a spacer arm sequence and a P7 sequence, wherein the 5' end of the P8 sequence is also modified with a fluorescent group, the P7 sequence is 4 bp-10 bp, and the P7 sequence of the embodiment is shown in the following table.

The spacer arm sequence between the P7 and P8 sequences of the invention can be selected from 2-10T, and the spacer arm used in the embodiment is 5T.

TABLE 5P 7 sequences of three-stage Signal amplification probes

SEQ ID NO.	P7 sequence (5 '→ 3')	SEQ ID NO.	P7 sequence (5 '→ 3')
				81	TCGTA	91	CTAGA
82	CTATG	92	TCGAG
				83	TGCTA	93	TCGAC
84	CTTAG	94	GACAC
				85	GTTCA	95	GACTA
86	CTAGG	96	GACTC
				87	GATCG	97	CTAGT
88	CGTAG	98	AGATG
				89	CTGAT	99	CTAGC
90	AGCGT	100	TGAGC

The sequence of P8 can be a polyT sequence of 3-10 bases, in this example, the sequence of P8 is a polyT sequence of 5 bases, when the technical scheme includes a three-stage signal amplification probe, the 5' end of the sequence of P8 carries a fluorophore label, and the fluorophore can be selected from: FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705 and AlexaFluor 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 in emission wavelength, so as to distinguish the different types of target nucleic acids.

4) 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 is sequentially provided with a specific sequence P1 sequence capable of being combined with the pairing in the target nucleic acid to be detected of different target genes, a spacer arm sequence and a P2 sequence capable of being combined with the complementary pairing of the primary signal amplification probe P3 sequence from the 5 'end to the 3' end, and the P3 sequence contains one or more base fragments which are reversely complementary with the P2 sequence; the P2 sequences aiming at different target genes are different from each other, and the P2 sequence is 8 bp-25 bp. The sequence of the P1 is 10 bp-25 bp, the GC content is 45% -65%, and no sequence with specific combination exists between the P1 sequence and other nucleic acids of the whole detection system; the spacer is used for spacing the capture probe P2 sequence from the P1 sequence, 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 2-10T, and in this embodiment preferably 5T.

TABLE 6P 2 sequences of capture probes

Example 2A detection kit for nucleic acid

The invention provides a nucleic acid detection KIT, which can detect the mRNA expression levels of target genes EGFR, KIT, B2M and the like, and specifically comprises the following steps:

1) capture probe

Aiming at mRNA of target genes such as EGFR, KIT, B2M and the like, designing capture probes, wherein the capture probes are connected with the mRNA of the target genes and a primary signal amplification probe, and the base sequence of each capture probe sequentially comprises from 5 'end to 3' end: the probe comprises a specific sequence P1 capable of being complementarily paired and combined with target gene mRNA to be detected, a spacer arm sequence and a P2 sequence capable of being complementarily paired with a primary signal amplification probe P3 sequence, wherein the P3 sequence contains one or more base sequences which are reversely complementary with the P2 sequence;

in this example, 5 capture probes are designed for each marker gene, the P2 sequence of the capture probe is selected from table 6 of example 2, and the P1 sequence of the capture probe is specifically shown in table 7, so as to improve the specificity of detection (in use, for each target gene, 1 or more capture probes are selected to complete detection, and the specificity and stability are good, as shown in example 6). The P1 sequence is selected from SEQ ID NO. 121-SEQ ID NO.135, and the P2 sequence is selected from: SEQ ID NO.101 to SEQ ID NO. 120; the sequences of P2 aiming at different target gene mRNA are different from each other, and the sequence of P1 is 15 bp-30 bp.

The spacer is used for spacing the capture probe P2 sequence from the P1 sequence, 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.

TABLE 7P 1 sequences for target Gene Capture probes

2) Components of the kit

In this embodiment, the fluorophore may be selected from: FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705 and Alexa Fluor 488, wherein the fluorophores aiming at different target genes mRNA are different from each other, namely the colors of the corresponding fluorophores are different from each other or the emission wavelengths are different from each other, so as to distinguish different types of marker genes.

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 8.

TABLE 8 detection kit for specific target genes

Example 3 detection of samples Using the kit of example 2

In this example, the kit of example 2 is used to detect samples from different cancer cell sources, and specifically, as shown in the following table, those skilled in the art can obtain related cell lines from existing products according to their cell line names. In this example, the same volume of cell preservation solution was collected from each of the cancer cell lines and used as a test sample in the following test.

Sample numbering	Cancer species	Cell line name
			1～5	Lung cancer	NCI-H1975
6～10	Prostate cancer	PC3
			11～15	Stomach cancer	KtaoIII
16～20	Breast cancer	MCF-7

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, cancer cell filtration to filter membrane

1. The cancer cell sample preserved with the preservation solution was centrifuged at 600 Xg for 5min in a sample preservation tube, 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.

Thirdly, digesting the cells, exposing the nucleic acid, and hybridizing it 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 nucleic acid sequence

1. The probe buffer and the color development buffer are preheated for 20min in a water bath at 40 ℃ before being used.

2. Preparing a probe working solution:

reagent composition	Dosage per sample
		Probe mixed solution	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.

Fifthly, amplifying hybridization and target nucleic acid sequence signal amplification

1. Preparing an amplification working solution:

reagent composition	Dosage per sample
		Signal amplification probe mixed liquid	2μL
Probe buffer (40 ℃ pre-heating)	48μL
		Total volume	50.0μL

Mixing by vortex, and packaging into 24-well plate with 50 μ l per well

2. And taking out the filter membrane, and reversely buckling the filter membrane onto the amplification 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.

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.

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 RI color development buffer solution in a 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. The liquid was removed and washed three times by adding 1ml RI washing solution per well, each time for 2 min. The filter membrane is kept in the washing liquid until the next experimental operation, and the soaking time of the sample in the RI washing liquid can not exceed 30 min.

Observation 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 mu LRI anti-quencher, covering with 18mm × 18mm cover glass, and directly performing microscopic examination or storing at-20 ℃.

2. The number of cancer cells with heterokaryon nuclei was counted by 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 9 excitation and emission wavelengths of fluorophores

Eighthly, judging and analyzing detection results

1. Positive cancer cell identification criteria

Cancer cells are enriched on the filter membrane, and the judgment standard of the cancer cells is as follows:

1) has an EGFR gene specific marker, and appears in the kit to show a red fluorescent signal spot under a Cy3 channel.

2) Has a KIT gene specific marker, and is expressed as green color on an Alexa Fluor 488 channel in the KIT

A fluorescent signal spot.

3) Has a B2M gene specific marker, and appears to show a purple fluorescent signal spot under a Cy5 channel in the kit.

4) Nuclear DAPI staining positive.

5) The shape of the cancer cell nucleus is irregular, the diameter is larger than 10 mu m, the diameter is 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; reading each sample separately according to the fluorescence signal intensity of target detection mRNA

The number of mRNA fluorescent points with corresponding colors of 50 cancer cells in the sample is calculated, and the average point number is calculated, and the specific result is as follows:

TABLE 10 sample test results (number of fluorescence signal points)

Sample(s)	Cy3	Alexa Fluor 488	Cy5	DAPI
					1	36	16	14	+
2	38	13	17	+
					3	35	12	8	+
4	37	17	12	+
					5	35	15	16	+
6	40	69	38	+
					7	48	74	40	+
8	38	68	34	+
					9	39	67	37	+
10	44	62	34	+
					11	8	26	11	+
12	9	31	3	+
					13	15	25	5	+
14	8	27	9	+
					15	17	31	13	+
16	23	11	9	+
					17	25	7	16	+
18	16	9	14	+
					19	29	19	8	+
20	24	12	10	+

From the results, the kit can realize detection, the detection results of the 5 samples from different cancer cell lines are similar, and the mRNA expression levels of the 4 samples from different cancer cell lines can be distinguished in detail, so that the kit has good repeatability and stable and reliable detection results.

EXAMPLE 4 detection comparison of three Signal amplification Components

1. Comparison of detection effects of three signal amplification Components in example 1

In the embodiment 1 of the present invention, three signal detection components are provided, and three signal amplification components can be detected, in this embodiment, EGRR gene mRNA detection is taken as an example, and three signal amplification schemes are designed, specifically, see table 11, to compare the detection effects of the three signal amplification components provided by the present invention. The relevant operation steps of this embodiment are shown in embodiments 1-3.

TABLE 11 marker gene detection kit consisting of three different probes

2. In this example, the same volume of uniform cell preservation solution derived from lung cancer cell line NCI-H1975 was taken as a detection sample, 30 samples (samples 21-50) were formed, 10 samples were used in each experimental group, and the detection method described in example 3 was used to perform detection and observation, where "-" or "+" used for DAPI staining of cell nuclei indicates whether fluorescence was detected; respectively reading the number of mRNA fluorescent points with corresponding colors of 50 cancer cells in each sample according to the fluorescent signal intensity of target detection mRNA, and calculating the average point number, wherein the specific result is as follows:

TABLE 12 detection results (number of fluorescent signal points) of three signal amplification schemes

From the above results, it can be seen that the detection can be realized by all three signal amplification components, however, as can be seen from the above results, the signal amplification system formed by the first-level signal amplification probe, the second-level signal amplification probe and the third-level signal amplification probe is used, the number of fluorescence signal points is more, the signal intensity is better, and the detection effect is better.

EXAMPLE 5 Effect of Capture probes

To verify the effect of the capture probe, this example uses EGFR and KIT gene markers as examples, and sets up a detection KIT containing the capture probe (Group 4) and not containing the capture probe (Group 5). Wherein the signal amplification probes selected by Group 4 are all selected from the signal amplification probes in example 1; in Group 5, the sequence of the primary signal amplification probe P3 for EGFR gene is selected from SEQ ID NO. 136-SEQ ID NO.140 in Table 13, the sequence of the primary signal amplification probe P3 for KIT gene is selected from SEQ ID NO. 141-SEQ ID NO.145 in Table 13, and the other probes are selected from the probes provided in examples 1-2.

The specific probe compositions are shown in Table 14. TABLE 13P 3 sequence of Primary Signal amplification Probe

TABLE 14 kit including and excluding capture probes

2. In this embodiment, uniform cell preservation solutions derived from lung cancer cell strains NCI-H1975 are respectively taken to have the same volume and are used as detection samples to form 20 samples (samples 51-70), 10 samples are used in each experimental group, and the detection method described in embodiment 3 is used to perform detection and observation, wherein "-" or "+" is used to indicate whether fluorescence is detected or not for DAPI staining of cell nuclei; respectively reading the number of mRNA fluorescent points with corresponding colors of 50 cancer cells in each sample according to the fluorescent signal intensity of target detection mRNA, and calculating the average point number, wherein the specific result is as follows:

TABLE 15 test results (number of fluorescence signal points)

From the above results, it can be seen that the signal amplification method of the present invention can also achieve direct detection of a target gene without using a capture probe, but from the above results, it can also be seen that the use of a capture probe followed by the signal amplification system of the present invention has a greater number of fluorescence signal points, a better signal intensity, and a better detection effect.

EXAMPLE 6 selection of the number of Capture probes for the target Gene

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

The invention provides a signal amplification system, and 5 capture probes are designed for different types of marker genes respectively (Table 7). In actual use, at least 1 corresponding capture probe can be selected for each marker gene to complete detection, and the specificity and the stability can meet the requirements.

In this embodiment, the number of capture probes for EGFR gene mRNA detection is selected as an example, see experimental groups 6-8, and 1, 3, and 5 capture probes are selected, and the first-level signal method probe, the second-level amplification probe, and the third-level signal amplification probe are selected as the signal amplification probes, so as to compare the detection effects.

TABLE 16 selection of different numbers of EGFR capture probes

2. In this embodiment, uniform cell preservation solutions derived from lung cancer cell strains NCI-H1975 are respectively taken to have the same volume and are used as detection samples to form 30 samples (samples 71-100), 10 samples are used in each experimental group, and the detection method described in embodiment 3 is used to perform detection and observation, wherein "-" or "+" is used to indicate whether fluorescence is detected or not for DAPI staining of cell nuclei; respectively reading the number of mRNA fluorescent points with corresponding colors of 50 cancer cells in each sample according to the fluorescent signal intensity of target detection mRNA, and calculating the average point number, wherein the specific result is as follows:

TABLE 17 comparison of the results of detection Using different numbers of capture probes (number of fluorescent signal points)

As can be seen from comparison of three groups of experiments, 1, 3 and 5 capture probes are used for detecting EGFR gene, and the specificity and the stability are good. When all 5 capture probes are used, the number of fluorescence signal points is more, the detection signal is stronger and more stable, and the detection effect is optimal.

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.

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 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 nucleic acid signal amplification detection kit is characterized by mainly comprising: at least one primary signal amplification probe and at least one secondary signal amplification probe for each target nucleic acid, or essentially comprising: at least one primary signal amplification probe and at least one secondary signal amplification probe for each target nucleic acid, and a capture probe, wherein

the secondary signal amplification probe sequentially comprises from the 5 'end to the 3' end: p5 sequence, spacer arm sequence, P6 sequence; the 3' end of the P6 sequence is further modified with a fluorescent group, the colors or emission wavelengths of the fluorescent groups aiming at different target nucleic acids are different, and the P5 sequence is 5 bp-15 bp;

the capture probes can be used for connecting target nucleic acid and primary signal amplification probes, and each capture probe comprises the following components from the 5 'end to the 3' end: the specific P1 sequence, the spacer arm sequence and the P2 sequence which can be complementarily matched and combined with target nucleic acid to be detected are different from each other aiming at the P2 sequences of different target genes, the P1 sequence is 15 bp-30 bp, and the P2 sequence is 8 bp-25 bp;

when the capture probe is contained in the kit, the P3 sequence contains at least one base fragment which is complementarily paired with the P2 sequence, and a spacer arm sequence is arranged between each base fragment which is complementarily paired with the P2 sequence; when the kit does not contain a capture probe, the P3 sequence is a specific base fragment which can be combined with the complementary pairing of the target nucleic acid to be detected;

2. A signal amplification detection kit is characterized by mainly comprising: at least one primary signal amplification probe, at least one secondary signal amplification probe, and at least one tertiary signal amplification probe for each target nucleic acid, or consisting essentially of: for each target nucleic acid there is at least one primary signal amplification probe, at least one secondary signal amplification probe, and at least one tertiary signal amplification probe, and a capture probe, wherein,

the capture probes are used for connecting target nucleic acid and primary signal amplification probes, each capture probe is provided with a specific sequence P1, a spacer arm sequence and a P2 sequence which can be complementarily paired and combined with the target nucleic acid to be detected from the 5 'end to the 3' end, and the P2 sequences aiming at different target genes are different; the sequence of the P1 is 15 bp-30 bp, and the sequence of the P2 is 8 bp-25 bp;

the three-stage signal amplification probe sequentially comprises from the 5 'end to the 3' end: the fluorescent probe comprises a P8 sequence, a spacer arm sequence and a P7 sequence, wherein the 5' end of the P8 sequence is also modified with fluorophores, the fluorophores aiming at different target nucleic acids are different, the colors of the corresponding fluorophores are different, or the emission wavelengths of the corresponding fluorophores are different, and the P7 sequence is 4 bp-10 bp;

3. The nucleic acid signal amplification detection kit of claim 1 or 2, wherein the P1 sequence of the capture probe is selected from SEQ ID NO. 121-SEQ ID NO.135, and the P2 sequence of the capture probe is selected from: SEQ ID No.101 to SEQ ID No. 120;

the sequence of the primary signal amplification probe P3 is selected from SEQ ID NO. 1-SEQ ID NO.20, and the sequence of P4 is selected from SEQ ID NO. 21-SEQ ID NO. 40;

the secondary signal amplification probe is selected from P5 sequences SEQ ID NO. 41-SEQ ID NO.60, and the P6 sequence is selected from SEQ ID NO. 61-SEQ ID NO. 80;

the sequence of the tertiary signal amplification probe P7 is selected from SEQ ID NO. 81-SEQ ID NO.100, the sequence of P8 is polyT, if more than one capture probe exists for each target nucleic acid, the P1 sequences are different, and the P2 sequences are the same or different.

4. The nucleic acid signal amplification detection kit according to claim 1 or 2, wherein the polyT is 3 to 10 tts.

5. The nucleic acid signal amplification detection kit according to claim 1 or 2, wherein the spacer arm sequence between the P4 sequence and the P3 sequence in the primary signal amplification probe is selected from the group consisting of 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 2-10T.

6. The nucleic acid signal amplification detection kit of claim 1 or 2, wherein when the capture probe is contained in the kit, the P3 sequence contains 2-5 base fragments complementary paired with the P2 sequence, and a spacer arm sequence is arranged between each base fragment complementary paired with the P2 sequence, and the spacer arm sequence is 4-10T.

7. The nucleic acid signal amplification detection kit according to claim 1 or 2, wherein the P4 sequence contains 2 to 4 base fragments complementarily paired with the P5 sequence, and a spacer sequence is provided between each base fragment complementarily paired with the P5 sequence, and the spacer sequence is 3 to 10T.

8. The nucleic acid signal amplification detection kit according to claim 1 or 2, wherein the sequence of P6 comprises 2-4 base fragments complementarily paired with the sequence of P7, and a spacer sequence is provided between each base fragment complementarily paired with the sequence of P7, and the spacer sequence may be 2-10T.

9. The nucleic acid signal amplification detection kit according to claim 1 or 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 AlexaFluor 488, and the fluorophores for different target nucleic acids are different from each other.

10. The nucleic acid signal amplification detection KIT according to claim 1 or 2, wherein the target nucleic acid is mRNA selected from at least one of the EGFR gene, the KIT gene and the B2M gene,

the P1 sequence aiming at the EGFR gene is selected from at least one of SEQ ID NO. 121-SEQ ID NO.125, and the P2 sequence is: SEQ ID NO.103, the P3 sequence is: SEQ ID NO.3, and the sequence of P4 is: the sequence of the P5 is SEQ ID NO.45, the sequence of the P6 is SEQ ID NO.67, and the sequence of the P7 is SEQ ID NO. 87;

the sequence of P1 aiming at the KIT gene is selected from SEQ ID NO. 126-SEQ ID NO.130, and the sequence of P2 is: SEQ ID NO.109, the P3 sequence is: SEQ ID NO.9, wherein the sequence of P4 is: 30, 50 is the sequence of P5, 74 is the sequence of P6, 94 is the sequence of P7; the P1 sequence aiming at the B2M gene is selected from SEQ ID NO. 131-SEQ ID NO.135, and the P2 sequence is: SEQ ID NO.115, the P3 sequence is: SEQ ID NO.15, the P4 sequence is: the sequence of the P5 is SEQ ID NO.55, the sequence of the P6 is SEQ ID NO.79, the sequence of the P7 is SEQ ID NO.99, and if more than one capture probe exists for each target gene, the P1 sequences are different, and the P2 sequences are the same or different.