CN114107446A

CN114107446A - Nucleic acid detection kit and detection method thereof

Info

Publication number: CN114107446A
Application number: CN202111547220.2A
Authority: CN
Inventors: 王岩; 林明芳; 王源舒; 杨浩; 江宁; 王寅; 吴�琳
Original assignee: Beijing Herui Exquisite Medical Laboratory Co ltd; Fujian Herui Gene Technology Co ltd
Current assignee: Beijing Herui Exquisite Medical Laboratory Co ltd; Fujian Herui Gene Technology Co ltd
Priority date: 2021-12-16
Filing date: 2021-12-16
Publication date: 2022-03-01

Abstract

The invention discloses a detection kit and a detection method of nucleic acid, and relates to the technical field of nucleic acid detection, wherein 8-150 nt of polynucleotide is connected with a nucleic acid sequence capable of being reversely complementary with target nucleic acid to form a capture probe for capturing specific RNA in a sample. Compared with the conventional probe capture method, the method can directly capture, separate and detect the specific target nucleic acid in the sample without extracting the total RNA. The invention is not limited by the type of the sample, can reduce the detection cost, simplify the library construction operation, and simultaneously improve the detection efficiency and stability of the specific RNA.

Description

Nucleic acid detection kit and detection method thereof

Technical Field

The invention relates to the technical field of nucleic acid detection, in particular to a nucleic acid detection kit and a detection method thereof.

Background

RNA is a genetic information carrier present in biological samples, and in addition to the most widely studied mRNA, many non-coding important functional RNAs have been discovered in recent years, including lncRNA, circRNA, snoRNA, miRNA, and the like. They can be used as diagnostic and prognostic biomarkers for various diseases, in particular extracellular free rna (cfrna) detectable in various biological fluids (such as plasma, serum, saliva and urine), and as non-invasive biomarkers for early cancer diagnosis, tumor progression monitoring, prediction of therapeutic response. Compared to DNA biomarkers, RNA biomarkers are more dynamic in reflecting cellular status and regulatory processes, and provide more information than DNA. Particularly, the detection rate of fusion genes carried out on RNA is higher than that of DNA layer detection. And some RNA with special structure, such as circRNA, has the potential of stable existence in plasma and/or serum.

The traditional RNA detection method comprises an RNA imprinting method and a PCR method, and the detection flux of the methods is low. The gene chip technology can greatly improve the detection flux, but the detection process is complicated and the cost is high. Finally, RNA sequencing technology has become a common approach in the field of RNA detection.

Currently, conventional RNA library construction methods are selected accordingly according to RNA quality and research direction. For example, investigation of mRNA and lncRNA will typically produce an enucleated ribosome transcriptome library, and this method will also yield a quantity of circRNA information. If the integrity of mRNA is good, mRNA pooling can be performed separately by polyA enrichment, but some of the IncRNA and circRNA information is lost. In addition, if more accurate analysis of circRNA is desired, a linear RNA removal step is added to achieve enrichment.

In addition to preparing transcriptome libraries, there are probe-based RNA capture sequencing methods that use probes to capture gene regions in transcriptome libraries for identification. The design of the probe is generally overlapped and fully covered, the number of the probes is large, the synthesis cost of the probe is increased, and the design of the probe is strict, for example, the probe for detecting the fusion gene must cover the fusion breakpoint, and the probe for detecting the circRNA must cover the reverse shearing site. In addition, the method requires a large input amount of RNA, and is particularly not suitable for detecting low-content circulating free RNA in plasma.

The detection method is characterized in that RNA is separated from a biological sample, total RNA is extracted from the biological sample by methods such as chromatographic column adsorption, magnetic bead adsorption or Trizol extraction in the existing RNA extraction method, then the RNA is correspondingly processed according to experimental requirements to obtain a product, and then downstream detection is carried out, so that the detection period and the labor cost are greatly increased. Moreover, the cost of removing some background RNA such as rRNA is high, and different species need to be distinguished by using different kits. The polyA enrichment for detecting mRNA depends on the integrity of the sample, and is particularly not suitable for degradable samples such as FFPE. In addition, the detection of the circRNA is also limited by unstable RNase R digestion, large enzyme activity batch difference, certain digestion capacity on partial circRNA and the like, so that the detection effect of the circRNA is unstable.

In view of this, the present application is presented.

Disclosure of Invention

The invention aims to provide a nucleic acid detection kit and a detection method thereof.

The invention is realized by the following steps:

in a first aspect, embodiments of the present invention provide a method for detecting a nucleic acid, comprising: mixing and hybridizing the capture probe combination with a sample to be detected to capture the target nucleic acid in the sample, and performing sequencing detection or PCR detection on the captured target nucleic acid; wherein the capture probe combination comprises a capture probe and a solid phase carrier. The capture probe is a single-stranded nucleic acid sequence and comprises a first sequence and a second sequence; the first sequence is a polynucleotide which mediates the combination with the solid phase carrier, and the length of the polynucleotide is 8-150 nt; the second sequence is a base sequence capable of being reverse complementary to the target nucleic acid sequence.

In a second aspect, the embodiments provide a kit for detecting a target nucleic acid, comprising: the capture probe combination used in the method for detecting nucleic acid described in the preceding examples.

In a third aspect, the embodiments of the present invention provide the use of a capture probe combination in the preparation of a kit for detecting a target nucleic acid, wherein the capture probe combination is used in the method for detecting a nucleic acid as described in the previous embodiments.

The invention has the following beneficial effects:

the invention forms a capture probe for capturing target nucleic acid in a biological sample by connecting a sequence reversely complementary with the target nucleic acid with 8-150 nt of polynucleotide, and the capture probe has the following advantages:

1. the capture probe is in a single-stranded structure, and a polynucleotide sequence of the capture probe does not form a complex secondary structure, so that the combination efficiency of the probe and the target nucleic acid is ensured.

2. The capture probe is not limited by the type of the sample, and is particularly suitable for some fluid samples such as degradable samples and plasma with low target nucleic acid content.

3. When the sample is subjected to complex extraction and lysis conditions such as protein degradation, the capture probe can also directly mediate the combination of the polynucleotide and the magnetic beads to complete the direct capture of the target nucleic acid.

4. The capture probe can directly capture specific RNA in a sample and establish a library without extracting total RNA, so that the interference of background RNA such as rRNA is avoided.

5. The capture probe can realize the capture and separation of target nucleic acid under the condition of less probe number, and has unique advantages particularly on the detection of fusion genes.

In conclusion, the detection method provided by the invention can reduce the detection cost, simplify the library construction operation process, and simultaneously remarkably improve the detection efficiency and the detection stability of the specific nucleic acid.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the results of the Qsep1 fragment analysis in example 5;

FIG. 2 shows the results of sequencing identification in example 5;

FIG. 3 shows the results of quality control of the library in validation example 1 using Qsep1 bioanalyzer;

FIG. 4 shows the results of determination of the concentration of PCR product in validation example 2;

FIG. 5 shows the results of the Qsep1 fragment analysis in validation example 2;

FIG. 6 shows the results of the Qsep1 fragment analysis in validation example 3;

FIG. 7 shows the results of the Qsep1 fragment analysis in validation example 4;

FIG. 8 shows the results of the Qsep1 fragment analysis in validation example 5;

FIG. 9 shows the results of the Qsep1 fragment analysis in validation example 6;

FIG. 10 shows the results of the Qsep1 fragment analysis in validation example 7;

FIG. 11 shows the results of the Qsep1 fragment analysis in validation example 8;

FIG. 12 shows the results of the Qsep1 fragment analysis in validation example 9;

FIG. 13 shows the results of the Qsep1 fragment analysis in validation example 10;

FIG. 14 shows the results of the Qsep1 fragment analysis in validation example 11;

FIG. 15 shows the results of the Qsep1 fragment analysis in validation example 12.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The embodiment of the invention provides a method for detecting nucleic acid, which comprises the following steps: and mixing and hybridizing the capture probe combination with a sample to be detected to realize capture and separation of the target nucleic acid in the sample, and performing sequencing detection or PCR detection on the captured target nucleic acid.

Specifically, the capture probe combination comprises a capture probe and a solid phase carrier. The capture probe is a single-stranded nucleic acid sequence comprising a first sequence and a second sequence.

The first sequence is a polynucleotide which mediates the combination with the solid phase carrier, and the length of the polynucleotide is 8-150 nt. In alternative embodiments, the length of the first sequence may be any length between 8nt and 150 nt. In some embodiments, the length of the first sequence may be 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt or 80nt, within which the effect of the assay is more excellent. Within this length range, the capture probes are able to bind to the magnetic beads more efficiently and capture the target nucleic acids. If the amount is less than 8bp, binding efficiency to magnetic beads may be low or non-specific binding may be caused.

Preferably, the polynucleotide of the first sequence is selected from any one of poly A, poly T, poly C and poly G. The solid phase carrier is immobilized or coated with oligonucleotide sequence, the first sequence is complementary combined with oligonucleotide on the solid phase carrier, for example, when the first sequence is poly A, the oligonucleotide on the solid phase carrier is oligo dT, when the first sequence is poly C, the oligonucleotide sequence on the solid phase carrier is oligo dG, and so on.

More preferably, the first sequence is any one of poly T, poly C and poly G. When the first sequence is poly A, the oligonucleotide on the solid phase carrier is oligo dT, and can be combined with mRNA with complete structure or partial other RNA containing poly A tail in the extraction process. When the first sequence is any one of poly T, poly C and poly G, the enrichment of RNA containing poly A tail, including housekeeping gene and other non-target genes, can be more effectively avoided, background interference which is difficult to remove can be caused if the subsequent second-generation sequencing is applied for analysis, and if the sequencing effect is to be achieved, the sequencing with extremely high depth flux can be only carried out, so that the cost is greatly increased.

The second sequence is a base sequence capable of being reverse complementary to the target nucleic acid sequence. In a preferred embodiment, the length of the second sequence is 10 to 120 nt; in this length range, the capture probe can bind to the magnetic bead more efficiently and capture the target nucleic acid, and if less than 10nt, the binding rate to the target nucleic acid may be low or the binding efficiency may not be high. In some embodiments, the second sequence may be 10nt, 20nt, 40nt, 60nt, 80nt, 100nt, or 120nt in length. More preferably, the length of the second sequence is 40-60 nt, and the detection effect is better in the range.

In an alternative embodiment, the captured target nucleic acid is subjected to sequencing detection or PCR detection, and the second generation sequencing and PCR detection method is suitable for RNA with the target nucleic acid length of more than 50nt, which can be derived from sequences existing in organisms, artificially synthesized sequences or from existing gene databases. Preferably, the target nucleic acid includes, but is not limited to, any one of mRNA, fusion gene RNA, circRNA and incrna, tRNA, rRNA, viral RNA, and ribozyme RNA.

The second sequence being capable of being reverse complementary to the target nucleic acid sequence means that it is capable of being reverse complementary to the target nucleic acid sequence or a part thereof, i.e.the length of the second sequence is ≦ the sequence length of the target nucleic acid. The second sequence is designed based on the target nucleic acid, and when the length of the target nucleic acid is more than 50nt, the second sequence can be designed according to any continuous 10-120 nt on the RNA sequence.

Preferably, when the target nucleic acid is a fusion gene RNA, the second sequence may be designed only at consecutive bases of the first gene of the fusion gene. The fusion gene is a chimeric gene formed by connecting two broken genes end to end and placing the two broken genes under the control of a set of regulatory sequences. The gene containing the kinase domain sequence or the protein functional sequence in the fusion gene is called a first gene, namely a tumor gene, and the gene containing the promoter sequence is called a second gene, namely a gene fused with the first gene; the connecting position of the first gene and the second gene is a fracture site.

Typically, the first gene type is fewer and the cleavage sites are more fixed, while the second gene type is more and the cleavage sites are randomly diverse. Therefore, in the conventional probe design, different probes are designed according to different fusion types, the probes generally cover the first gene and the second gene in a imbricate manner, the number of the probes is large, and the synthesis cost of the probes is increased. The probe of the invention can be designed only at the continuous bases of the first gene, and can realize the detection of the fusion gene with a plurality of fusion forms of the same first gene and different second genes.

The CircRNA is a non-coding RNA, and is a covalent closed loop formed by reverse variable splicing of mRNA precursors, and the splicing position of the loop is called a reverse splicing site. The conventional CircRNA probe is designed at a reverse cleavage site, and the design range of a probe sequence is limited. Preferably, when the target nucleic acid is a circRNA, the method provided by the invention can be designed to increase the design range of the probe sequence at any continuous base including the reverse cleavage site.

The invention mixes the capture probe and the sample to be detected, so that the second sequence on the capture probe can specifically identify and combine with the target nucleic acid (such as RNA) to form a capture probe-target RNA hybrid, and in the solid phase separation process, the hybrid is combined with the solid phase carrier through the mediation of the polynucleotide sequence on the capture probe, and is adsorbed on the surface of the solid phase carrier, and when the solid phase carrier is magnetic beads, the hybrid can be separated from the biological sample together with the target RNA through magnetic adsorption. The captured product can be used for subsequent PCR amplification or second-generation sequencing library construction after being digested by proper DNase. The detection method has the advantages of simplicity, convenience, rapidness and low cost, and total RNA does not need to be extracted, so that the factors of large sample demand and unstable background RNA removal in the traditional library construction process are avoided, and the high-efficiency detection of specific RNA is realized.

The detection method provided by the present invention is not directed to the diagnosis and treatment of diseases, but is directed to the qualitative or quantitative detection of RNA in a sample.

Optionally, in the sequence of the capture probe, the first sequence is located at the 3 'end and/or the 5' end of the second sequence.

In alternative embodiments, the capture probe sequence has a blocking modification at the 3' end, which may be at least one of a phosphorylation modification, a C3 or C6 Spacer modification, a dideoxycytosine nucleoside, and an amino group. In other alternative embodiments, the 3' end of the capture probe sequence may not be modified by blocking. In the case of blocking modification, non-specific amplification is hardly caused, and in the case of blocking modification, the capture efficiency of the capture probe is not reduced.

When the target nucleic acid is two or more kinds of RNA, the detection method comprises: the capture probes corresponding to each target nucleic acid are prepared into a probe pool for simultaneous capture detection of two or more RNAs, and the detection steps are as described in the previous examples.

In alternative embodiments, the solid support is selected from at least one of magnetic beads, nitrocellulose filter, nylon membrane, latex particles, gel microbeads, and microwell plates. Preferably, when the solid phase carrier is a magnetic bead, the magnetic bead is an oligo dN magnetic bead, such as an oligo dT magnetic bead. The oligo dT magnetic bead is a magnetic bead coupled with a segment of oligonucleotide-thymidylate (T) on the surface of the magnetic bead, and the oligo dT magnetic bead can be hybridized and combined with polyA on the capture probe through the oligo dT coupled on the surface of the oligo dT magnetic bead to complete the capture and separation of the target nucleic acid.

In an alternative embodiment, the capture probe combination and the sample to be tested may be mixed in any one of the following three ways: the capture probe can be mixed with the magnetic beads firstly and then mixed with the sample to be detected; or the capture probe can be mixed with the sample to be detected and then mixed with the magnetic beads; or the three are mixed at the same time. Preferably, the capture probe combination and the sample to be tested are mixed in the following manner: mixing the capture probe with a sample to be detected to enable a second sequence on the capture probe to identify and combine with target nucleic acid to form a capture probe-target nucleic acid hybrid; under the limitation of this mixing sequence, the binding efficiency of the capture probe to the target nucleic acid is significantly better than other mixing sequences or formats. The capture probe-target nucleic acid hybrid is then hybridized to a solid support mixture to complete enrichment for the target.

When the solid phase carrier is magnetic beads, the detection method further comprises the steps of adsorbing the magnetic beads by a magnetic device after the solid phase carrier is captured, removing supernatant, carrying out series of washing and purification to remove impurities adsorbed on the surfaces of the magnetic beads, and finally eluting by ribozyme-free water to obtain target nucleic acid.

Preferably, before mixing the sample to be tested with the capture probe, the detection method further comprises: and mixing the sample to be detected, the extracting solution and the proteinase K, and cracking the sample to release various RNAs in the sample. If the sample to be detected is a solid sample such as biological tissue, the sample to be detected and the extracting solution can be mixed and ground to be in a homogenate state, and then the proteinase K is added. And then diluting the sample by using a diluent, and purifying the sample by using a impurity washing solution.

The extracting solution comprises the following components: rnase inhibitors, surfactants, thiol reagents, and buffers. The RNase inhibitor can be one or more of guanidine hydrochloride, urea, guanidine isothiocyanate, vanadyl riboside complex and 8-hydroxyquinoline, and the action concentration range of the RNase inhibitor can be between 0.05 and 8 mol/L. The extractive solution contains 0.05-20% surfactant, which can be one or more of PEG200, Triton X-100, Tween 20, SDS, LDS, SLS, and NP-40. The sulfhydryl reagent can be one or more of dithiothreitol, cysteine, glutathione, 2-mercaptoethanol and tris (2-formylethyl) phosphine hydrochloride. The buffer solution for preparing the extracting solution is prepared from one or more of commercial Tris-HCl, NaCl, PBS and NaOH salt solutions, the concentration range of the buffer solution can be between 0.01 and 10mol/L, and the pH value range of the buffer solution is between pH 6.0 and 9.0.

The detection method also comprises diluting and purifying the cracked product.

The diluent for dilution is prepared from one or more salt solutions of commercial Tris-HCl, NaCl, LiCl, KCl, EDTA disodium, PBS and NaOH, the concentration range of the diluent can be between 0.001 and 5.0mol/L, and the pH value range of the diluent is between pH 6.0 and 9.0;

the impurity washing liquid for purification comprises the following components: the impurity washing solution I is prepared from one or more reagents of commercial Tris-HCl, LiCl, EDTA disodium, NaCl, NaOH, LDS, SDS, dithiothreitol and 2-mercaptoethanol, the concentration range of the impurity washing solution I can be 0.001-2mol/L, and the pH value range of the impurity washing solution I is 7.0-8.5. The impurity washing liquid II is prepared from one or more salt solutions of commercial Tris-HCl, LiCl, EDTA disodium, NaCl, NaOH, LDS and SDS, the concentration range of the impurity washing liquid II can be 0.001-1mol/L, and the pH value range of the impurity washing liquid II is 7.0-8.5. The impurity washing liquid III is prepared from one or more salt solutions of commercial Tris-HCl, LiCl, EDTA disodium, NaCl and NaOH, the concentration range of the impurity washing liquid III can be 0.001-1mol/L, and the pH value range of the impurity washing liquid III is 7.0-8.5. The impurity washing solution IV is prepared from commercial Tris-HCl, NaCl and MgCl₂KCl, dithiothreitol and 2-mercaptoethanol, wherein the concentration range of the salt solution is 0.001-0.1mol/L, and the pH value range of the salt solution is 7.0-8.5.

Preferably, the sample to be tested is selected from: a biological sample selected from the group consisting of a cell sample, a saliva sample, a whole blood sample, a serum sample, a plasma sample, a urine sample, a tissue sample, a microorganism sample, and a plant sample.

The embodiment of the invention also provides a detection kit for detecting the target nucleic acid, which comprises: a capture probe combination for use in a method of detecting nucleic acid as described in any preceding example.

Preferably, the detection kit further comprises: at least one of an extracting solution, proteinase K, a diluent and a washing solution. It should be noted that the components of the extracting solution, the diluting solution and the impurity washing solution provided in this embodiment are the same as those of the extracting solution, the diluting solution and the impurity washing solution adopted in the detection method described in any of the above embodiments, and are not described again here.

In addition, the embodiment of the present invention also provides an application of a capture probe combination in preparing a kit for detecting a target nucleic acid, wherein the capture probe combination is used in the method for detecting a nucleic acid described in any of the foregoing embodiments.

It is understood that the target nucleic acid in the application and detection kit is the same as any corresponding embodiment or implementation mode, and will not be described herein again.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Extracting solution: 1.00M guanidinium isothiocyanate, 61.54mM Tris-HCl pH 8.0, 1.00% SLS, 5.00mM DTT;

diluting liquid: 35mM Tris-HCl pH 8.0, 1.28M LiCl, 12.8mM EDTA;

washing impurity liquid I: 100mM Tris-HCl pH 7.5, 500mM LiCl, 10mM EDTA, 0.1% LDS, 5mM DTT;

washing impurity liquid II: 10mM Tris-HCl pH 7.5, 150mM LiCl, 1mM EDTA, 0.1% LDS;

washing impurity liquid III: 10mM Tris-HCl pH 7.5, 150mM LiCl, 1mM EDTA;

and (4) impurity washing liquid IV: 50mM Tris-HCl pH 8.3, 3mM MgCl₂，75mM KCl，10mM DTT。

Example 2

Two types of typical target RNAs were selected as study subjects in this example: 2 species of circRNA: circRNA-CDYL (hsa _ circ _0008285), circRNA-ZKSCAN1(hsa _ circ _0001727), mRNA of 2 fusion genes: EML (13) -ALK (20) and EML (06) -ALK (20), wherein the 4 RNAs are respectively provided with capture probes circ-ZKSCAN1-SP40, circ-CDYL-SP40 and ALK (20) -SP40, the polyA base sequence of the capture probes is 25nt, the polyA (first sequence) is positioned at the 3 end of the reverse complementary sequence (second sequence) and is a thick underlined part in the table 1, the length of the reverse complementary base sequence is 40nt, the reverse complementary sequence of the circRNA capture probe is designed at a non-reverse shearing site, and the reverse complementary sequences of the EML (13) -ALK (20) fusion gene and the EML (06) -ALK (20) fusion gene capture probe are designed at the first gene which is common to the EML (13) -ALK (20) exon. The specific sequence is shown in dotted line in Table 1, and the 3-end of the capture probe is not modified by blocking. The specific sequences of the probes are shown in Table 1.

TABLE 1 Capture probes

Example 3

The embodiment provides a method for capturing and extracting specific RNA of plasma or biological fluid, which comprises the following steps.

(1) Taking plasma, adding the extracting solution and proteinase K according to the sample volume shown in the table below, mixing uniformly, and placing in a metal bath at 65 ℃ for incubation for 20 min.

TABLE 2 extract and proteinase K volume

(2) According to the volume shown in the table below, the diluent and the nucleic acid probe were added, mixed well and incubated at 65 ℃ for 10 min. The beads were then added to the sample mixture at the volumes shown in the table below and incubated at room temperature for 30min with inversion.

TABLE 3 Diluent and bead solution volumes

(3) The sample was placed on a magnetic stand for adsorption and the supernatant was discarded.

(4) 1.0mL of the impurity washing solution I was added, after resuspension, the mixture was transferred to a 1.5mL centrifuge tube, and the centrifuge tube was placed on a magnetic frame for adsorption, and the supernatant was discarded.

(5) Adding 1.0mL of impurity washing liquid II, placing the mixture on a magnetic frame for adsorption after heavy suspension, and discarding the supernatant. The washing with the impurity washing solution II was repeated 1 time.

(6) 1.0mL of the impurity-washing solution III was added, and after resuspension, the mixture was placed on a magnetic frame for adsorption, and the supernatant was discarded. 0.2mL of the impurity washing solution III was added, resuspended, transferred to a PCR tube, placed on a magnetic rack for adsorption, and the supernatant was discarded.

(7) Adding 50 μ L of impurity washing solution IV, suspending, placing on a magnetic frame for adsorption, and discarding the supernatant.

(8) Referring to the volume of the following table, the eluent was added, heated at 75 ℃ for 2min, placed on a magnetic frame for adsorption, and then transferred to a new PCR tube to obtain RNA eluted products. The product can be used for downstream PCR and library construction.

TABLE 4 volume of eluent

Example 4

This example provides a method for capturing and extracting specific RNA from a tissue, comprising the steps of: 5mg of the tissue was weighed into a 5mL centrifuge tube, and 628. mu.L of the extract was added and ground to a homogenized state. Then, 2mL of ribozyme-free water and 80. mu.L of proteinase K were added thereto, and the mixture was cleaved on a metal bath at 65 ℃ for 20 min.

The subsequent steps were as in (2) to (8) in the procedure of example 3 except that the amount of the magnetic beads was 200. mu.L and the volume of the eluent was 52. mu.L.

Example 5

This example provides a method for detecting PCR after capturing specific RNA, which comprises the following steps:

(1) RNA extraction: 2mL of plasma was collected and extracted according to the procedure of example 3. Except that one or more capture probes were added for the purpose of the study.

(2) DNA digestion: DNA digestion was performed using a commercial kit HiScript III RT Supermix for qPCR (+ gDNA wiper) kit (Vazyme, R323-01), and the system and reaction were configured as shown in Table 5.

TABLE 5 DNA digestion reaction System and conditions

(3) And (3) cDNA synthesis: the cDNA was synthesized using HiScript III RT Supermix for qPCR (+ gDNA wiper) kit (Vazyme, R323-01), and the system and reaction were configured as shown in Table 6.

TABLE 6 cDNA Synthesis reaction System and conditions

Components	Volume/. mu.L
		5×HiScript III qRT Super Mix	4
The DNA-digested RNA described above	16
		Total volume	20
Temperature of	Time
		50℃	15min
85℃	5sec
		4℃	∞

(4) And (3) PCR detection: 2 μ L of cDNA was taken for PCR detection, amplified using a commercial kit, and the system and reaction were configured as shown in Table 7. Wherein F and R respectively correspond to upstream and downstream amplification primers of circRNA-CDYL and circRNA-ZKSCAN1, and the amplification primers comprise a primer sequence of Circ-CDYL-F1: 5'-ACCCACTAGTGCCTCAGGTG-3', respectively; Circ-CDYL-R1: 5'-AGCCTTTCCACCGAACCAAA-3'; Circ-ZKSCAN 1-F2: AGGTCTGGCTGCAGGAATACCG, respectively; Circ-ZKSCAN 1-R2: CTCACCTTTATGTCCTGGGAGGT, respectively; the sizes of the corresponding amplicon fragments are 142bp and 292bp respectively.

TABLE 7 PCR reaction systems and conditions

(5) Qsep1 band identification: taking 1 μ L of PCR product, using Qsep1 bioanalyzer to perform fragment analysis and rough concentration measurement, the final library may have a deviation of about 10bp in main peak distribution from the theoretical value due to the influence of Qsep1 mobility. Qsep1 quality control chart As shown in FIG. 1, the size of the band conforms to the theoretical value.

(6) First generation verification: and performing Sanger sequencing on the product, and detecting whether the amplification product contains a circular RNA characteristic structure, namely a reverse splicing site sequence. The Sanger sequencing results are shown in FIG. 2, and the reverse cleavage site sequences of the 2 circular RNAs were confirmed to be correct. Therefore, the capture probe provided by the invention can realize the capture of specific circular RNA in a sample.

Example 6

The embodiment provides a method for detecting library construction sequencing after specific RNA capture, which comprises the following steps:

(1) RNA extraction: 2mL of plasma was collected and extracted according to the procedure of example 3. Except that one or more capture probes are added for the purpose of the study.

(2) DNA digestion: DNA digestion was performed using a commercial kit HiScript III RT Supermix for qPCR (+ gDNA wiper) kit (Vazyme, R323-01) with the configuration system and reactions as in Table 8.

TABLE 8 DNA digestion reaction System and conditions

Components	Volume/. mu.L
		4×gDNA wiper Mix	5
RNA	15
		Total volume	20
Temperature of	Time
		42℃	2min
4℃	∞

(3) And (3) RNA recovery: RNA Clean Beads (Vazyme, N412-01) were used for recovery, and the specific RNA recovery procedure was as follows: 1. adding 1.8 XRNA Clean Beads, and incubating at room temperature for 5 min; 2. placing the PCR tube on a magnetic frame for magnetic attraction, and removing the supernatant; 3. keeping the PCR tube on the magnetic frame all the time, and adding 80% ethanol to wash for 2 times; 4. keeping the PCR tube on the magnetic frame all the time, opening the cover and air-drying the magnetic beads until no ethanol remains.

(4) Building a library: library construction was performed using a Vazyme library construction kit (Vazyme, NR611) by first adding a cleavage solution to the magnetic bead products in step (3), followed by the following procedures as described in the specification.

Verification example 1

Compared with the traditional column extraction method, the method specifically comprises the following steps:

(1) the same plasma was extracted using commercial column extraction with reference to kit instructions and the extraction procedure of the invention with reference to example 3. Both methods extracted 2mL of plasma and finally eluted with 16 μ L of ribozyme-free water. Two parallel controls were run for each experiment.

(2) The products extracted by the two methods are subjected to library building according to the steps (2) to (4) in the example 6. And finally finishing sequencing on an Illumina sequencing platform. The results are shown in Table 9.

TABLE 9 sequencing assay results

As can be seen from FIG. 3, the library insert was of a qualified size, with a single peak at around 370 bp.

After bioinformatics analysis of sequencing data, the results are shown in table 9, and in contrast, under the condition that rRNA is not removed, the method provided by the invention can effectively remove rRNA in a plasma sample, the residual ratio of the rRNA is less than 1%, while the residual ratio of the rRNA in a column extraction method is 57%, so that data amount is seriously wasted. Therefore, the capture and enrichment capacity of the invention to specific circRNA is more than 10 times of that of the conventional column extraction method, and the sensitivity is superior to that of the common column extraction method. It can thus be demonstrated that the method used in the present invention allows a significant enrichment of circRNA in plasma. Similarly, the extraction detection method developed by the invention is also suitable for other biological samples.

Therefore, compared with the traditional RNA extraction and circRNA library construction detection method, the method has the following advantages: 1. the method is suitable for other complex biological samples such as blood plasma or tissues and the like without changing the formula of an extraction reagent; 2. total RNA extraction is not required; 3. rRNA and other linear RNA removal is not required; 4. aiming at the requirements, enrichment detection of specific circRNA can be realized, and compared with the traditional column extraction method, the method not only can improve the sensitivity and reduce the library construction cost, but also simplifies the library construction operation process.

Verification example 2

And verifying the influence of the length of the capture probe polyA on the detection effect.

Eight experiments A-1, A-2, A-3, A-4, A-5, A-6, A-7 and A-8 are designed on the same plasma, and the extraction and detection method thereof refers to the steps (1) - (5) of example 5, except that probes of different circ-CDYL and circ-ZKSCAN1 are added in each experiment, the polyA length of the capture probe corresponding to each experiment is 50nt, 25nt, 20nt, 15nt, 10nt, 8nt, 6nt and 80nt, the reverse complementary sequences are the same sequences, and the lengths are 22-23 nt.

PCR products of the A-1 to A-8 panels were purified and recovered using AMPure XP Beads magnetic Beads (Beckman, A63882), and then subjected to Qubit recovery^TMAs a result of concentration measurement using 1 XDsDNA HS Assay Kits (Thermo, Q33231), as shown in FIG. 4, PCR concentration showed a significant decrease when the polyA length was < 20nt or when the polyA length was > 50 nt. Indicating a decrease in probe capture efficiency. The results of the fragment analysis and concentration determination of Qsep1 are shown in figure 5. Particularly, when the length of polyA is 15nt or less, the amplification band has a significantly reduced tendency, and when the length is less than 10nt, the objective product is substantially absent. Therefore, the polyA length of the probe is suitably 15nt to 80 nt. Between 20 and 50nt is a preferred length.

Verification example 3

And verifying the influence of the length of the reverse complementary sequence of the capture probe on the detection effect.

Five sets of experiments B-1, B-2, B-3, B-4 and B-5 were designed from the same plasma, with the extraction and detection methods referred to in steps (1) - (5) of example 5, except that probes of different circ-CDYL and circ-ZKSCAN1 were added to each set of experiments, with polyA lengths of 25nt for each probe and 22-23nt, 40nt, 50nt, 60nt and 80nt for the reverse sequences, respectively.

The results of the fragment analysis and concentration determination of Qsep1 are shown in figure 6. The length of the reverse sequence can be captured to target RNA between 20nt and 80nt, and the capture effect is optimal particularly when the reverse sequence is about 40nt to 50 nt.

Verification example 4

And verifying the influence of the position of the polyA on the capture probe on the detection effect.

Two sets of experiments, C-1 and C-2, were designed with the same plasma, and the extraction and detection methods were as described in steps (1) - (5) of example 5, except that each set of experiments added probes for different circ-CDYL and circ-ZKSCAN1, with the polyA sequences of the probes located at the 5 'and 3' ends of the reverse complement, respectively.

The Qsep1 band identification results are shown in FIG. 7. The difference between the amplified bands of group C-1 and group C-2 was not great, and it was found that the capture effect of the target circRNA was not great by the polyA sequence at the 3 'end and 5' end of the capture probe.

Verification example 5

And verifying the influence of the terminal modification condition of the capture probe on the detection.

Two sets of experiments, D-1 and D-2, were designed with the same plasma, and the extraction and detection methods were as described in (1) - (5) of example 5, except that each set of experiments was spiked with different probes for circ-CDYL and circ-ZKSCAN1, whose capture probes were modified with non-blocking and phosphate-blocking bases at the 3' end, respectively.

The Qsep1 band identification results are shown in FIG. 8. The amplified bands of the D-1 group and the D-2 group have little difference in intensity and have no obvious non-specific amplification, so that the 3 'end of the capture probe has no blocking modification and can not cause non-specific amplification, and the blocking modification of the 3' end can not cause the capture efficiency to be reduced. Therefore, the presence or absence of a blocking modification at the 3' end of the capture probe is applicable, and it is preferable to select a non-blocking modification of the probe in order to reduce the synthesis cost of the probe.

Verification example 6

The designed region of the reverse complement sequence of the capture probe in the circRNA was verified.

Two sets of experiments, namely E-1 and E-2, are designed for the same plasma, and the extraction and detection methods refer to the steps (1) to (5) of example 5, except that different capture probes are added in the extraction process, reverse complementary sequences of the capture probes are respectively designed at a non-reverse shearing site and a reverse shearing site, and specific sequences of the probes are shown in a table 10.

TABLE 10 Capture probes

The Qsep1 band identification results are shown in FIG. 9. For circRNA-ZKSCAN1, the capture effect of the two capture probes was comparable, while for circRNA-CDYL, the capture effect of the reverse sequence design of the capture probe at the cleavage site was significantly less than that at the non-cleavage site. It was thus demonstrated that the reverse complement of the capture probe can be designed at any position of the gene sequence of interest. Preferably, the design works better at non-cleavage sites.

Verification example 7

The specificity of the capture probe is verified.

Two groups of experiments of F-1 and F-2 are designed by adopting the same plasma, and the extraction and detection method refers to example 5(1) - (5), except that no capture probe is added in the extraction process of the F-2 group.

The results are shown in FIG. 10, and the F-1 group amplified the corresponding target band, while the F-2 group did not amplified the target band, indicating that the extraction detection system is specific.

Verification example 8

And verifying the effect of the capture probe in other extracting solutions.

Two groups of experiments, G-1 and G-2, are designed for the same plasma:

the extraction and detection methods of group G-1 were identical to steps (1) to (5) of example 4;

the extraction method of group G-2 is as follows: extraction was performed using a commercial extraction kit with oligo dT magnetic beads (NEB, S1550): (1) mixing 2mL of plasma with 628. mu.L of NEB extract, adding proteinase K, mixing well, and incubating in 65 deg.C metal bath for 20 min. (2) Adding the same amount of probe as that of G-1, mixing well, and incubating at 65 deg.C for 10 min. (3) Adding the same amount of magnetic beads as G-1, mixing well, and incubating at room temperature for 30 min. (4) The subsequent purification washing steps were performed as described in the specification.

The results are shown in FIG. 11. No band was amplified in group G-2, indicating that the capture probe only served as a capture in the lysates provided by the present invention.

Verification example 9

And verifying the application of the capture probe in mRNA fusion gene detection.

Two sets of experiments, H-1 and H-2, were designed with the same plasma, in which H-1 was incorporated into the EML4(13) -ALK (20) fusion reference and H-2 was incorporated into the EML4(06) -ALK (20) reference. Extraction and detection methods ALK (20) -SP40-3 was added during extraction in both experiments, and then PCR amplification was performed using primers corresponding to EML4(13) -ALK (20) and EML4(06) -ALK (20), respectively, with reference to steps (1) - (5) of example 5. The sizes of the corresponding amplification products of EML4(13) -ALK (20) are 180bp, and the sizes of the corresponding amplification products of EML4(06) -ALK (20) are 120bp and 153 bp.

The results are shown in FIG. 12, and both H-1 and H-2 amplified the target band, which shows that only one capture probe is needed to detect multiple fusion types of the fusion breakpoint in the first gene, i.e., ALK gene exon 20, and the second gene of multiple fusion breakpoints, i.e., EML4 gene, compared with the conventional capture probe method, the number of probes is greatly reduced.

Verification example 10

Verifying the reverse complementary sequence design region of the capture probe in the mRNA fusion gene.

Two experiments I-1 and I-2 were designed with the same plasma, in which an equal amount of EML4(13) -ALK (20) fusion reference was incorporated, and the extraction and detection methods were as described in steps (1) to (5) of example 5, except that different capture probes were added during the extraction in the two experiments, the second sequence of the capture probe in group I-1 was designed at the position of the exon 20 of the ALK gene, which is the first gene of the fusion gene, and the second sequence of the capture probe in group I-2 was designed at the position of the junction of the exon 20 of the ALK gene and the exon 13 of the EML4 gene. Then, PCR amplification was performed using primers corresponding to EML4(13) -ALK (20). The corresponding amplification product size is 180 bp.

The result is shown in FIG. 13, and both I-1 and I-2 amplify the target band, which indicates that the capture probe of the present invention is used, the second sequence is only designed at the exon of the first gene, and the capture of the fusion gene can be realized, compared with the conventional probe which is designed at the junction of two genes, the present invention can design the corresponding probe without knowing the position of the fusion breakpoint, and the design of the probe is simpler.

Verification example 11

The first sequence of the capture probe is verified.

Two sets of experiments, namely J-1 experiments and J-2 experiments, are designed on the same plasma, and the extraction and detection method refers to the steps (1) to (5) of example 5, except that a first sequence added with a capture probe in the extraction process is PolyT, and a solid phase carrier is Oligo dA magnetic beads. The capture probe was added to group J-1 and not to group J-2. The specific sequence is shown in Table 11.

TABLE 11 Capture probes

The Qsep1 band identification results are shown in FIG. 14. The J-1 group amplifies a corresponding target band, and the J-2 group does not amplify a target band, which shows that when the first sequence is any one of poly T, poly C and poly G, the oligo dA, dG or dC magnetic beads on the solid phase carrier can realize the capture of the target nucleic acid in the sample, and the extraction detection system has specificity.

Verification example 12

And (3) verifying the removal effect of the non-target gene.

Two sets of experiments, K-1 and K-2, were designed from the same plasma, and the extraction and detection methods were as in steps (1) - (5) of example 5, except that after extraction, the two sets of experiments were PCR amplified using the primers corresponding to the ACTB gene (non-target gene), and the corresponding amplicon fragment size was 370 bp. In the K-1 group, a first sequence added with a capture probe is PolyA in the extraction process, and a solid phase carrier is Oligo dT magnetic beads. The first sequence in group K-2 is PolyT, and the solid phase carrier is Oligo dA magnetic beads.

The results are shown in FIG. 15. The corresponding target band is amplified by the K-1 group, and the target peak signal of the K-2 group is obviously weakened, which shows that when the first sequence is any one of poly T, poly C and poly G, the enrichment of the non-target gene RNA with the PolyA structure can be obviously weakened relative to the capture probe with the first sequence being the PolyA.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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Claims

1. A method for detecting a nucleic acid, comprising: mixing and hybridizing the capture probe combination with a sample to be detected to capture the target nucleic acid in the sample, and performing sequencing detection or PCR detection on the captured target nucleic acid;

wherein the capture probe combination comprises a capture probe and a solid phase carrier;

the capture probe is a single-stranded nucleic acid sequence and comprises a first sequence and a second sequence;

the first sequence is a polynucleotide which mediates the combination with the solid phase carrier, and the length of the polynucleotide is 8-150 nt;

the second sequence is a base sequence capable of being reverse complementary to the target nucleic acid sequence.

2. The method for detecting a nucleic acid according to claim 1, wherein the polynucleotide is selected from any one of polyA, polyT, polyC and polyG;

preferably, the length of the first sequence is 8-150 nt;

preferably, the length of the first sequence is 20-50 nt;

preferably, in the sequence of the capture probe, the first sequence is located at the 3 'end and/or the 5' end of the second sequence.

3. The method for detecting a nucleic acid according to claim 1, wherein the length of the second sequence is 10 to 120 nt;

preferably, the length of the second sequence is 40-60 nt.

4. The method for detecting a nucleic acid according to claim 1, wherein the solid support is at least one selected from the group consisting of magnetic beads, nitrocellulose filter, nylon membrane, latex particles, gel microbeads and microwell plates;

preferably, when the solid phase carrier is a magnetic bead, the magnetic bead is an oligo dN magnetic bead, and N is selected from any one of A, T, C and G.

5. The method for detecting nucleic acid according to any one of claims 1 to 4, wherein the capture probe combination and the sample to be detected are mixed in a manner that:

mixing the capture probe and a sample to be detected to enable a second sequence on the capture probe to identify and combine with the target nucleic acid to form a capture probe-target nucleic acid hybrid;

hybridizing the capture probe-target nucleic acid hybrid with the solid support;

preferably, before mixing the sample to be tested with the capture probe, the detection method further comprises: and mixing the sample to be detected, the extracting solution and the proteinase K, and performing cracking, dilution and purification on the sample.

6. The method for detecting a nucleic acid according to any one of claims 1 to 4, wherein the target nucleic acid is RNA having a length of 50nt or more;

preferably, the target nucleic acid is selected from any one of mRNA, fusion gene RNA, circRNA, lncRNA, tRNA, rRNA, viral RNA, and ribozyme RNA;

preferably, when the target nucleic acid is two or more kinds of RNA, the detection method comprises: the capture probes corresponding to each target nucleic acid are prepared into a probe pool for simultaneously capturing and detecting two or more RNAs.

7. The method for detecting a nucleic acid according to any one of claims 1 to 4, wherein the sample to be detected is selected from the group consisting of: any one of a cell sample, a saliva sample, a whole blood sample, a serum sample, a plasma sample, a urine sample, a tissue sample, a microorganism sample, and a plant sample.

8. A kit for detecting a target nucleic acid, comprising: a capture probe set for use in a method for detecting a nucleic acid according to any one of claims 1 to 7.

9. The kit for detecting RNA of claim 8, further comprising: at least one of an extracting solution, proteinase K, a diluent and a washing solution.

10. Use of a capture probe combination for the preparation of a kit for the detection of a target nucleic acid, wherein the capture probe combination is the capture probe combination used in the method for the detection of a nucleic acid according to any one of claims 1 to 7.