Patent Application
20230212660
The present disclosure relates to the field of biotechnology, in particular, to a combination product for detecting DNA.
After decades of development and rapid maturity in recent years, nucleic acid detection technology has been widely used in various aspects, such as infectious disease pathogen detection (including bacteria, mycoplasma, viruses, etc.), tumor mutation detection, genetic disease detection, and individual genotypes detection, etc. The nucleic acid detection technology has much better sensitivity and specificity in many application fields, such as infectious disease diagnosis, than colloidal gold/immunofluorescence method, and it is the gold standard for various diagnoses. Among the numerous nucleic acid detection techniques, the probe hybridization-based detection technology is well known and can be used in combination with a variety of techniques.
The existing probes usually have no capability of signal amplification, but problems such as inconvenient detection, low sensitivity, and so on.
The present disclosure relates to a combination product for DNA detection, including a ribonuclease H II and a probe, wherein the probe is a single-stranded probe, and has a sequence which can be partially or entirely complementary to a target DNA molecule to be detected, and one or more RNA bases are embedded in a complementary region of the probe and divide the complementary region of the probe into at least two segments, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 DNA bases and base substitutions that do not interfere with a hybridization of a nucleic acid strand in total.
The present disclosure also relates to a kit including the combination product as described above.
The present disclosure also relates to a use of the combination product as described above in the detection of SNP, wherein the probe has one RNA base.
The present disclosure also relates to a use of the combination product as described above in the detection of a target DNA obtained from amplification with a LAMP, NEAR or RPA primer.
The beneficial effects of the present disclosure are as follows:
(1) the cleaved probes have an increased percentage in the complementary strand at the same concentration and thus an intensified signal;
(2) the product fragment is shorter after cleavage with the probe (≤13 bases), and easier to dissociate from the complementary strand, avoiding extension of the cleaved product as a primer and blockage of the complementary strand, so that the complementary strand binds to a new probe, thereby improving the sensitivity of the system;
(3) the system with good compatibility may be used in combination with various isothermal amplification methods such as LAMP, NEAR, RPA, etc., and can be incorporation with a reporting system before the reaction, so as to achieve result output without additional operations and reactions; and
(4) in contrast with fluorescent dyes and color indicators commonly used in other isothermal amplification, the probe can ensure the specificity for the detection result by identifying the amplification product when used in combination with isothermal amplification methods such as LAMP, NEAR, and RPA, etc.
To describe the technical solutions of the embodiments of the present disclosure or the prior art more clearly, the following briefly introduces the accompanying drawings required for describing the embodiments or the prior art. Apparently, the accompanying drawings in the following description show some embodiments of the present disclosure, and persons of ordinary skill in the art may also derive other drawings from these accompanying drawings without creative efforts.
Reference to the embodiments of the present disclosure will now be provided in detail, one or more examples of which are described below. Every example is provided as an explanation rather than a limitation of the present disclosure. In fact, it is apparently to those skilled in the art that various modifications and changes may be made to the present disclosure without departing from the scope or spirit of the present disclosure. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.
All technical and scientific terms used herein have the same meaning as commonly understood by skilled person in the art to which this disclosure belongs, unless otherwise defined. The terms used in the specification of the present disclosure herein are for the purpose of describing specific embodiments only and are not intended to limit the present disclosure. The term “and/or” used herein includes any and all combinations of one or more of the associated listed items.
The present disclosure relates to a combination product for DNA detection, including a ribonuclease H II and a probe,
wherein, the probe is a single-stranded probe and has a sequence which can be partially or entirely complementary to a target DNA molecule to be detected, and one or more RNA bases are embedded in a complementary region of the probe and divide the complementary region of the probe into at least two segments, each of which independently has DNA bases and base substitutions that do not interfere with a hybridization of a nucleic acid strand that is less than or equal to 13 in total.
Ribonuclease HII (RNase HII) is an endoribonuclease that is suitable for cleaving the 5′ end of a ribonucleic acid inside a double-stranded DNA, resulting in a 5′ phosphate and a 3′ hydroxyl end.
The term “base substitution” refers to a structure that contains no base, and connects the upstream and downstream of the nucleic acid strand to maintain the integrity of the probe as a whole, without interfering with the hybridization of the nucleic acid strand. For example, when a polymorphic site that does not need to be detected appears in a target sequence, a degeneracy analysis may be achieved by replacing the base in the probe at the corresponding position with a base substitution. The common base substitutions are a deoxynucleotide spacer (dSpacer) or a C3 spacer (Spacer). There may be 1, 2, 3, 4, 5, or 6 base substitutions.
There may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or more RNA bases, which may be dispersed in DNA bases individually or in continuous fragments (each of the contiguous fragments contains at least two RNA bases), and further may be inserted fully and continuously into the middle of the probe.
In some embodiments, each segment has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 DNA bases or base substitutions.
In some preferred embodiments, the RNA bases are distributed in the probe as discrete as possible to increase the efficiency of the cleaved product being dissociating into single strands.
In some preferred embodiments, RNase HII reacts at a temperature in a range from 55° C. to 65° C., and the RNA bases are distributed in the probe at intervals of 3 to 8 bases.
In some embodiments, the probe includes only RNA bases and DNA bases.
In some embodiments, each segment has the DNA bases or base substitutions between 0 and 10.
In some embodiments, each segment has the DNA bases or base substitutions of less than or equal to 6.
The fragmentation of the probe may be detected by methods known to those skilled in the art, such as observing the size of the bands of a nucleic acid fragment by means of electrophoresis and the like. In some preferred embodiments, the probe is labeled with a label to determine whether the probe is cleaved.
In some embodiments, the probe has a Tm value expressed as a temperature that is greater than or equal to a value obtained by subtract 10° C. from the reaction temperature of the cleavage reaction with the ribonuclease H II.
In some embodiments, at least two segments are labeled with different detectable identification elements, which are a specific nucleic acid sequence or a label labeled on bases and/or nucleic acid backbones. The label produces a detectable signal when the probe is cleaved at the RNA base with the ribonuclease H II.
In some embodiments, the label is labeled on bases of the probe at both ends.
In some embodiments, the label includes a fluorophore and a quencher, and at least one fluorophore and at least one quencher are located in different segments.
, The fluorophore will be separated from the quencher upon the cleavage of the probe, which results in an enhanced fluorescence, indicating the presence of a target nucleic acid. The fluorophore may include FAM, HEX, CY3, CY5, FITC, and the like, and the quencher may include BHQ1, BHQ2, BHQ3, TAMRA, Dabcyl, Dabsyl, and the like.
In some preferred embodiments, multiple modifications may be added on multiple RNA bases to achieve a reduced background and an enhanced signal. For example, “BHQ1-RNA base-FAM-RNA base-BHQ1” is labeled along the probe in 5′ to 3′ direction to reduce the negative background signal.
In other embodiments, the probe has an end fixed on the surface of a solid phase carrier, or bound to the solid phase carrier after the reaction is completed, and has the other end is coupled with a signal substance.
Whether the probe is cleaved is determined by the presence or absence of the signal substance.
The solid phase carrier may be a material, such as polystyrene, plastic, cellulose, polyacrylamide, polyethylene, polypropylene, cross-linked dextran, glass, silicone rubber, agarose gel, etc. The carrier may be in a form of test tubes, EP tubes, multiwell plates (especially ELISA plates), wells of microreaction plates, beads (especially magnetic beads), small discs, etc.
In some embodiments, the combination product further includes a test strip, wherein, the test strip includes a sample pad, a reaction membrane provided with a quality inspection region and a detection region along a liquid flow direction, and an absorption pad:
the label includes a first label and a second label;
the sample pad is coated with a signal substance, which is labeled with a first anti-label against the first label;
the quality inspection region is constantly coated with a second anti-label against the second label;
the detection region is constantly coated with a secondary antibody against the first anti-label;
the first label and the first anti-label, the second label and the second anti-label can form different label-anti-label complexes;
alternatively, the test strip comprises a sample pad, a reaction membrane provided with a quality inspection region and a detection region along a liquid flow direction, and an absorption pad;
the label includes a first label and a second label;
the sample pad is coated with a signal substance, which is labeled with a first anti-label against the first label;
the detection region is constantly coated with a second anti-label against the second label;
the quality inspection region is constantly coated with a secondary antibody against the first anti-label;
the first label and the first anti-label, the second label and the second anti-label can form different label-anti-label complexes.
“Secondary antibody” refers to an antibody against the first anti-label.
In some embodiments, the signal substance is selected from the group consisting of a fluorophore, a colorimetric label, a quantum dot, a colloidal gold, an alkyne group for Raman diffraction imaging, a cycloalkene for click reaction, a polymer-labeling initiating group, a polypeptide/protein molecule, an LNA/PNA, a non-natural amino acid and analog thereof, a non-natural nucleic acid and analog thereof, and a nanostructure; the nanostructure includes an inorganic nanoparticle, a NV-center, an aggregation/assembly-induced luminescent molecule, a rare-earth ion ligand molecule, and a polyoxometalate.
The degradation products from the probe are detected by means of immunochromatography, and the integrity of the probe can be indicated by a signal substance such as colloidal gold or quantum dots.
In some embodiments, the combination of the label/anti-label in the label-anti-label complexes is selected from the group consisting of biotin or derivative thereof/streptavidin, biotin or derivative thereof/avidin, biotin or derivative there of/neutravidin, hapten/antibody, antigen/antibody, receptor/ligand, digoxigenin/digoxigenin ligand, carbohydrate/agglutinin, and polynucleotide/complementary polynucleotide;
wherein the derivative of biotin is any one of D-biotin, activated biotin, biocytin, ethylenediamine biotin, cadaverine biotin and desthiobiotin; and wherein the antigen and hapten may be a polypeptide, or protein or protein subunit, and such protein or protein subunit may also be an antibody or an antibody fragment.
The term “antibody” includes polyclonal antibodies and monoclonal antibodies. The term “antibody fragment” includes the antigen binding fragments of these antibodies, including Fab, F(ab′)2, Fd, Fv, scFv, bispecific antibodies and the minimum recognition unit of antibodies, as well as single-chain derivatives of these antibodies and fragments, for example, scFv-Fc, etc. The type of the antibody may select from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. In addition, the term “antibody” includes naturally-occurring antibodies and non-naturally-occurring antibodies, including, for example, chimeric, bifunctional, humanized antibodies and human antibodies, and related synthetic isoforms thereof.
In some preferred embodiments, the nucleic acid sequences at the two flank of the RNA base of the probe are labeled with biotin and digoxigenin respectively, the gold nanoparticles are labeled with digoxigenin monoclonal antibody, and the streptavidin is immobilized on the nitrocellulose membrane. The probe is detected for its integrity by means of immunochromatography. The presence of the target leads to cleavage of the probe to be cut, and eventually the elimination of the colour on in the streptavidin line.
In some embodiments, the 3′ end of the probe includes a non-extendable blocking moiety.
The blocked group may be selected from amino, phosphorylation, spacer, biotin, and other modification, and blockage may be achieved by labeling with a fluorophore, a quencher, etc., at the 3′ end. It is also possible to extend the probe at one ends of the 5′ and 3′ ends, with introduced 3 to 8 base sequences that are reverse complementary to the other end, or extend the probe at both of the 5′ and 3′ ends to introduce 3 to 8 base sequences that are reverse complementary to the other end, making the probe itself form a hairpin structure, and the two ends are labeled with a fluorophore and a quencher to reduce the negative background signal.
In some embodiments, blockage of the 3′ end is achieved by introducing a nucleic acid sequence that is unrelated to the target sequence at the 3′ end of the probe.
In some embodiments, the spacer modification is any one selected from ethylene glycol, C9 spacer (Spacer 9), C18 spacer (Spacer 18), dideoxy spacer [1′, 2′-Dideoxyribose (dSpacer)], or C3 Spacer (Spacer 3).
In some embodiments, the spacer modification is the C3 spacer.
In some embodiments, the probe has one RNA base and has a length of less than 25 nt.
In some embodiments, the combination product further includes a LAMP primer, a NEAR primer, or an RPA primer.
According to a further aspect of the present disclosure, the present disclosure also relates to a kit including the combination product as described above.
In some embodiments, the kit further comprises a ribonuclease HII digestion buffer, a DNA polymerase, dNTPs, an amplification buffer and other components.
The present disclosure further relates to a use of the combination product as described above in the detection of SNP.
When used for SNP detection, the probe has one RNA base which is paired with the SNP site (paired with the wild type or mutant type).
RNase HII targets double-stranded DNA, which has certain requirements on the base pairing in the double-strand. The mismatched bases in the double-strand will affect the cleavage efficiency. The closer the mismatched bases are to the cleaving position, the lower the cleavage efficiency. When the RNA base position is mismatched, the cleavage efficiency is only about 1/10 of the level. Benefit from this and the presence of signal amplification in the reporting system, a single DNA single-stranded molecule may induce the cleavage of multiple probes, and the signal difference between the perfect match and the mismatch may be further increased by amplification, and eventual results may effectively distinguish differences between single bases and allows SNP detection.
In some embodiments, in SNP detection, the reaction temperature of the cleavage reaction with the ribonuclease H II is less than or equal to the temperature shown in the Tm value plus 10° C.
The system includes multiple probes for different alleles of the same SNP site. Each probe is labeled with a fluorophore corresponding to a different fluorescence channel, and with a quencher pairing with the fluorophore, which enables genotyping of samples in a single tube reaction.
The present disclosure also relates to a use of the combination product as described above in the detection of a target DNA obtained from amplification with a LAMP, NEAR or RPA primer.
The reporting system may follow the existing amplification method, to detect the end-point product. For example, NEAR, LAMP, RPA and other primers are directly added to the reaction system including RNase HII and probe for reaction and result determination. The reporting system, on the premise of being compatible with the amplification, may be directly incorporated into the amplification technology to identify and cleave the probe as the occurrence of the target nucleic acid, thereby achieving the fastest and easiest detection. The single-stranded target DNA produced by amplification may be directly detected, and for the system where the product is double-stranded DNA, the probe may be bond to the target sequence by denaturizing, annealing, etc., and thereby allows detection.
In some embodiments, the above applications specifically includes: 1. amplifying nucleic acid; 2. adding part or all of the reaction product to the RNase HIT system after the reaction is completed, and melting the double-stranded DNA product by thermal denaturation, then followed by probe cleavage and detection.
In some embodiments, in the above applications, RNase HII and the probe are added to the amplification system, the amplification and probe cleavage are performed simultaneously, and then the probe is detected for its integrity. For example, the signal is detected in real time by fluorescence, or the end-point fluorescence value is determined after the reaction is completed, or the integrity of the probe is confirmed by means of immunochromatography and the like after the reaction is completed.
In some embodiments, in the above application, the reaction is carried out at a temperature in a range from 35° C. to 45° C., and further preferably, the RNase HIT derived from E. coli is used.
In some embodiments, the reaction temperature of the above application is in a range from 55° C. to 65° C., using a thermophilic RNase, such as the RNase HII derived from Thermus thermophilus or Pyrococcus abyssi.
In some embodiments, each segment has the DNA bases or base substitutions of less than or equal to 6, and the amplification is performed at a temperature in a range from 35° C. to 45° C. (which may be 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., or 44° C.).
By shortening length of the DNA segment, the reporting system can operate at a lower reaction temperature, and can be used in lower temperature amplification systems such as RPA, and has a wide range of applications.
The principle provided by the present disclosure is as follows: RNase HII may specifically recognize the RNA bases in the DNA double-strand, and break the phosphodiester bond connecting the DNA bases in the 5′ direction of the RNA bases, resulting in a nick in the DNA double-strand in the 5′ direction of the RNA base. In organisms, DNA repair is triggered by this cleavage, which can remove RNA bases in the double-stranded DNA and ensure the accuracy for the genetic information. Based on the characteristic of RNase HII in strictly pairing with substrates, a single-stranded probe is designed in the present disclosure with one or more RNA bases embedded in the probe, and RNase HII is applied to the reporting system of the amplification system. Since RNase HII can only recognize and cleave double strands, it will not act on single-stranded probes. The probe will hybridize with a single-stranded DNA that is complementary thereto (if any) in the environment at a suitable temperature and be cleaved at a specific position by RNase HII. As the melting temperature of the cleaved probe drops, the stable binding with complementary single-stranded DNA cannot be kept and the probe will be dissociated from the complementary single-stranded DNA. The complementary single-stranded DNA may bind to a new probe and a new cycle starts to achieve signal amplification. In the process, the detection result is obtained by cleaving with RNase HIT, which converts the input of single-stranded DNA complementary to the probe into the output of probe breakage, base-pairing to ensure the specificity of the generated product and to enable the signal amplification, and detecting the breakage of the probe.
The embodiments of the present disclosure will be described in detail below in combination with examples.
The effect of probe length on the detection effect was tested at different reaction temperatures.
1. Design of Primer and Probe
In Table 1, Complement is the target sequence, Probe-Tm-59.5 and Probe-Tm-54.8 are two probes with different Tm values, have sequences which are both reverse complementary to Complement. In a direction from 5′ to 3′, the underlined bases indicated that they were modified, in which T in the Probe-Tm-59.5 probe was modified with a quencher, G was a RNA base, and C was modified with a fluorescent reporter group; T in the Probe-Tm-54.8 probe was modified with a quencher, G was a RNA base, and C was modified with a fluorescent reporter group.
2. Cleavage Reaction
25 μL of reaction system: 20 mM Tris-HCl, 70 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween 20, 9 mM MgSO4, 1 μg RNase HII, 0.02 μM Complement, 0.2 μM Probe-Tm-59.5 or Probe-Tm-54.8.
Reaction conditions: 65° C./60° C., 1 min, 35 cycles (fluorescence was collected every 1 min).
3. Results Analysis
The result is shown in
A variety of probes were designed for a target sequence. The probes have the same basic sequences, except DNA base segment length separated by RNA bases. Probe: target sequence was added at a molar concentration of 100:1, and cleavage was performed in the presence/absence of DNA polymerase, respectively, to compare the effect of DNA base segment length on the cleavage.
1. Design of Primer and Probe
In Table 2, the probe-segments≤2, ≤5, ≤10, ≤13, and ≤15 are five probes with different DNA base segment lengths separated by RNA bases, and have sequences which are all reverse complementary to the target sequence. In a direction from 5′ to 3′, the underlined bases indicated that they were modified, wherein A and U of probe-segment≤2 were RNA bases; A and U of probe-segment≤5 were RNA bases; A and U of probe-segment≤10 were RNA bases; A of probe-segment≤13 was RNA base; C of probe-segment≤15 was RNA base; in addition, the 5′ ends of the five probes were all modified with a quencher, and the 3′ ends were all modified with a fluorescent reporter group and a C3 spacer.
2. Cleavage Reaction
25 μL reaction system: 20 mM Tris-HCl, 70 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween 20, 1.7 mM dNTPs, 9 mM MgSO4, 8 U Bst 2.0 WarmStart, 1 μg RNase HII, 20 nM target sequence, five probes each at 200 nM: probe-segment≤2, probe-segment≤5, probe-segment≤10, probe-segment≤13, probe-segment≤15.
Reaction conditions: 60° C., 1 min, 60 cycles (fluorescence was collected every 1 min).
3. Result Analysis
The result is shown in
The effect of probes with different DNA base segment length on cleavage was compared at 37° C.
1. Design of Primer and Probe
In Table 3, Tm-645 is the target sequence, and N-probe-1R and N-probe-3R are probes with different DNA base segment lengths separated by RNA bases, and have sequences which are all reverse complementary to the target sequence. In a direction from 5′ to 3′, the underlined bases indicated that they were modified, wherein the 5′ end of N-probe-1R was modified with a fluorophore, U was an RNA base, and the 3′ end was modified with both quenching reporter group and C3 spacer; the 5′ end of N-probe-3R was modified with a fluorophore, A, U, and A were RNA bases, and the 3′ end was modified with both quenching reporter group and C3 spacer.
2. Cleavage Reaction
25 μL of reaction system: 20 mM Tris-HCl, 70 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween 20, 1 μg RNase HII, 20 nM Tm-645, 200 nM N-probe-1R or N-probe-3R.
Reaction conditions: 37° C., 1 min, 15 cycles (fluorescence was collected every 1 min).
4. Result Analysis
The result is shown in
The target was amplified by LAMP with a set of six primers, and the generation of specific products was reported in real time with RNase HII and probes in the system.
1. Design of Primer and Probe
In Table 4, SARS-CoV-2-B3, SARS-CoV-2-BIP, SARS-CoV-2-LB, SARS-CoV-2-F3, SARS-CoV-2-FIP, SARS-CoV-2-LF were amplification primers for SARS-CoV-2, and SARS-CoV-2-Probe-F was a detection probe for SARS-CoV-2. In a direction from 5′ to 3′, the underlined bases indicated that they were modified, T of the probe SARS-CoV-2-Probe-F was modified a quencher, A was an RNA base, C was modified with a reporter fluorophore, and the 3′ end was modified with a C3 spacer.
2. Isothermal Amplification Reaction
25 μL of reaction system: 20 mM Tris-HCl, 70 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween 20, 9 mM MgSO4, 1.7 mM dNTPs, 8 U Bst 2.0 WarmStart, 1.6 μM SARS-CoV-2-BIP/SARS-CoV-2-FIP, and 0.4 μM SARS-CoV-2-LB/SARS-CoV-2-LF/SARS-CoV-2-B3/SARS-CoV-2-F3. The detection system of RNase HII further included 1 μg of RNase HII, and 0.2 μM of SARS-CoV-2-Probe-F, and the system for the dye method further included 0.5×LAMP fluorescent dye.
Reaction conditions: 63° C., 1 min, 40 cycles (fluorescence was collected every 1 min).
3. Results Analysis
The result is shown in
For the same set of LAMP amplification primers, probes with the same target sequence but different DNA segment lengths were designed respectively, and compared for effect of segment length on the reaction sensitivity was.
1. Design of Primer and Probe
In Table 5, SARS-CoV-2-B3, SARS-CoV-2-BIP, SARS-CoV-2-LB, SARS-CoV-2-F3, SARS-CoV-2-FIP, SARS-CoV-2-LF were amplification primers for SARS-CoV-2, and SARS-CoV-2-Probe-12 and SARS-CoV-2-Probe-6 were detection probes for SARS-CoV-2. In a direction from 5′ to 3′, the underlined bases indicated that they were modified, wherein the 5′ end of the probe SARS-CoV-2-Probe-12 was modified with a quencher group, A was an RNA base, and the 3′ end was modified with a reporter fluorophore and C3 spacer; the 5′ end of SARS-CoV-2-Probe-6 was modified with a quencher, G, A, and G were RNA bases, and the 3′ end was modified with a reporter fluorophore and C3 spacer.
2. Isothermal Amplification Reaction
25 μL of reaction system: 20 mM Tris-HCl, 70 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween 20, 9 mM MgSO4, 1.7 mM dNTPs, 8 U Bst 2.0 WarmStart, 1.6 μM SARS-CoV-2-BIP/SARS-CoV-2-FIP, and 0.4 μM SARS-CoV-2-LB/SARS-CoV-2-LF/SARS-CoV-2-B3/SARS-CoV-2-F3. The detection system of RNase HII further included 1 μg of RNase HII, and 0.2 μM of SARS-CoV-2-Probe-12 or SARS-CoV-2-Probe-6.
Reaction conditions: 63° C., 1 min, 60 cycles (fluorescence was collected every 1 min).
3. Results Analysis
The result is shown in
The target was amplified by LAMP with a set of six primers, and the probe was cleaved by the RNase HII in the system synchronously as the product increased. After the LAMP reaction, if there was no target, the probe remained intact, and if there was a target, the probe was cleaved. In the colloidal gold test strip, the gold conjugate pad contained digoxigenin antibody-labelled gold nanoparticles, streptavidin was immobilized in the quality control line, and the secondary antibody was immobilized in the detection line. When the probe was intact, the antibody and streptavidin of the gold nanoparticles recognized the two labels in the probe respectively, and thereby forms a sandwich therewith, so that the gold nanoparticles were captured in the quality control line for color development. At the same time, since most of the gold nanoparticles were captured in the quality control line, the detection line did not develop; when the probe was cleaved, streptavidin could capture only the cleaved halves of-probe, but no gold nanoparticles. The more cleaved probes, the less gold nanoparticles captured by the quality control line, the more gold nanoparticles flowing to the detection line and captured by the secondary antibody, and the darker the color of the detection line. The detection line developed in the presence of the target sequence, but did not develop in the absence of the target sequence.
1. Design of Primer and Probe
In Table 6, SARS-CoV-2-B3, SARS-CoV-2-BIP, SARS-CoV-2-LB, SARS-CoV-2-F3, SARS-CoV-2-FIP, SARS-CoV-2-LF were amplification primers for SARS-CoV-2, and SARS-CoV-2-Probe-LFB was a detection probe for SARS-CoV-2. In a direction from 5′ to 3′, the underlined bases indicated that they were modified, the 5′ end of the probe SARS-CoV-2-Probe-LFB was modified with digoxigenin, A was an RNA base, and the 3′ end was modified with biotin.
2. Isothermal Amplification Reaction
25 μL of reaction system: 20 mM Tris-HCl, 70 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween 20, 9 mM MgSO4, 1.7 mM dNTP, 8 U Bst 2.0 WarmStart, 1 μg RNase HII, 1.6 μM SARS-CoV-2-BIP/SARS-CoV-2-FIP, 0.4 μM SARS-CoV-2-LB/SARS-CoV-2-LF/SARS-CoV-2-B3/SARS-CoV-2-F3, 0.2 μM SARS-CoV-2-Probe-LFB.
Reaction conditions: 63° C., 30 min.
3. Colloidal Gold Detection
The sample pad of a test strip was inserted at an end into an EP tube containing the amplification product. The chromatography was carried out for 10 minutes before interpretation of the result.
4. Result Analysis
The result is shown in
The target was amplified by LAMP with a set of six primers, and the probe was cleaved by the RNase HII in the system synchronously as the product increased. After the LAMP reaction, if there was no target, the probe remained intact, and if there was a target, the probe was cleaved. In the colloidal gold test strip, the gold conjugate pad contained digoxigenin antibody-labelled gold nanoparticles, streptavidin was immobilized in the detection line, and the secondary antibody was immobilized in the quality control line. When the probe was intact, the antibody and streptavidin on the gold nanoparticles recognized the two labels of the probe respectively, and thereby forms a sandwich therewith, so that the gold nanoparticles were captured in the detection line for color development; when the probe was cleaved, streptavidin could capture only the cleaved halves of probe, but no gold nanoparticles, thus the detection line did not develop. The detection line developed in the absence of the target sequence, and disappeared in the presence of the target sequence. The gold conjugate pad had an excess amount of gold nanoparticles immobilized thereon. Whether or not the detection line capture the gold nanoparticles, surplus gold nanoparticles could be combined with the secondary antibody in the quality control line for color development, to confirm that the test strip functions normally in the reaction.
In the examples, two probes, i.e. SARS-CoV-2-Probe-LFB and SARS-CoV-2-Probe-multiR, were used respectively. The former had a single RNA base, and the latter had multi-RNA bases divided the probe into shorter spaced DNA segments, to compare the benefits of the short segment design.
1. Design of Primer and Probe
In Table 7, SARS-CoV-2-B3, SARS-CoV-2-BIP, SARS-CoV-2-LB, SARS-CoV-2-F3, SARS-CoV-2-FIP, SARS-CoV-2-LF were amplification primers for SARS-CoV-2, and SARS-CoV-2-Probe-LFB and SARS-CoV-2-Probe-multiR were detection probes for SARS-CoV-2. In a direction from 5′ to 3′, the underlined bases indicated that they were modified, wherein the 5′ end of the probe SARS-CoV-2-Probe-LFB was modified with digoxigenin, C was an RNA base, and the 3′ end was modified with biotin; the 5′ end of the SARS-CoV-2-Probe-multiR probe was modified with digoxigenin, U, C and U were RNA bases, and the 3′ end was modified with biotin.
2. Isothermal Amplification Reaction
25 μL of reaction system: 20 mM Tris-HCl, 70 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween 20, 1.7 mM dNTP, 9 mM MgSO4, 8 U Bst 2.0 WarmStart, 1 μg RNase HIT, 1.6 SARS-CoV-2-BIP/SARS-CoV-2-FIP, 0.4 SARS-CoV-2-LB/SARS-CoV-2-LF/SARS-CoV-2-B3/SARS-CoV-2-F3, 20 nM SARS-CoV-2-Probe-LFB or SARS-CoV-2-Probe-multiR.
Reaction conditions: 63° C., 30 min.
3. Colloidal Gold Detection
The sample pad of a test strip was inserted at an end into an EP tube containing the amplification product. The chromatography was carried out for 10 minutes before interpretation of the result.
4. Result Analysis
The result is shown in
The recognition effect of single base changes in target nucleic acid was tested with different probe lengths.
In Table 8, Complement-25 and Mism were target sequences which were reverse complementary to the two probes, but the base in Mism corresponding to the probe RNA base G was T; Probe-Tm-59.5 and Probe-extended were two probes with different lengths, and have sequences which were reverse complementary to the target sequence, respectively. In a direction from 5′ to 3′, the underlined bases indicated that they were modified, T of the Probe-Tm-59.5 probe was modified with a quencher, G was RNA bases, and C was modified with a fluorescent reporter group; T of the Probe-extended probe was modified with a quencher, G was RNA bases, and C was modified with a fluorescent reporter group.
2. Cleavage Reaction
25 μL of reaction system: 20 mM Tris-HCl, 70 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween 20, 9 mM MgSO4, 1 μg RNase HII, 0.02 μM Complement or Mism, and 0.2 μM Probe-Tm-59.5 or Probe-extended.
Reaction conditions: 60° C., 1 min, 60 cycles (fluorescence was collected every 1 min).
3. Results Analysis
The result is shown in
The target was amplified by LAMP with a set of six primers, and the genotype of the SNP site was reported in real time through RNase HII and probes in the system. The reaction system included both of two probes targeting two alleles at the SNP site and labelled with two different fluorophores respectively. The base at SNP site of the target sequence could only trigger the cleavage of the perfectly paired probe, to produce fluorescence corresponding to the channel. The genotype of the sample could be obtained by determining whether the fluorescence generates in the two channels.
1. Design of Primer and Probe
In Table 9, 181-F3, 181-B3, 181-FIP, 181-BIP, 181-LF and 181-LB were amplification primers for the rs1815739 site, 181-C was a detection probe for the allele C, and 181-T was a detection probe for the T allele. In a direction from 5′ to 3′, the underlined bases indicated that they were modified, wherein C of the 181-C probe was modified with a reporter fluorescent HEX group, G was an RNA base, T was modified with a quencher, and the 3′ end was modified with C3 spacer; C of 181-T probe was modified with a reporter fluorescent FAM group, A was an RNA base, T was modified with a quencher, and the 3′ end was modified with C3 spacer.
2. Isothermal Amplification Reaction
25 μL of reaction system: 20 mM Tris-HCl, 70 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween 20, 1.7 mM dNTP, 9 mM MgSO4, 8 U Bst 2.0 WarmStart, 1 μg RNase HIT, 1.6 μM 181-BIP/181-FIP, 0.4 μM 181-LB/181-LF/181-B3/181-F3, 0.2 μM 181-C/181-T.
Reaction conditions: 63° C., 1 min, 40 cycles (fluorescence was collected every 1 min).
3. Results Analysis
The result is shown in
The target was amplified by LAMP with a set of six primers, and the probe for the genotype at the corresponding SNP site was cleaved by RNase HII in the system as the amplification product increased. In the presence of the target sequence, bases at the SNP site could trigger the cleavage of the perfectly paired probe. Two probes were designed to target two genes at the SNP site respectively. The probes were modified with labels that could be recognized by colloidal gold test strips. The genotype in the sample could be determined by the color development in the two colloidal gold test strips. The probe would be cleaved and the detection line in the test strip would develop, in the presence of perfectly paired gene at the SNP site in the target sequence; the probe would not be cleaved, the quality control line rather than the detection line in the test strip would develop, in the absence of paired gene at the SNP site.
1. Design of Primer and Probe
In Table 10, 181-F3, 181-B3, 181-FIP, 181-BIP, 181-LF and 181-LB were amplification primers for the rs1815739 site, 181-C-LFB was a detection probe for the allele C, and 181-T-LFB was a detection probe for the T allele. In a direction from 5′ to 3′, the underlined bases indicated that they were modified, wherein the 5′ end of the 181-C-LFB probe was modified with digoxigenin, C was an RNA base, and the 3′ end was modified with biotin; the 5′ end of the 181-T-LFB probe was modified with digoxigenin, A was an RNA base, and the 3′ end was modified with biotin.
2. Isothermal Amplification Reaction
25 μL of reaction system: 20 mM Tris-HCl, 70 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween 20, 1.7 mM dNTP, 9 mM MgSO4, 8 U Bst 2.0 WarmStart, 1 μg RNase HIT, 1.6 μM 181-BIP/181-FIP, 0.4 μM 181-LB/181-LF/181-B3/181-F3, 0.2 μM 181-C-LFB/181-T-LFB.
Reaction conditions: 63° C., 1 min, 40 cycles (fluorescence was collected every 1 min).
3. Colloidal Gold Detection
The sample pad of a test strip was inserted at an end into an EP tube containing the amplification product. The chromatography was carried out for 10 minutes before interpretation of the result.
4. Result Analysis
The result is shown in
Each of the technical features of the above-mentioned embodiments may be combined arbitrarily. To simplify the description, not all the possible combinations of each of the technical features in the above embodiments are described. However, all of the combinations of these technical features should be considered as within the scope of this disclosure, as long as such combinations do not contradict with each other.
The above-mentioned embodiments are merely illustrative of several embodiments of the present disclosure, which are described specifically and in detail, but it cannot be understood to limit the scope of the present disclosure. It should be noted that, for those ordinary skilled in the art, several variations and improvements may be made without departing from the concept of the present disclosure, and all of which are within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110170603.6 | Feb 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/116861 | 9/7/2021 | WO |
