CN113755558A - Nucleic acid detection method based on liquid chip technology - Google Patents
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Abstract
The invention discloses a nucleic acid detection method based on a liquid chip technology, which comprises the following steps of 1) PCR amplification of a nucleic acid sequence to be detected: 2) preparing a liquid chip: 3) nucleic acid hybridization: 4) incubation at constant temperature; and 5) analyzing results; wherein, the forward primer used in the step 1) is a common primer with a section of specific binding sequence, and the specific binding sequence in the forward primer is defined as an Anti-Probe sequence; the forward primer is equal to the reverse primer; the obtained PCR amplification product is a double-stranded DNA product, the double-stranded DNA product is provided with a mark, the Anti-Probe sequence exists in a free single-stranded state, and one end of the double-stranded DNA product is subjected to multiple PCR to realize the detection of multiple targets, and the enrichment of multiple nucleic acid target sequences is realized by using specific primers. After the specific Anti-Probe binding sequence carried by the 5' end of the enriched product is combined with the Probe which is connected on the coding microsphere and can be complementary with the Anti-Probe binding sequence, the classification detection of the sequences of a plurality of check counting tables is realized.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a nucleic acid detection method based on a liquid chip technology.
Background
Nucleic acid detection generally requires two processes, namely PCR amplification and nucleic acid hybridization, of nucleic acid to be detected, and then analysis of the product. Among them, PCR (polymerase chain reaction) is a molecular biology technique for in vitro amplification of specific DNA fragments by replication; the principle of PCR is: DNA is denatured at 95 ℃ in vitro to form single strands; combining the primer and the single strand according to the base complementary pairing principle at the temperature of 60 ℃; the DNA polymerase synthesizes a complementary strand at-72 ℃ in the direction from phosphate to the five carbon sugar (5'-3'), so that the PCR product is usually double-stranded DNA. Nucleic acid hybridization is the process by which complementary nucleotide sequences (DNA to DNA, DNA to RNA, RNA to RNA, etc.) form stable homoduplexes or heteroduplexes through base pairing. Conventional hybridization procedures include: denaturation of nucleic acid (nucleotide probe is denatured at 95 ℃) and hybridization (overnight hybridization at 55-62 ℃), double strands of double-stranded nucleic acid molecules are untied under the action of external factors, and double-stranded structures can be formed according to the base complementary pairing principle after conditions are recovered. The traditional hybridization reaction temperature is determined by G, C base content of the sequence, and the hybridization temperature is easier to optimize for a single-weight hybridization system; for the multiple hybridization system, the optimization of hybridization temperature is difficult.
In the prior art, because viruses have very high genetic variation rate and polymorphism and different types of viruses have certain structural difference, the multiple PCR technology is combined with nucleic acid hybridization and used for specifically detecting the enriched target sequence to simultaneously distinguish different types of viruses or different viruses with the same infection phenotype. However, at present, there are few kits for hybridization detection on a flow fluorescence technology platform in the market, and a temperature-changing denaturation step is required before hybridization, which is complicated and has high requirements on instruments. In contrast, the previously published CN113186257A patent proposes a hybridization method after PCR amplification based on liquid chip, which can perform hybridization and incubation under constant temperature condition without pre-denaturation, thereby reducing the time consumption of detection and simplifying the operation procedure of detection. However, for different nucleic acids to be detected, probes matched with the nucleic acids to be detected still need to be designed and synthesized, the project still consumes a long time, and the research and development cost still is high.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a nucleic acid detection method based on a liquid chip technology, which combines a multiple PCR technology, a constant-temperature hybridization and detection technology based on the liquid chip technology, a specific sequence and coding microsphere principle, can realize hybridization and incubation under a constant temperature condition, save detection time, simplify detection flow, effectively reduce the research and development period and cost of products, and quickly realize the productization of projects.
In order to solve the above technical problems, the nucleic acid detection method based on the liquid chip technology provided by the invention comprises the following steps:
1) PCR amplification of target nucleic acid sequences to be detected: amplifying a target nucleic acid sequence to be detected by using a labeled primer and a common primer, wherein the common primer is provided with a section of specific binding sequence, and the specific binding sequence is defined as an Anti-Probe sequence; the obtained PCR amplification product is a double-stranded DNA product which is provided with a mark, and the Anti-Probe sequence exists at one end of the double-stranded DNA product in a free single-stranded state; in a preferred embodiment, the common primers with Anti-Probe sequences are 17-30nt in length, and the Anti-Probe sequences are 17-30nt in length; more preferably, the Anti-Probe sequence is linked to the 5' end of the common primer, and dSpacer, but not limited to dSpacer, is used between the Anti-Probe sequence and the common primer.
2) Preparing a liquid chip: connecting a single-stranded DNA Probe to a microsphere with a coding function, wherein the nucleotide sequence of the single-stranded DNA Probe and the Anti-Probe sequence are specific complementary sequences; the microspheres are provided with a marker; in a preferred embodiment, the single-stranded DNA probe is a DNA oligonucleotide probe, the length of the DNA oligonucleotide probe is 17-30nt, and the 5 'end or the 3' end of the DNA oligonucleotide probe is provided with a plurality of poly-T nucleotides or spacers, and the 5 'end or the 3' end of the poly-T nucleotides or spacers is provided with a functional group capable of combining with a functional group on the surface of the microsphere with a coding function, and the functional group can be amino, hydroxyl, or thiol; the label of the microsphere includes but is not limited to luminescent materials such as biotin, digoxin or dyes.
3) Nucleic acid hybridization: mixing the double-stranded DNA product amplified by PCR in the step 1) with the liquid chip obtained in the step 2), and performing specific recombination hybridization on the single-stranded DNA Probe and the Anti-Probe sequence under a constant temperature condition to obtain a hybridization product; preferably, the hybridization buffer consists of, but is not limited to, the following components in the following concentrations: TMAC 5M, SLS 0.1.1%, Tris-HCL 10Mm, and the pH value of the hybridization buffer is 7.5.
4) Adding a color developing agent into the hybridization product obtained in the step 3), and incubating at constant temperature;
5) analyzing the result of the product incubated at the constant temperature in the step 4) by using a flow cytometry analyzer.
The scheme can rapidly and accurately analyze whether the target fragment exists in the detected sample in a homogeneous liquid phase, and can detect a plurality of targets at the same time, wherein the time consumption of the whole hybridization process is less than 45 min. The traditional temperature-dependent hybridization method needs complex temperature-variable hybridization processes such as heating denaturation, cooling annealing, base pairing renaturation and the like, generally takes about 2-3 hours, and if multiple detection development is carried out, Tm values of different probes need to be considered so as to ensure the consistency of hybridization temperature; meanwhile, the DNA Probe is finally combined with the Anti-Probe sequence instead of the nucleic acid to be detected, so that different DNA probes do not need to be designed and synthesized aiming at different nucleic acids, the research and development program of the DNA Probe in the research and development process is omitted, the research and development period is shortened, and the research and development time and the research and development cost are saved.
Specifically, in the above method, when the nucleic acid to be detected contains different types, multiplex PCR amplification is used.
Specifically, in the above method, the labeled primer in step 1) is a forward primer, and the common primer is a reverse primer; or the labeled primer is a reverse primer, and the common primer is a forward primer. Preferably, the labeled primer is a reverse primer, and the common primer is a forward primer.
Specifically, in the method, the single-stranded DNA probe in the step 2) is connected with the microsphere in a manner of forming a covalent bond between functional groups; preferably, the functional group is a combination of carboxyl and amino (the two react to form an amide bond), or a combination of carboxyl and hydroxyl (the two react to form an aliphatic bond), or a combination of sulfhydryl and sulfhydryl (the two react to form a disulfide bond), or a combination of aldehyde group and amino (the two react to form an aldehyde-amine condensation reaction); more preferably, the functional group is a combination of a carboxyl group and an amino group (which react to form an amide bond).
Specifically, the microspheres in the step 2) are fluorescence encoding microspheres or quantum dot encoding microspheres. Understandably, the microspheres selected herein are encodable microspheres, which are not limited to the two listed above, and the two types of encodable microspheres are only preferred; understandably, the surface of the microsphere is further provided with a sealing agent, an antibacterial agent and the like, and the skeleton of the microsphere is mostly made of polystyrene material, polyacrylic acid material and the like, but not limited to the two materials.
Specifically, in the method, the temperature of the constant temperature condition in the step 3) is in the range of 48-52 ℃; the preferred temperature is 50 ℃.
Specifically, in the method, the temperature range of the constant-temperature incubation in the step 4) is 48-52 ℃; preferably, the temperature for isothermal hybridization in step 3) is kept the same as the temperature for isothermal incubation in step 4).
Specifically, the method for analyzing by using the flow cytometer in the step 5) comprises the following steps: simultaneously detecting the fluorescence value of the label on the double-stranded DNA product labeled in the step 1) and the optical signal on the microsphere with the coding function in the step 3) by a flow cytometer.
The method provided by the invention combines a multiplex PCR technology, a constant temperature hybridization and detection technology based on a liquid chip technology, a specific sequence and a coding microsphere principle, wherein a double-stranded DNA product amplified by the multiplex PCR has a label, one end of the double-stranded DNA product is an Anti-Probe sequence in a free single-stranded state, the Anti-Probe sequence in the single-stranded state and a DNA Probe are subjected to constant temperature hybridization to obtain a final product, and finally, a flow cytometry is used for testing a signal of the final product. The invention simplifies the experimental operation flow, reduces the instrument burden, increases the safety of the experimental operation, simultaneously saves the research and development procedure of the DNA probe in the research and development process, shortens the research and development period, saves the research and development time and the research and development cost, and can effectively and rapidly deal with the project development of various nucleic acid detection requirements.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the present invention are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of the detection principle and steps of the present invention.
FIG. 2 is an agarose gel electrophoresis gel of the PCR amplification product of example two of the present invention.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
The embodiment provides a nucleic acid detection method based on a liquid chip technology, which combines a multiplex PCR technology, constant-temperature hybridization based on the liquid chip technology, a detection technology, a specific sequence and a coding microsphere principle, designs a typing detection kit for different mutant strains of a novel coronavirus, can realize the reverse transcription of a novel coronavirus nucleic acid RNA template (a Bio-Rad reverse transcription reagent is adopted, and a reverse transcription system is configured in a table 2), obtains a double-stranded DNA product after PCR, and realizes the hybridization and signal detection of a probe system under a constant temperature condition. The detection process comprises the following steps:
1) a pseudovirus synthesis template sequence of mutation sites of the novel coronavirus HV69-70del, N501Y and P681H and a pseudovirus synthesis template sequence of the internal reference beta-actin gene are amplified by using a primer with a label and a common primer (specific primer sequences are shown in Table 1) connected with an Anti-Probe sequence specifically, reverse transcription and PCR programs are respectively shown in Table 3 and Table 5, and double-stranded DNA products of HV69-70del, N35501 501Y, P681H and beta-actin are obtained, wherein the sequences have free single-stranded Anti-Probe sequences and carry labels.
TABLE 1 PCR primers
TABLE 2 preparation of reverse transcription System
| Components | 20 μ L system |
| Reverse transcription reagent | 4μL |
| Reverse transcriptase | 1μL |
| RNA template | 5μL |
| ddH2O | The volume is fixed to 20 mu L |
TABLE 3 PCR procedure
| Step (ii) of | Temperature (. degree.C.) | Time |
| Reverse transcription | 42 | 30min |
| Enzyme denaturation | 95 | 1min |
TABLE 4 PCR System formulation
| Components | 25 μ L System |
| 2x Taq Mix | 12.5μL |
| Form panel | 5μL |
| F + R primer | 5μL |
| ddH2O | The volume is determined to be 25 mu L |
TABLE 5 PCR procedure
2) Connecting single-stranded DNA probes for distinguishing HV69-70del, N501Y, P681H and beta-actin to fluorescent microspheres or quantum dot microspheres or other types of microspheres with coding functions by using carboxyl and amino or other functional groups to generate covalent bonds through chemical reaction, wherein the probes comprise nucleic acid sequences which are complementary to the Anti-Probe sequences in the free single-stranded state connected to the single-stranded product sequences with the labels in the step 1);
TABLE 6 Probe sequences
| Target genes | Probe amplification sequence | Serial number |
| HV69-70del | 5’-GTTGCTCAAGTGTCCGACTTACTTTTTTTTTT-NH2-3’ | SEQ ID NO：9 |
| N501Y | 5’-CTACTATAGATCTCTGTCATCCTGTTTTTTTTTT-NH2-3’ | SEQ ID NO：10 |
| P681H | 5’-CCGATAGAGATCGTCACAGCTTTTTTTTTT-NH2-3’ | SEQ ID NO：11 |
| β-actin | 5’-AGCGAGACGATTGCAGGTTTTTTTTTT-NH2-3’ | SEQ ID NO：12 |
3) Adding the Probe coupled with the microsphere with the coding function in the step 2) into a double-stranded amplification product with a label and an Anti-Probe sequence in a free single-stranded state in the step 1), and carrying out constant-temperature hybridization, wherein the hybridization procedure is shown in the table 7;
TABLE 7 hybridization procedure
| Step (ii) of | Temperature (. degree.C.) | Time | Number of cycles |
| Hybridization of | 50 | 20min | - |
4) Adding a color developing agent (SA-PE is adopted in the embodiment) into the hybridization product in the step 3), incubating at constant temperature, and performing incubation procedures shown in the table 8;
TABLE 8 hybridization procedure
| Step (ii) of | Temperature (. degree.C.) | Time | Number of cycles |
| Incubation | 50 | 10min | - |
5) Analyzing the result of the incubation product in the step 4) by using a flow cytometry analyzer.
The results are shown in Table 9:
TABLE 9 hybridization signals
From the above results, the signal value of hybridization of the amplification product corresponding to each template was known. The positive signals of HV69-70del, N501Y, P681H and beta-actin can be distinguished obviously (the hybridization signal Cut-off set by the inventor in the method is more than or equal to 150 as a positive value), and the PCR product obtained by the multiplex PCR amplification mode can be effectively hybridized with the probe to obtain the hybridization signal and can effectively judge the negative and positive of the tested sample.
Example two
The embodiment provides a nucleic acid detection method based on a liquid chip technology, which combines a multiplex PCR technology, a constant-temperature hybridization and detection technology based on the liquid chip technology, a specific sequence and coding microsphere principle, designs a kit for Human Papillomavirus (HPV) typing detection, can realize the enrichment of a multiple PCR target sequence of a human papillomavirus DNA template to obtain a double-stranded DNA product, and realizes the hybridization and signal detection of a probe system under a constant temperature condition. The detection process comprises the following steps:
1) amplifying HPV16, HPV18 and an internal reference beta-Globin template (the template is a DNA sequence searched and synthesized according to NCBI) by using a primer with a label and a common primer (the specific primer sequence is shown in Table 10) connected with an Anti-Probe sequence specifically coded, wherein the PCR program is shown in Table 12, the PCR system is prepared as shown in Table 11, and detection target double-stranded DNA products of HPV16, HPV18 and internal reference beta-Globin with the Anti-Probe sequence in a label and free single-stranded state are obtained; the PCR amplification electrophoresis detection result is shown in FIG. 2, wherein M represents DNA Mark, 16 represents DNA extracted from HPV16 type positive cells, 18 represents DNA extracted from HPV18 type positive cells, and the internal reference represents beta-Globin.
TABLE 10 PCR primers
TABLE 11 PCR System formulation
| Components | 25 μ L System |
| 2x Taq Mix | 12.5μL |
| Form panel | 5μL |
| F + R primer | 1μL |
| ddH2O | To constant volume25μL |
TABLE 12 PCR procedure
2) Connecting single-stranded DNA probes for distinguishing HPV16, HPV18 and internal reference beta-Globin to fluorescent microspheres or quantum dot microspheres or other types of microspheres with coding functions by adopting a mode that carboxyl and amino or other functional groups generate covalent bonds through chemical reaction, wherein the probes in the table 13 comprise nucleic acid sequences which are complementary to Anti-Probe sequences in a free single-stranded state connected to the labeled single-stranded product sequences in the step 1);
TABLE 13 Probe sequences
| Target genes | Probe amplification sequence | Serial number |
| HPV16 | 5’-AATGGGACGATGGTGGTGTTTTTTTTTT-NH2-3’ | SEQ ID NO：19 |
| HPV18 | 5’-TACACACCATTCATCATAACTAACTTTTTTTTTT-NH2-3’ | SEQ ID NO：20 |
| β-actin | 5’-CGTGGGCACATAGAGCGATTTTTTTTTT-NH2-3’ | SEQ ID NO：21 |
3) Adding the Probe coupled with the microsphere with the coding function in the step 2) into a double-stranded amplification product with a label and an Anti-Probe sequence in a free single-stranded state in the step 1), and carrying out isothermal hybridization, wherein the hybridization procedure is shown in the table 14;
TABLE 14 hybridization procedure
| Step (ii) of | Temperature (. degree.C.) | Time | Number of cycles |
| Hybridization of | 50 | 20min | - |
4) Adding a color developing agent (SA-PE is adopted in the embodiment) into the hybridization product in the step 3), incubating at constant temperature, and incubating according to the procedure shown in the table 15;
TABLE 15 incubation procedures
| Step (ii) of | Temperature (. degree.C.) | Time | Number of cycles |
| Incubation | 50 | 10min | - |
5) Analyzing the result of the incubation product in the step 4) by using a flow cytometry analyzer. The results are shown in Table 16.
TABLE 16 hybridization signals
From the above results, the signal value of hybridization of the amplification product corresponding to each template was known. Positive signals of HPV16, HPV18 and internal reference beta-Globin can be obviously distinguished (the hybridization signal Cut-off set by the inventor in the method is more than or equal to 150 as a positive value), and a free Anti-Probe sequence on a product can be effectively hybridized with a Probe to obtain a hybridization signal through a PCR product obtained in a multiple PCR amplification mode, and the negative and positive of a detected sample can be effectively judged.
In summary, the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A nucleic acid detection method based on a liquid chip technology is characterized by comprising the following steps:
1) PCR amplification of target nucleic acid sequences to be detected: amplifying a target nucleic acid sequence to be detected by using a labeled primer and a common primer, wherein the common primer is provided with a section of specific binding sequence, and the specific binding sequence is defined as an Anti-Probe sequence;
the obtained PCR amplification product is a double-stranded DNA product which is provided with a mark, and the Anti-Probe sequence exists at one end of the double-stranded DNA product in a free single-stranded state;
2) preparing a liquid chip: connecting a single-stranded DNA Probe to a microsphere with a coding function, wherein the nucleotide sequence of the single-stranded DNA Probe and the Anti-Probe sequence are specific complementary sequences; the microspheres are provided with a marker;
3) nucleic acid hybridization: mixing the double-stranded DNA product amplified by PCR in the step 1) with the liquid chip obtained in the step 2), and performing specific recombination hybridization on the single-stranded DNA Probe and the Anti-Probe sequence under a constant temperature condition to obtain a hybridization product;
4) adding a color developing agent into the hybridization product obtained in the step 3), and incubating at constant temperature;
5) analyzing the result of the product incubated at the constant temperature in the step 4) by using a flow cytometry analyzer.
2. The method for detecting nucleic acid according to claim 1, wherein a multiplex PCR amplification procedure is used in step 1).
3. The method for detecting nucleic acid according to claim 2, wherein the labeled primer in step 1) is a forward primer, and the common primer is a reverse primer; or
The labeled primer is a reverse primer, and the common primer is a forward primer.
4. The method for detecting nucleic acid according to claim 1, wherein the single-stranded DNA probe and the microsphere in step 2) are linked by forming a covalent bond between functional groups.
5. The method for detecting nucleic acid according to claim 4, wherein the functional group is a combination of carboxyl and amino, or a combination of carboxyl and hydroxyl, or a combination of thiol and thiol, or a combination of aldehyde and amino.
6. The method for detecting nucleic acid according to claim 1, wherein the microspheres in step 2) are fluorescence-encoded microspheres or quantum dot-encoded microspheres.
7. The method for detecting a nucleic acid according to claim 1, wherein the temperature of the isothermal condition in step 3) is in a range of 48 ℃ to 52 ℃.
8. The method for detecting nucleic acid according to claim 1, wherein the incubation temperature in step 4) is in the range of 48 ℃ to 52 ℃.
9. The method for detecting nucleic acid according to claim 1, wherein the temperature for the isothermal hybridization in step 3) is the same as the temperature for the isothermal incubation in step 4).
10. The method for detecting a nucleic acid according to claim 1, wherein the analysis in step 5) by a flow cytometer is: simultaneously detecting the fluorescence value of the label on the double-stranded DNA product labeled in the step 1) and the optical signal on the microsphere with the coding function in the step 3) by a flow cytometer.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114645073A (en) * | 2022-02-24 | 2022-06-21 | 万子健生物技术(上海)有限公司 | Microsphere coated with multiple probes as well as preparation method and application thereof |
| CN115058493A (en) * | 2022-06-07 | 2022-09-16 | 浙江大学 | DNA probe for multiple nucleic acid detection, CRISPR-reverse dot hybrid nucleic acid detection system and application |
| CN115125290A (en) * | 2022-08-04 | 2022-09-30 | 山东鲁抗好丽友生物技术开发有限公司 | Amplification method of multiple single strands and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952798A (en) * | 2012-11-07 | 2013-03-06 | 龙岩九健生物芯片技术研究所 | Design method of PCR (Polymerase Chain Reaction) primers |
| CN105316398A (en) * | 2014-07-30 | 2016-02-10 | 益善生物技术股份有限公司 | Amplification primer for detecting food-borne pathogenic microorganisms and liquid chip kit |
| CN112575125A (en) * | 2021-01-19 | 2021-03-30 | 季华实验室 | Primer group, probe microsphere group, kit and method for detecting respiratory tract pathogenic microorganisms |
| CN113186257A (en) * | 2021-05-28 | 2021-07-30 | 杭州深度生物科技有限公司 | Constant-temperature hybridization method after PCR amplification based on liquid chip technology |
| CN113215312A (en) * | 2021-04-28 | 2021-08-06 | 山东莱博生物科技有限公司 | Coronavirus SARS-CoV-2 digital PCR multiple detection kit and its application |
| CN114107572A (en) * | 2022-01-26 | 2022-03-01 | 潮州凯普生物化学有限公司 | Primer probe set for detecting different new coronavirus mutant strains based on multiplex PCR technology, detection kit and application of detection kit |
-
2021
- 2021-08-27 CN CN202110992820.3A patent/CN113755558A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952798A (en) * | 2012-11-07 | 2013-03-06 | 龙岩九健生物芯片技术研究所 | Design method of PCR (Polymerase Chain Reaction) primers |
| CN105316398A (en) * | 2014-07-30 | 2016-02-10 | 益善生物技术股份有限公司 | Amplification primer for detecting food-borne pathogenic microorganisms and liquid chip kit |
| CN112575125A (en) * | 2021-01-19 | 2021-03-30 | 季华实验室 | Primer group, probe microsphere group, kit and method for detecting respiratory tract pathogenic microorganisms |
| CN113215312A (en) * | 2021-04-28 | 2021-08-06 | 山东莱博生物科技有限公司 | Coronavirus SARS-CoV-2 digital PCR multiple detection kit and its application |
| CN113186257A (en) * | 2021-05-28 | 2021-07-30 | 杭州深度生物科技有限公司 | Constant-temperature hybridization method after PCR amplification based on liquid chip technology |
| CN114107572A (en) * | 2022-01-26 | 2022-03-01 | 潮州凯普生物化学有限公司 | Primer probe set for detecting different new coronavirus mutant strains based on multiplex PCR technology, detection kit and application of detection kit |
Non-Patent Citations (3)
| Title |
|---|
| FEI ZHAO: "A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology", MICROBIOLSPECTRUM, vol. 9, no. 3, 17 November 2021 (2021-11-17), pages 1 - 10 * |
| HSING-YI CHUNG: "Emergency SARS-CoV-2 Variants of Concern: Novel Multiplex Real-Time RT-PCR Assay for Rapid Detection and Surveillance", MICROBIOLSPECTRUM, vol. 10, no. 1, 23 February 2022 (2022-02-23), pages 1 - 10 * |
| JOLENE YIN LING FU: "SARS-CoV-2 multiplex RT-PCR to detect variants of concern (VOCs) in Malaysia, between January to May 2021", JOURNAL OF VIROLOGICAL METHODS, vol. 301, 10 January 2022 (2022-01-10), pages 1 - 3, XP086964564, DOI: 10.1016/j.jviromet.2022.114462 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114645073A (en) * | 2022-02-24 | 2022-06-21 | 万子健生物技术(上海)有限公司 | Microsphere coated with multiple probes as well as preparation method and application thereof |
| CN115058493A (en) * | 2022-06-07 | 2022-09-16 | 浙江大学 | DNA probe for multiple nucleic acid detection, CRISPR-reverse dot hybrid nucleic acid detection system and application |
| CN115058493B (en) * | 2022-06-07 | 2023-09-19 | 浙江大学 | DNA probe for multiple nucleic acid detection, CRISPR-reverse dot hybridization nucleic acid detection system and application |
| CN115125290A (en) * | 2022-08-04 | 2022-09-30 | 山东鲁抗好丽友生物技术开发有限公司 | Amplification method of multiple single strands and application thereof |
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