CN116949142B - Amplification method for RNA target detection and application of kit - Google Patents
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Abstract
The invention belongs to the field of molecular biology, and particularly relates to an amplification method for RNA target detection and application of a kit. The RNA amplification system comprises a reverse transcription primer T, an upstream amplification primer F, a downstream amplification primer R and a specific probe P; the reverse transcription primer T comprises two partial sequences T1 and T2, wherein the near 3 'end is T1, the near 3' end comprises a base specifically combined with a target sequence, and an RNA base is introduced in the middle of the sequence T1; the near 5' end is a T2 sequence; the upstream amplification primer F is consistent with the reverse transcription primer T2 in sequence; the 5 'end of the specific probe P and the 3' end of the reverse transcription primer T1 sequence have overlapping bases. The method has the advantages of high detection speed, good specificity, high accuracy and the like, can be compatible with various PCR instrument models, and is widely applied to various application scenes of clinical detection.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to an amplification method for RNA target detection and application of a kit.
Background
Ribonucleotides (RNAs) are a class of biological macromolecules with important biological functions and clinical value. In cells, RNA is mainly classified into three types, namely tRNA, rRNA and mRNA, according to different structural functions, and the RNA is involved in important body activities such as gene expression and regulation. RNA is also an important mark of pathogens such as viruses and bacteria, and for some viruses, RNA is a unique genetic information carrier, and for other pathogens such as bacteria, the RNA has the characteristics of poor stability and easy degradation, and can reflect pathogen activity and metabolic state in real time relative to the detection of RNA of DNA.
Respiratory Syncytial Virus (RSV) and mycoplasma pneumoniae are common pathogens that can cause respiratory infections. About 50% of infant pneumonia and 90% of infant bronchitis are caused by infection with RSV; adults with immune deficiency and the elderly are also susceptible people to RSV. Mycoplasma pneumoniae is frequently found in teenagers, and can cause common pneumonia, and also cause severe bilateral pneumonia and other complications, which seriously endanger the physical health of patients. Currently, the molecular biological method for detecting RSV and mycoplasma pneumoniae clinically mainly comprises a fluorescent quantitative reverse transcription PCR method. The technical method not only can detect respiratory syncytial virus with RNA as genetic information, but also can detect RNA nucleic acid targets in mycoplasma pneumoniae.
Quantitative reverse transcription PCR (quantitative reverse transcription PCR, RT-qPCR) is currently one of the most commonly used RNA detection techniques. In this method, total RNA or messenger RNA (mRNA) is first reverse transcribed into complementary DNA (cDNA) by reverse transcriptase. Subsequently, qPCR reactions were performed using cDNA as template. However, it is difficult to distinguish DNA information in the target in this way. At present, the technology for detecting RNA is relatively few, the application and development are mature, the technology is an RNA isothermal amplification technology, but the technology has certain limitations in the aspect of the commercialization application, such as the need of combining a special extraction mode, complex operation and the like, and the application of the requirement of simultaneously detecting some DNA targets and RNA targets is limited by different systems and amplification programs. Therefore, there is a need to develop an RNA detection method that is easy to operate, has good compatibility, high sensitivity, and strong specificity, so as to monitor the occurrence and development of diseases and evaluate the therapeutic effect.
Disclosure of Invention
In order to solve the problems, the invention provides an RNA amplification method, which can effectively amplify target RNA and distinguish DNA targets in targets by designing reverse transcription primers, amplification primers and specific probes so as to realize amplification of the RNA targets.
In one aspect, the invention provides an RNA amplification method, wherein the RNA amplification system at least comprises a reverse transcription primer T, an upstream amplification primer F, a downstream amplification primer R and a specific probe P; the reverse transcription primer T comprises two partial sequences T1 and T2, wherein the near 3 'end is T1, the near 3' end comprises a base specifically combined with a target sequence, and an RNA base is introduced in the middle of the sequence T1; the near 5' end is a T2 sequence; the upstream amplification primer F is consistent with the reverse transcription primer T2 in sequence; the 5 'end of the specific probe P and the 3' end of the reverse transcription primer T1 sequence have overlapping bases.
Specifically, the T1 may comprise 10-25 bases that specifically bind to a target sequence.
Specifically, RNA bases are introduced at intervals of 3-12 bases in the T1 sequence.
Specifically, the T2 sequence may comprise 16-28 bases, and the sequence may be an artificially designed sequence which is not homologous to the target sequence.
Specifically, the specific probe P may comprise 20 to 35 bases.
Further specifically, the 5 '-end of the specific probe P overlaps 3-15 bases with the 3' -end of the reverse transcription primer T1 sequence.
Specifically, the fluorescent group marked on the 5' end of the specific probe P can be one or more of FAM, VIC, TET, CAL Gold 540, JOE, HEX, TAMRA, ROX, CY3 and CY 5; the 3' -end labeled quenching group can be one or more of DABCYL, BHQ-1, BHQ-2, BHQ-3 and ECLIPE.
Specifically, the downstream amplification primer R may comprise 16-35 bases.
Specifically, the Tm of the T1 is 40-50 ℃; the Tm of the T2 is 50-60 ℃; the Tm of the upstream amplification primer F is 50-60 ℃; the Tm of the downstream amplification primer R is 50-60 ℃; the Tm of the specific probe P is 55-70 ℃.
In another aspect, the present invention provides an RNA amplification kit, wherein the kit comprises at least one reverse transcription primer T, an upstream amplification primer F, a downstream amplification primer R, and a specific probe P; the reverse transcription primer T comprises two partial sequences T1 and T2, wherein the near 3 'end is T1, the near 3' end comprises a base specifically combined with a target sequence, and an RNA base is introduced in the middle of the sequence T1; the near 5' end is a T2 sequence; the upstream amplification primer F is consistent with the reverse transcription primer T2 in sequence; the 5 'end of the specific probe P and the 3' end of the reverse transcription primer T1 sequence have overlapping bases.
Specifically, the kit can also comprise reverse transcriptase, endonuclease RNase HII, polymerase and buffer solution.
In particular, the kit can be used to detect a nucleic acid RNA target in a pathogen of interest.
Further specifically, the pathogen may be respiratory syncytial virus or mycoplasma pneumoniae.
Preferably, the sequence of the reverse transcription primer T for detecting the respiratory syncytial virus is SEQ ID NO.1; the sequence of the upstream primer F is SEQ ID NO.2; the sequence of the downstream primer R is SEQ ID NO.3; the sequence of the specific probe P is SEQ ID NO.4.
The sequence of the reverse transcription primer T for mycoplasma pneumoniae detection is SEQ ID NO.8; the sequence of the upstream primer F is SEQ ID NO.9; the sequence of the downstream primer R is SEQ ID NO.10; the sequence of the specific probe P is SEQ ID NO.11.
Specifically, the detecting step includes:
(1) Extracting pathogen RNA;
(2) Amplifying the RNA in the step (1) by the amplification method or the kit;
(3) And judging negative and positive according to the amplification curve threshold.
Further specifically, the RNA OD extracted in step (1) 260 /OD 280 The value of (2) should be 2.0-2.2 and the concentration should be between 2-50 ng/. Mu.L.
The invention has the technical effects that: in respiratory syncytial virus and mycoplasma pneumoniae RNA detection, the disclosed methods are capable of specifically detecting target RNAs, and the amplification methods have the ability to distinguish DNA targets in a target.
Drawings
FIG. 1 is a graph showing the amplification of a sample of respiratory syncytial virus detected by the method of the present disclosure.
FIG. 2 is an amplification graph of a sample of respiratory syncytial virus detected by a conventional Taqman probe method.
FIG. 3 is a graph showing amplification using a DNA plasmid containing an amplicon fragment as a template in example 1.
FIG. 4 is an amplification plot of a sample of Mycoplasma pneumoniae detected by the methods disclosed herein.
FIG. 5 is an amplification graph of a traditional Taqman probe method for detecting a mycoplasma pneumoniae sample.
FIG. 6 is a graph showing amplification using a DNA plasmid containing an amplicon fragment as a template in example 2.
FIG. 7 is a flow chart of the amplification principle of the disclosed method.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
The information of all reagents in the invention is shown in Table 1:
TABLE 1
Example 1 detection of respiratory syncytial Virus RSV
1.1 materials and instruments
A sample of a throat swab from a clinical patient infected with respiratory syncytial virus RSV is collected and stored in a conventional sampling tube or swab holder at 4 ℃.
Ultraviolet spectrophotometry (NanoDrop 2000, thermo fisher), real-time fluorescence PCR instrument (ABI 7500).
1.2 primers and probes
According to the gene sequence corresponding to respiratory syncytial virus RSV, the specific detection primer is designed by utilizing oligo 7.0 software, and the specific design mode is as follows: comprises a reverse transcription primer T, an upstream amplification primer F, a downstream amplification primer R and a specific probe P; the reverse transcription primer T (26-48 bp) comprises two partial sequences T1 and T2, wherein the near 3' end is T1 and comprises 10-25 bases which are specifically combined with a target sequence, tm is 40-50 ℃, and an RNA base is introduced into the T1 sequence at intervals of 3-12 nt; the near 5' end is a T2 sequence which contains 16-28 bases and has Tm of 50-60 ℃, and the partial sequence is an artificial design sequence and is not homologous with a target sequence; the upstream amplification primer F comprises 16-28 bases, is consistent with the sequence of the reverse transcription primer T2, and has a Tm of 50-60 ℃; the downstream amplification primer R comprises 16-35 bases, and the Tm is 50-60 ℃ consistent with the target sequence; the length of the amplicons formed by the upstream and downstream amplification primers is 60-150bp; the specific probe P comprises 20-35 bases, tm is 55-70 ℃, and the 5 'end of the specific probe P overlaps 3-15 bases with the 3' end of the reverse transcription primer T1 sequence.
The primers and probes were synthesized by Shanghai chemical company, and the specific sequences are shown in Table 2:
TABLE 2
Note that: the bold font is the RNA base introduced in the primer; r, Y degenerate bases, where R represents A/G and Y represents C/T.
The reverse transcription primer used in this example was a primer for introducing RNA base, the fluorescent group modified at the 5 'end of the probe P was FAM, and the quenching group modified at the 3' end was BHQ1.
1.3 detection of respiratory syncytial Virus RSV
Detection principle: reverse transcription primer T containing RNA base is specifically combined with target RNA single-stranded sequence to guide reverse transcription to synthesize cDNA first strand to generate RNA-cDNA hybrid strand, and target RNA single strand in the hybrid strand is digested and degraded by RNase H. The downstream primer R is specifically combined with the cDNA strand digested in the previous step to generate a cDNA second strand, and extends out of the complementary sequence of T2 in the reverse transcription primer, and the generated cDNA second strand with the T2 complementary sequence can be used as an amplification template of the upstream primer and the downstream primer in the subsequent PCR flow. The amplification process is characterized in that the fluorescent probe preferentially binds to the target sequence and the upstream amplification primer F initiates amplification extension, during which the exo-activity of the polymerase specifically cleaves the probe P to generate a fluorescent signal. In the reverse transcription process, when a reverse transcription primer T containing RNA bases is specifically combined with a DNA template sequence, endonuclease RNase HII can specifically cut the RNA bases in the primer, so that a downstream primer cannot be reversely extended to generate a T2 complementary sequence, and therefore DNA cannot become a PCR amplification template; in the PCR amplification process, the reverse transcription primer is partially overlapped with the probe position, and only the amplification with the upstream amplification primer can trigger fluorescent signal generation; thus, the aim of amplifying only RNA targets is achieved.
1.3.1 extraction of sample RNA
Nucleic acid extraction of clinical pharyngeal swab sample with commercial nucleic acid extraction kit, concentration and purity determination with ultraviolet spectrophotometer, and RNA OD thereof 260/ OD 280 The value of (2) is 2.0-2.2, the concentration is 2-50 ng/. Mu.L, the sample nucleic acid is unqualified and is not used for detection, and the sample nucleic acid is preserved at 20 ℃ for standby after being qualified.
1.3.2 quantitative reverse transcription PCR
The experiment was divided into the following three experiments, and the specific amplification reaction system is shown in table 3, and three replicates were set for each experiment.
TABLE 3 Table 3
(1) Experiment 1-1 is a test sample of the method of the invention:
primers and probes are shown in Table 2.
Blank control: the template is water;
experimental group: the template is collected RNA of a throat swab sample of a clinical patient infected by RSV;
positive control: the template is a self-constructed RSV pseudovirus containing target RNA sequence, the vector of the RSV pseudovirus is pET 32a-MS2, the insertion site is BamHI/NotI, and the target RNA sequence is SEQ ID NO.5.
(2) Experiment 1-2 is a traditional Taqman probe method for detecting samples:
both primers and probes were synthesized by Shanghai, see Table 4.
TABLE 4 Table 4
| Primer probe type | SEQ ID NO | Sequence(s) |
| Upstream primer F | SEQ ID NO.6 | AGATCAACTTCTRTCATCCAGCA |
| Downstream primer R | SEQ ID NO.3 | TGCACATCATAATTAGGAGTRTCA |
| Probe P | SEQ ID NO.4 | AYACCATCCAACGGAGCACAGGAGA |
Blank control: the template is water;
experimental group: the template is collected RNA of a throat swab sample of a clinical patient infected by RSV;
positive control: the template is a self-constructed RSV pseudovirus containing the RNA sequence of interest.
(3) Experiments 1-3 verify the detection effect of the DNA sample for the method of the invention:
primers and probes are shown in Table 2.
Experimental group: the template is a DNA plasmid (Ct 20-25) comprising an amplicon fragment, wherein the plasmid is a PUC plasmid; the DNA sequence of the amplified fragment inserted in the plasmid is SEQ ID NO.7.
The PCR reaction tube was placed on a real-time fluorescence PCR instrument for testing, and the reaction program settings are shown in Table 5:
TABLE 5
And judging positive results according to the amplification curve of the target, and judging positive if the amplification curve of the FAM channel appears. The results of experiments 1-1 and 1-2 show that the same clinical sample of respiratory syncytial virus is detected by the traditional Taqman probe method and the method disclosed by the invention, and the results of the two detection methods are consistent, and are positive, as shown in figures 1 and 2. The results of experiments 1-3 show that the detection method of the invention has the capability of distinguishing DNA targets in targets, as shown in figure 3.
EXAMPLE 2 detection of Mycoplasma pneumoniae RNA
2.1 materials and instruments
The sterilization container collects clinical sputum samples of mycoplasma pneumoniae infected patients, and the samples are preserved at 4 ℃ and extracted and detected as soon as possible.
Instrument is 1.1
2.2 primers and probes
The design method of the primer and the probe is the same as 1.2, and the specific sequences are shown in Table 6:
TABLE 6
| Primer probe type | Name of the name | SEQ ID NO | Sequence(s) |
| Reverse transcription primer T | MP-T | SEQ ID NO.8 | TACACAGGCATCCACAGACATGACCGCGAACTAT |
| Upstream primer F | MP-F | SEQ ID NO.9 | TACACAGGCATCCACAGAC |
| Downstream primer R | MP-R | SEQ ID NO.10 | TGGTTTGGAGCAAAACATCGC |
| Probe P | MP-P | SEQ ID NO.11 | TATCCGCCCAGTTGAAGAACGCCCAAGT |
Note that: the bold font is the RNA base introduced in the primer.
The reverse transcription primer used in this example was a primer for introducing RNA base, the fluorescent group modified at the 5 'end of the probe P was FAM, and the quenching group modified at the 3' end was BHQ1.
2.3 detection of Mycoplasma pneumoniae RNA
2.3.1 extraction of sample RNA
Liquefying and extracting nucleic acid from clinical sample with commercial nucleic acid extraction kit, measuring concentration and purity with ultraviolet spectrophotometer, and determining RNA OD 260/ OD 280 The value of (2) is 2.0-2.2, the concentration is 2-50 ng/. Mu.L, the sample nucleic acid is unqualified and is not used for detection, and the sample nucleic acid is preserved at 20 ℃ for standby after being qualified.
2.3.2 quantitative reverse transcription PCR
The experiment was divided into the following three experiments, and the specific amplification reaction system is shown in table 7, and three replicates were set for each experiment.
TABLE 7
(1) Experiment 2-1 is a test sample of the method of the invention:
primers and probes are shown in Table 6.
Blank control: the template is water;
experimental group: the template is collected clinical sputum sample RNA of a patient infected by mycoplasma pneumoniae;
positive control: the template was an in vitro transcribed RNA target fragment (SEQ ID NO. 12) using the kit Transcriptaid T7High Yield Transcription, thermoFisher K0441.
(2) Experiment 2-2 is a sample detected by a traditional Taqman probe method:
both primers and probes were synthesized by Shanghai, see Table 8.
TABLE 8
| Primer probe type | SEQ ID NO | Sequence(s) |
| Upstream primer F | SEQ ID NO.13 | TTGGATCCCAAGGYSATGACC |
| Downstream primer R | SEQ ID NO.10 | TGGTTTGGAGCAAAACATCGC |
| Probe P | SEQ ID NO.11 | TATCCGCCCAGTTGAAGAACGCCCAAGT |
Note that: s represents a degenerate base, representing C/G; y represents C/T.
Blank control: the template is water;
experimental group: the template is collected clinical sputum sample RNA of a patient infected by mycoplasma pneumoniae;
positive control: the template was an in vitro transcribed RNA target fragment, using the kit transdaid T7High Yield Transcription, thermosusher K0441.
(3) Experiments 2-3 verify the detection effect of the DNA sample for the method of the invention:
primers and probes are shown in Table 6.
Experimental group: the template is a DNA plasmid (Ct 20-25) comprising an amplicon fragment, wherein the plasmid is a PUC plasmid; the DNA sequence of the amplified fragment inserted in the plasmid is SEQ ID NO.14.
The PCR reaction tube was placed on a real-time fluorescence PCR instrument for testing, and the reaction program settings are shown in Table 9:
TABLE 9
And judging positive results according to the amplification curve of the target, and judging positive if the amplification curve of the FAM channel appears. The results of experiment 2-1 and experiment 2-2 show that the same clinical sample of mycoplasma pneumoniae is detected by the traditional Taqman probe method and the method disclosed by the invention, and the results of the two detection methods are consistent, and are determined to be positive, as shown in fig. 4 and 5. The results of experiments 2-3 show that the detection method of the invention has the capability of distinguishing DNA targets in targets, as shown in FIG. 6.
Comparative example
Comparative examples were set up and run with reference to the procedure of experiments 2-3 in example 2, and the comparative examples were set up in the manner set forth in Table 10:
table 10
Experimental results show that none of comparative examples 1-3 can effectively amplify target RNA while distinguishing DNA targets in the targets, further illustrating that the design methods disclosed in the present application for reverse transcription primers, amplification primers and specific probes can only amplify target RNA.
Claims (4)
1. A non-diagnostic RNA amplification method is characterized in that an RNA amplification system at least comprises a reverse transcription primer T, an upstream amplification primer F, a downstream amplification primer R and a specific probe P; the reverse transcription primer T comprises two partial sequences T1 and T2, wherein the near 3 'end is T1, the near 3' end comprises a base specifically combined with a target sequence, and an RNA base is introduced in the middle of the sequence T1; the near 5' end is a T2 sequence; the upstream amplification primer F is consistent with the reverse transcription primer T2 in sequence; the 5 'end of the specific probe P and the 3' end of the reverse transcription primer T1 sequence are provided with overlapping bases;
the T1 comprises 10-25 bases which are specifically combined with a target sequence;
introducing RNA bases at intervals of 3-12 bases in the T1 sequence;
the T2 sequence contains 16-28 bases, is a designed sequence and is not homologous with a target sequence;
the specific probe P comprises 20-35 bases;
the 5 'end of the specific probe P is overlapped with the 3' end of the reverse transcription primer T1 sequence by 3-15 bases;
the downstream amplification primer R comprises 16-35 bases;
the Tm of the T1 is 40-50 ℃; the Tm of the T2 is 50-60 ℃; the Tm of the upstream amplification primer F is 50-60 ℃; the Tm of the downstream amplification primer R is 50-60 ℃; the Tm of the specific probe P is 55-70 ℃;
the RNA amplification system also comprises reverse transcriptase, endonuclease RNase HII, polymerase and buffer solution.
2. The method of amplifying RNA according to claim 1, wherein the fluorescent group marked on the 5' -end of the specific probe P is one of FAM, VIC, TET, CAL Gold 540, JOE, HEX, TAMRA, ROX, CY3 and CY 5; the 3' -end marked quenching group is one of DABCYL, BHQ-1, BHQ-2, BHQ-3 and ECLIPE.
3. A kit for detecting a nucleic acid RNA target in respiratory syncytial virus, which is characterized in that the sequence of a reverse transcription primer T for respiratory syncytial virus detection is SEQ ID NO.1; the sequence of the upstream primer F is SEQ ID NO.2; the sequence of the downstream primer R is SEQ ID NO.3; the sequence of the specific probe P is SEQ ID NO.4;
the kit also comprises reverse transcriptase, endonuclease RNase HII, polymerase and buffer solution.
4. A kit for detecting a nucleic acid RNA target in mycoplasma pneumoniae, which is characterized in that the sequence of a reverse transcription primer T for mycoplasma pneumoniae detection is SEQ ID NO.8; the sequence of the upstream primer F is SEQ ID NO.9; the sequence of the downstream primer R is SEQ ID NO.10; the sequence of the specific probe P is SEQ ID NO.11;
the kit also comprises reverse transcriptase, endonuclease RNase HII, polymerase and buffer solution.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003012142A1 (en) * | 2001-08-01 | 2003-02-13 | Nederlands Instituut Voor Zuivelonderzoek | Detection of nucleic acids by real-time pcr using chimeric rna-dna primers |
| CN1537954A (en) * | 2003-04-18 | 2004-10-20 | ������������ʽ���� | Expressing genetic analysis method and probe reagent box for expressing genetic analysng |
| CN109576352A (en) * | 2018-11-25 | 2019-04-05 | 江苏宏微特斯医药科技有限公司 | Single tube detects method, probe and its kit of multiple object to be measured nucleic acid sequences |
| CN113005181A (en) * | 2020-12-22 | 2021-06-22 | 广州血康陆道培生物技术有限公司 | Primer group for detecting non-coding small RNA (ribonucleic acid) by using multiplex fluorescent quantitative PCR (polymerase chain reaction) based on stem-loop method |
| WO2022117816A1 (en) * | 2020-12-03 | 2022-06-09 | The University Of Birmingham | Target rna detection |
-
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- 2023-06-20 CN CN202310732065.4A patent/CN116949142B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003012142A1 (en) * | 2001-08-01 | 2003-02-13 | Nederlands Instituut Voor Zuivelonderzoek | Detection of nucleic acids by real-time pcr using chimeric rna-dna primers |
| CN1537954A (en) * | 2003-04-18 | 2004-10-20 | ������������ʽ���� | Expressing genetic analysis method and probe reagent box for expressing genetic analysng |
| CN109576352A (en) * | 2018-11-25 | 2019-04-05 | 江苏宏微特斯医药科技有限公司 | Single tube detects method, probe and its kit of multiple object to be measured nucleic acid sequences |
| WO2022117816A1 (en) * | 2020-12-03 | 2022-06-09 | The University Of Birmingham | Target rna detection |
| CN113005181A (en) * | 2020-12-22 | 2021-06-22 | 广州血康陆道培生物技术有限公司 | Primer group for detecting non-coding small RNA (ribonucleic acid) by using multiplex fluorescent quantitative PCR (polymerase chain reaction) based on stem-loop method |
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