CN112322705A - Isothermal amplification fluorescence RMA method for multiple nucleic acid detection - Google Patents
Isothermal amplification fluorescence RMA method for multiple nucleic acid detection Download PDFInfo
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Abstract
The application belongs to biological detection technology, in particular to a constant temperature amplification fluorescence RMA method for multiple nucleic acid detection, which comprises the following preparation steps: (1) extracting total RNA of a sample to be detected as a template; (2) designing primers and probe sets for multiplex detection of pathogen nucleic acids; (3) preparing a fluorescent RMA reaction system, adding the extracted total RNA into the reaction system, and carrying out reverse transcription and RMA amplification reaction; (4) and (3) judging a detection result: and analyzing whether the sample to be detected contains a certain pathogen nucleic acid or not according to whether a corresponding amplification curve is generated or not. The corresponding amplification curve shows that the sample to be detected contains the nucleic acid of the pathogen and shows a positive result; the absence of the corresponding amplification curve or the amplification curve being lower than the detection threshold indicates that the pathogen nucleic acid is not detected in the sample to be detected, and a negative result is obtained.
Description
Technical Field
The application belongs to biological detection technology, and particularly relates to a constant temperature amplification fluorescence RMA method for multiplex nucleic acid detection.
Background
The existing experimental methods for detecting pathogens mainly comprise fluorescent quantitative PCR, enzyme-linked immunosorbent assay, indirect immunofluorescence, culture method and the like. The PCR technology has high detection sensitivity, wide application range, simple and convenient operation and the like, but has complex procedures, needs precise instruments and has longer detection time, thereby being not beneficial to field detection in non-laboratory environment and popularization and application in basic laboratories. Although the ELISA is simple and convenient to operate, the sensitivity is low, the influence factors are more, and the clinical application of the ELISA is limited by the factors such as false negative or false positive easily caused by low sensitivity and more influence factors. The indirect immunofluorescence method aims at specific antibodies generated by organisms caused by pathogens, the antibody detection has a window period, the missed diagnosis is easy to occur at the early stage of the disease, the sensitivity and the specificity are low, the judgment result needs to be judged by means of a fluorescence microscope, and the operation is complex. The culture method is always considered as the 'gold standard' for pathogen detection and is favored by clinicians, but is difficult to popularize and apply in primary hospitals due to the defects of complex operation steps, long culture time, great technical difficulty, low positive rate and the like.
Recombinase polymerase Amplification (RMA) is a nucleic acid isothermal Amplification technique that is considered as a nucleic acid detection technique that can replace PCR, and mainly relies on three enzymes, Recombinase, single-stranded DNA binding protein (SSB), and strand-displacement DNA polymerase. The principle is that a recombinase is combined with a primer to form a complex, and a homologous sequence is recognized in double-stranded DNA; then recombinase is used for releasing double chains, the primer and the homologous sequence are subjected to chain exchange reaction and DNA synthesis is started, and exponential amplification is carried out on the template; the replaced DNA strand binds to SSB, preventing further replacement. The RMA technique has the advantages of being fast, simple, convenient, and free of special instruments, and is currently applied to detection of a plurality of important disease pathogens.
Aiming at the problems in the prior art, the isothermal amplification fluorescent RMA method for multiplex nucleic acid detection is developed and evaluated, and can be used for rapidly detecting nucleic acids of various pathogens.
Disclosure of Invention
In order to solve the problems of the prior art, the present application provides an isothermal amplification fluorescent RMA method for multiplex nucleic acid detection, which is achieved by the following scheme:
an isothermal amplification fluorescence RMA method for multiplex nucleic acid detection comprises the following preparation steps: (1) extracting total RNA of a sample to be detected as a template; (2) designing primers and probe sets for multiplex detection of pathogen nucleic acids; (3) preparing a fluorescent RMA reaction system, adding the extracted total RNA into the reaction system, and carrying out reverse transcription and RMA amplification reaction; (4) and (3) judging a detection result: and analyzing whether the sample to be detected contains a certain pathogen nucleic acid or not according to whether a corresponding amplification curve is generated or not. The corresponding amplification curve shows that the sample to be detected contains the nucleic acid of the pathogen and shows a positive result; the absence of the corresponding amplification curve or the amplification curve being lower than the detection threshold indicates that the pathogen nucleic acid is not detected in the sample to be detected, and a negative result is obtained.
Preferably, the primer length of the nucleic acid in the step (2) is generally 30-35 bp, the GC content is 40-60%, the 3-5 nucleotides at the 5 'end cannot be more than two G, the 3' end should be G and C, and the consecutive three nucleotides cannot be identical.
Preferably, the nucleic acid probe in the step (2) has a length of 46-52 bp, and comprises thymine nucleotide (T) carrying a fluorescent group and thymine nucleotide (T) carrying a quenching group, wherein the thymine nucleotide (T) carrying the fluorescent group and the thymine nucleotide (T) carrying the quenching group are separated by one tetrahydrofuran base (THF), and the fluorescent group and the quenching group are separated by 2-5 bases; the fluorescent group is modified by FAM, HEX, ROX and the like, and the quenching group is modified by BHQ; the 3' end of the probe is labeled with a modifying group for inhibiting polymerase extension or amplification. When the probe is combined with the amplification product, the exonuclease III recognizes the THF base site, and separates the fluorescent group from the quenching group, so that the fluorescent signal and the accumulation of the amplification product are synchronized, and the effect of real-time monitoring is achieved.
Preferably, the amplification reaction reagent in step (3) comprises the following components:
| reagent | Final concentration |
| Tris | 50mM |
| Magnesium acetate | |
| Polyethylene oxide | |
| 10 %（w/v） | |
| Trehalose | 2mM |
| Mannitol | 2.5mM |
| ATP | 10mM |
| dNTPs | 2mM |
| Creatine kinase | 1000ng/mL |
| Creatine phosphate | 25mM |
| Each |
10 µM |
| Each |
10 µM |
| M-MLV reverse transcriptase | 200ng/µL |
| Escherichia coli RecA protein | 100ng/µL |
| UvsY protein | 40ng/µL |
| Single chain binding protein GP32 | 800ng/µL |
| Bst polymerase | 60ng/µL |
| Exonuclease III | 80ng/µL |
The concentrations of the components are as shown in the table above.
Preferably, the amplification reaction in step (3) is performed in a fluorescence detector set at 42 ℃ for 20 min.
Preferably, the method can be used for detection of nucleic acids in serum, plasma, whole blood, oropharyngeal swabs, nasopharyngeal swabs, bacteria, fungi, various tissue cells, and urine.
A kit for isothermal amplification fluorescent RMA method for multiplex nucleic acid detection, which is provided with: an amplification reaction reagent, a detection tube of the amplification reaction reagent, a buffer solution, a magnesium acetate solution, a positive quality control product and a negative quality control product; the amplification reaction reagent is packaged in a single tube and is in a dry powder form.
Preferably, the positive quality control product contains a recombinant plasmid of an amplification gene sequence of a pathogen to be detected.
Preferably, the negative quality control material is sterile double distilled water; the kit can be used for simultaneously and rapidly detecting a plurality of pathogens.
Has the advantages that: (1) can save the reaction time and reduce the reaction temperature: the RMA reaction only needs 5-20min, and the whole detection process can be completed within 1h by adding sample treatment and preparation time, compared with the conventional PCR, the reaction can be carried out only at the constant temperature of 38-42 ℃ without temperature change;
(2) easy preservation: the enzyme, primer probe and the like required by amplification are freeze-dried into powder and can be stored at normal temperature;
(3) the operation is simpler and more portable: the detection reagent is not required to be prepared, the sample is only required to be added once, and the result can be detected on a computer without the operation of a skilled professional;
(4) single-tube typing: the multiple pathogens can be distinguished according to different fluorescent signals in one detection tube;
(5) the specificity is strong, the sensitivity is high, and the detection result is real and reliable;
(6) the pollution is not easy to occur: exonuclease III is added into the amplification reaction reagent to cut the amplification product, so that the possibility of product pollution is reduced.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 shows sensitivity experiments for detecting FluA-H1N1 by fluorescent RMA method;
FIG. 2 sensitivity assay for FluA-H3N2 by fluorescent RMA;
FIG. 3 sensitivity assay for FluA-H5N1 by fluorescent RMA;
FIG. 4 fluorescent RMA assay for detecting FluA-H1N 1;
FIG. 5 fluorescent RMA assay for detecting FluA-H3N 2;
FIG. 6 fluorescent RMA assay for detecting FluA-H5N1 specificity.
Detailed Description
To make the objects, technical solutions and advantages of the present application more clear, embodiments of the present application will be described in further detail below with reference to the accompanying drawings.
Example 1
A kit for multiple detection of influenza A virus subtypes based on a fluorescent RMA technology and establishment of a method are disclosed:
a kit for multiple detection of influenza A virus subtypes based on a fluorescent RMA technology is provided with: an amplification reaction reagent, a detection tube of the amplification reaction reagent, a buffer solution, a magnesium acetate solution, a negative quality control product (sterile double distilled water), an A H1N1 positive quality control product (a plasmid cloned with an HA gene segment of an A H1N1 influenza virus), an A H3N2 positive quality control product (a plasmid cloned with an HA gene segment of an A H3N2 influenza virus) and an A H5N1 positive quality control product (a plasmid cloned with an HA gene segment of an A H5N1 influenza virus).
1. Preparing a positive quality control product:
(1) extracting influenza A virus (FluA) RNA according to the instruction of a virus DNA/RNA extraction kit, and carrying out reverse transcription according to the instruction of a reverse transcription kit to obtain cDNA; (2) performing PCR amplification on HA gene segments of FluA (H1N 1, H3N2 and H5N 1) by taking cDNA as a template, performing 1% agarose gel electrophoresis on PCR amplification products, tapping and recovering, cloning and connecting to a pMD18-T vector, converting to escherichia coli competent cells, screening blue and white spots, selecting white colonies, and performing colony PCR verification; (3) sequencing the positive recombinant bacteria, culturing the recombinant bacteria with correct sequencing overnight, extracting plasmid DNA and obtaining a positive quality control product.
2. Designing fluorescent RMA primers and probes:
downloading HA gene sequences of different influenza A virus subtypes (H1N 1, H3N2 and H5N 1) from an NCBI database, comparing by adopting ClustalX software to find out highly conserved regions, designing primers and probes by taking the HA gene sequences as templates according to an RMA amplification reaction principle, wherein the sequences of fluorescent RMA detection primers and probes are shown in the following table 1:
TABLE 1 primer and Probe sequences
| Primer Probe name | Primer Probe sequence (5 '-3') |
| H1N1-F | ACGCATCAATGCATGAGTGTAACACGAAGTGTCAA |
| H1N1-R | CGGCAATGGCTCCAAATAGACCTCTGGATTGAATG |
| H1N1-P | ACACCCCTGGGAGCTATAAACAGCAGTCTCCC(FAM-dT)(THF)(BHQ-dT)CCAGAATATACACC(C3spacer) |
| H3N2-F | TGCATCACTCCAAATGGAAGCATTCCCAATGACAA |
| H3N2-R | CTTGAGTGCTTTTGAGATCTGCTGCTTGTCCTGTT |
| H3N2-P | GGTTGGGAGGGAATGGTAGACGGTTGGTACGG(HEX-dT)(THF)(BHQ-dT)CAGGCATCAAAATTCT(C3spacer) |
| H5N1-F | CCCAAAGTAAACGGGCAAAGTGGAAGAATGGAGTT |
| H5N1-R | TGTGGAATGGCATACTAGAGTTTATCGCCCCCATT |
| H5N1-P | GCCGAATGATGCCATCAATTTCGAGAGTAATGGAAA(ROX-dT)(THF)(BHQ-dT)CATTGCTCCAGAATA(C3spacer) |
Wherein, the fluorescent group of the H1N 1-probe is modified by FAM, the fluorescent group of the H3N 2-probe is modified by HEX, the fluorescent group of the H5N 1-probe is modified by ROX, all quenching groups are modified by BHQ, and the 3' end is modified by a blocking group C3-spacer.
3. Establishment of a fluorescent RMA reaction system:
the reaction system in the RMA isothermal amplification reaction reagent is shown in the following table:
| reagent | Dosage (mu L) |
| Constant temperature amplification reaction tube (freeze-dried powder containing primer probe enzyme) | 1 tube |
| Buffer solution | 42.5 |
| Magnesium acetate (280 mM) | 2.5 |
| Form panel | 5 |
| |
50 |
42.5 mul of buffer solution and 5 mul of extracted virus RNA template are added into a constant temperature amplification reaction tube containing the freeze-dried powder of primer probe enzyme and mixed evenly. Finally, 2.5. mu.L of 280mM magnesium acetate solution was added to the tube and mixed well. The reaction tube was placed in a fluorescence detector and reacted at 42 ℃ for 20 min. The positive quality control is used as a positive control in each reaction, and sterile double distilled water is used as a negative control.
4. Sensitivity experiment for detecting FluA (H1N 1, H3N2 and H5N 1) by fluorescence RMA method
Using FluA (H1N 1, H3N2 and H5N 1) positive quality control substances as templates, and performing 10-fold gradient dilution to make the final concentration after dilution be 104 -100Copying/reacting, performing fluorescent RMA reaction under the above reaction system conditions with sterile double distilled water as negative control, repeating the test 3 times at each concentration, and obtaining the results shown in FIGS. 1-3 and 104 -101The results are positive, namely the sensitivity of the fluorescent RMA detection kit reaches 10 copies/reaction.
5. Specific experiment for detecting FluA (H1N 1, H3N2 and H5N 1) by using fluorescent RMA method:
respectively detecting virus nucleic acid samples such as FluA (H1N 1, H3N2 and H5N 1), influenza B virus (FluB), parainfluenza virus (HPIV), Respiratory Syncytial Virus (RSV) and the like by using the established fluorescent RMA method, evaluating the specificity of the method, repeatedly detecting 3 times in each test by using sterile double distilled water as a negative control, obtaining figures 4-6 according to the result, detecting as H1N1 positive only when the target pathogen is influenza A virus FluA-H1N1, and detecting as H3N2, H5N1 and other viruses as negative by using the fluorescent RMA method; only when the target pathogen is influenza a virus FluA-H3N2, the test is positive for H3N2, but negative for H1N1, H5N1 and other viruses; only when the target pathogen was influenza a virus FluA-H5N1, the assay was positive for H5N1, but negative for H1N1, H3N2 and other viruses. The fluorescent RMA method is proved to have good detection effect and specificity.
The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (9)
1. An isothermal amplification fluorescence RMA method for multiplex nucleic acid detection is characterized by comprising the following preparation steps: (1) extracting total RNA of a sample to be detected as a template; (2) designing primers and probe sets for multiplex detection of pathogen nucleic acids; (3) preparing a fluorescent RMA reaction system, adding the extracted total RNA into the reaction system, and carrying out reverse transcription and RMA amplification reaction; (4) and (3) judging a detection result: and analyzing whether the sample to be detected contains a certain pathogen nucleic acid or not according to whether a corresponding amplification curve is generated or not. The corresponding amplification curve shows that the sample to be detected contains the nucleic acid of the pathogen and shows a positive result; the absence of the corresponding amplification curve or the amplification curve being lower than the detection threshold indicates that the pathogen nucleic acid is not detected in the sample to be detected, and a negative result is obtained.
2. The isothermal amplification fluorescent RMA method for multiplex nucleic acid detection according to claim 1, wherein the primer length of the nucleic acid in step (2) is generally 30-35 bp, the GC content is 40-60%, the number of 3-5 nucleotides at the 5 'end is not more than two G's, the number of G's and C's at the 3 'end is G's and the consecutive three nucleotides are not the same.
3. The isothermal amplification fluorescent RMA method for multiplex nucleic acid detection according to claim 1, wherein the nucleic acid probe in step (2) has a length of 46-52 bp, and comprises thymine (T) carrying a fluorophore and thymine (T) carrying a quencher, which are separated by a tetrahydrofuran base (THF), and the fluorophore and the quencher are separated by 2-5 bases; the fluorescent group is modified by FAM, HEX, ROX and the like, and the quenching group is modified by BHQ; the 3' end of the probe is labeled with a modifying group for inhibiting polymerase extension or amplification.
4. The isothermal amplification fluorescent RMA method according to claim 1, wherein the amplification reaction reagents in step (3) comprise the following components:
the concentrations of the components are as shown in the table above.
5. The isothermal amplification fluorescent RMA method for multiplex nucleic acid detection according to claim 1, wherein in step (3), the amplification reaction is performed in a fluorescence detector set at 42 ℃ for 20 min.
6. The isothermal amplification fluorescent RMA method for multiplex nucleic acid detection according to claim 1, wherein the method is used for detection of nucleic acids in serum, plasma, whole blood, oropharyngeal swab, nasopharyngeal swab, bacteria, fungi, various tissue cells, and urine.
7. The kit for the isothermal amplification fluorescent RMA method according to claim 1, wherein: an amplification reaction reagent, a detection tube of the amplification reaction reagent, a buffer solution, a magnesium acetate solution, a positive quality control product and a negative quality control product; the amplification reaction reagent is packaged in a single tube and is in a dry powder form.
8. The kit for isothermal amplification fluorescent RMA method for multiplex nucleic acid detection according to claim 7, wherein said positive quality control contains a recombinant plasmid having an amplified gene sequence of a pathogen to be detected.
9. The kit for isothermal amplification fluorescent RMA method for multiplex nucleic acid detection according to claim 7, wherein said negative quality control substance is sterile double distilled water; the kit can be used for simultaneously and rapidly detecting a plurality of pathogens.
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