CN111778361A - High-sensitivity pathogenic nucleic acid rapid detection technology for non-diagnosis and treatment purposes - Google Patents
High-sensitivity pathogenic nucleic acid rapid detection technology for non-diagnosis and treatment purposes Download PDFInfo
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Abstract
The invention relates to the technical field of virus detection. The invention provides a high-sensitivity pathogenic nucleic acid rapid detection technology for non-diagnosis and treatment purposes, which comprises the following steps: (1) recombinase isothermal amplification; (2) the colloidal gold developed color. The application discloses high sensitive pathogenic nucleic acid rapid detection technique uses isothermal nucleic acid amplification-colloidal gold color development for pathogen spot detection, does not need special equipment and reagent, saves pathogen extraction step simultaneously, can limit capture and enlarge pathogen nucleic acid signal, sample such as direct detection patient's phlegm, blood, excrement and urine, be applicable to very much the spot detection, the bedside of hospital diagnoses, the application of crowd gathering life scene and other resource rare circumstances such as school, airport, especially need to obtain the scene of result fast urgently. Can solve the defects that the detection of pathogen nucleic acid depends on large-scale equipment and reagents, the detection period is long, the sensitivity is high and the pathogen needs to be extracted in the prior art.
Description
Technical Field
The invention relates to the technical field of virus detection, in particular to a high-sensitivity pathogen nucleic acid rapid detection technology for non-diagnosis and treatment purposes.
Background
The most important basis for molecular diagnostics is nucleic acid amplification technology. At present, the mainstream DNA amplification technology needs precise temperature-changing equipment, a complex kit (high requirements on storage and transportation conditions), a complex sample processing flow and professionals, so that the application in scenes such as field application, crowd detection and the like is very limited.
At present, the nucleic acid detection of the new coronavirus pneumonia mostly adopts a reverse transcription PCR fluorescence method, the detection period is long, at least 48-72 hours are needed, large, special and expensive equipment, reagents and professionals are relied on, and the detection sensitivity is about 30-50%. Meanwhile, after a sample of the new coronavirus pneumovirus is collected, viral nucleic acid (RNA) extraction and enrichment must be carried out, and the existing virus collection method is difficult to successfully reach the detection lower limit of the existing method, so that another important technical bottleneck is formed.
Disclosure of Invention
The object of the present invention is to provide a rapid detection technique for highly sensitive pathogenic nucleic acids for non-diagnostic and therapeutic purposes, which can bypass the pathogen extraction step.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a high-sensitivity pathogenic nucleic acid rapid detection technology for non-diagnosis and treatment purposes, which comprises the following steps:
(1) recombinase isothermal amplification;
(2) the colloidal gold developed color.
Preferably, the recombinase isothermal amplification process amplifies the ORF gene and the S gene separately.
Preferably, the forward ORF gene primer and the forward S gene primer used in the recombinase isothermal amplification process both contain a biotin label, and the reverse ORF gene primer and the reverse S gene primer both contain a FAM label or a digoxigenin label.
Preferably, the recombinase isothermal amplification time is 15-25 min.
Preferably, the temperature for the recombinase isothermal amplification is 40-44 ℃.
Preferably, the colloidal gold is developed by using a PCRD colloidal gold test paper.
Preferably, the amplified product of ORF gene and the amplified product of S gene are simultaneously detected on the same colloidal gold test paper during the color development of the colloidal gold.
Preferably, PCR of recombinase amplification products is further included between the step (1) and the step (2).
The invention also provides a primer pair of the ORF gene of the novel coronavirus, which comprises an ORF gene forward primer and an ORF gene reverse primer, wherein the sequence of the ORF gene forward primer is shown as SEQ ID NO: 1, the reverse primer sequence of the ORF gene is shown as SEQ ID NO: 2, respectively.
The invention also provides a novel primer pair of the S gene of the coronavirus, which comprises an S gene forward primer and an S gene reverse primer, wherein the sequence of the S gene forward primer is shown as SEQ ID NO: 3, the sequence of the S gene reverse primer is shown as SEQ ID NO: 4, respectively.
The invention provides a high-sensitivity pathogenic nucleic acid rapid detection technology for non-diagnosis and treatment purposes, which comprises the following steps: (1) recombinase isothermal amplification; (2) the colloidal gold developed color. The application discloses a high sensitive pathogenic nucleic acid rapid detection technique uses isothermal nucleic acid amplification-colloidal gold color development for pathogen spot detection, does not need special equipment and reagent, saves pathogen extraction step simultaneously, can limit capture and enlarge pathogen nucleic acid signal, sample such as direct detection patient's phlegm, blood, excrement and urine, be applicable to very much the spot detection, the bedside of hospital diagnoses, the application of crowd gathering life scene and other resource condition such as school, airport, especially need to obtain the scene of result fast urgently. Can solve the defects that the detection of pathogen nucleic acid depends on large-scale equipment and reagents, the detection period is long, the sensitivity is high and the pathogen needs to be extracted in the prior art.
Drawings
FIG. 1 is a photograph of agarose gel electrophoresis of example 4;
FIG. 2 shows the results of colloidal gold detection of single-gene detection of S gene, ORF gene and control group;
FIG. 3 shows the results of colloidal gold detection of double gene detection of S gene and ORF gene.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1 pathogen nucleic acid sequence determination and primer design
(1) Take the example of a novel coronavirus
New coronavirus sequences were obtained from https:// www.ncbi.nlm.nih.gov/nuccore/MN 908947.3.
(2) Design of new coronavirus ORF gene and S gene marker primer
ORF forward primer labeling: /5biotin/CG AAG TTG TAG GAG ACA TTA TAC TTAAACC
ORF reverse primer labeling: 56-FAM/TA GTA AGA CTA GAA TTG TCT ACA TAA GCA GC
ORF reverse primer labeling: 2/56-digoxigenin/TA GTA AGA CTA GAA TTG TCT ACA TAAGCA GC
Labeling of S forward primer: /5biotin/AG GTT TCA AAC TTT ACT TGC TTT ACA TAG A
Marking an S reverse primer: 56-FAM/TC CTA GGT TGA AGA TAA CCC ACA TAA TAAG
Marking an S reverse primer: 2/56-digoxigenin/TC CTA GGT TGA AGA TAA CCC ACA TAATAAG
Example 2 pathogen S and ORF Gene sequence amplification templates
(1) An RNA template;
(2) the heavy polymerase amplification kit is purchased from TwistDx, https:// www.twistdx.co.uk/en/products/product/twistamp-nfo;
(3) reverse transcriptase: protoSciptII reverse transfer, M0368L, New England Biotinlabs, https:// www.neb.com/products/M0368-protoscript-ii-reverse-transfer # product 20 information;
(4) the system of the Repolymerization (RPA) reaction is shown in Table 1;
(5) after the system was assembled, water bath was carried out at 42 ℃ for 20 min.
TABLE 1
The amplification is carried out separately in six groups, wherein four groups are used as experimental groups, the systems used for amplification are respectively numbered as a system 1, a system 2, a system 3 and a system 4, two groups are used as blank controls and are respectively numbered as a control group 1 and a control group 2, and no virus sequence RNA and ddH are added in the control group2The amount of O was adjusted to 12. mu.L, as shown in Table 1. The forward and reverse primer markers of the ORF gene used in the system 1 are biotin and FAM respectively; the forward and reverse primer markers of the ORF gene used in the system 2 are biotin and digoxigenin respectively; the forward and reverse primers of the S gene used in the system 3 are biotin and FAM respectively; the forward and reverse primers of the S gene used in the system 4 are biotin and digoxigenin respectively; the S gene forward and reverse primer markers used in the control group 1 are biotin and FAM respectively; the ORF gene forward and reverse primer markers used in control group 2 were biotin and FAM, respectively. The amplified product can be directly subjected to a colloidal gold color development step.
Example 3 PCR of recombinase amplification products
In the embodiment 2, the amplification products of the systems 1 to 4 are subjected to PCR re-amplification and then are subjected to colloidal gold color development.
(1) The PCR enzyme ExTaq (Takara);
(2) the PCR reaction system is shown in Table 2;
(3) after the system is assembled, the temperature is 95 ℃ for 3 min; 30s at 95 ℃, 30s at 55 ℃, 10s at 72 ℃ and 28 cycles; 5min at 72 ℃.
TABLE 2
EXAMPLE 4 agarose gel electrophoresis of PCR products
Taking 5 mu L of each amplification product from the PCR products of the systems 1 to 4 in the embodiment 3, carrying out agarose gel electrophoresis (2% agarose gel) at 120V for 30min, and imaging by using an imager, wherein M represents Marker I DNAader, 1 to 4 are electrophoresis results of the systems 1 to 4 respectively, and the result shows that the primer markers of different combinations in the systems 1 to 4 can amplify corresponding gene bands.
Example 5 Single Gene colloidal gold color development
Mixing the amplification products of the system 1, the system 3, the control group 1 and the control group 2 which are remained in the embodiment 2 with PCRDBuffer respectively, fixing the volume to 80 mu L, whirling and mixing uniformly, centrifuging by an instant centrifuge, dripping the mixed solution into a round sample application hole of PCRD colloidal gold test paper, standing for no more than 3min, and recording a band as shown in figure 2. The results of control 1 are shown in FIG. 2-1, the results of system 3 are shown in FIG. 2-2, the results of control 2 are shown in FIG. 2-3, and the results of system 1 are shown in FIG. 2-4. The results show that the primer marker selected by the application has excellent specificity.
Example 6 multigenic colloidal gold development
And (3) carrying out double-gene detection on the S gene and the ORF gene, amplifying the S gene and the ORF gene simultaneously, and simultaneously detecting the double genes on the same colloidal gold test paper to obtain the positive result.
(1) An RNA template;
(2) the heavy polymerase amplification kit is purchased from TwistDx, https:// www.twistdx.co.uk/en/products/product/twistamp-nfo;
(3) reverse transcriptase: protoSciptII reverse transfer, M0368L, New England Biotinlabs, https:// www.neb.com/products/M0368-protoscript-ii-reverse-transfer # product 20 information;
(4) the system of the Repolymerization (RPA) reaction is shown in Table 3;
(5) after the system was assembled, water bath was carried out at 42 ℃ for 20 min.
TABLE 3
Amplification was carried out separately in two separate sets, and the systems used for amplification were numbered as System 5 and System 6, respectively. The forward and reverse primer markers of the ORF gene used in the system 5 are biotin and digoxigenin, respectively, and the forward and reverse primer markers of the S gene are biotin and FAM, respectively; the forward and reverse primer markers of the ORF gene used in the system 6 are biotin and FAM, respectively, and the forward and reverse primer markers of the S gene are biotin and digoxigenin, respectively. And mixing the amplified products with 30 mu L of PCRD buffer respectively, uniformly mixing by vortex, centrifuging by an instant centrifuge, dripping the mixed solution into a circular sample application hole of a PCRD colloidal gold test paper, standing for no more than 3min, and recording strips. The results of system 5 are shown in FIG. 3-A, where band 1 detected the ORF gene, band 2 detected the S gene, band 3 indicated that the liquid had completely crossed the colloidal gold test strip. The results for system 6 are shown in FIG. 3-B. Wherein, the strip 1 detects S gene, the strip 2 detects ORF gene, the strip 3 shows that the liquid completely overflows the colloidal gold test strip.
As can be seen from the above examples, the present invention provides a rapid detection technique for highly sensitive pathogenic nucleic acids for non-diagnostic and therapeutic purposes, comprising the steps of: (1) recombinase isothermal amplification; (2) the colloidal gold develops color. The application discloses high sensitive pathogenic nucleic acid rapid detection technique uses isothermal nucleic acid amplification-colloidal gold color development for pathogen spot detection, does not need special equipment and reagent, saves pathogen extraction step simultaneously, can limit capture and enlarge pathogen nucleic acid signal, sample such as direct detection patient's phlegm, blood, excrement and urine, be applicable to very much the spot detection, the bedside of hospital diagnoses, the application of crowd gathering life scene such as school, airport and other resource condition fewly, especially the scene that needs to obtain the result fast urgently. Can solve the defects of dependence on large-scale equipment and reagents, long detection period, sensitivity and the need of extracting pathogens in the prior art for detecting pathogen nucleic acid.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Shenzhen Biotech (Shenzhen) Limited
<120> a rapid detection technique for highly sensitive pathogenic nucleic acids for non-diagnostic and therapeutic purposes
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tagtaagact agaattgtct acataagcag c 31
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aggtttcaaa ctttacttgc tttacataga 30
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<213> Artificial Sequence (Artificial Sequence)
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tcctaggttg aagataaccc acataataag 30
Claims (10)
1. A rapid detection technique for highly sensitive pathogenic nucleic acids for non-diagnostic and therapeutic purposes, comprising the steps of:
(1) recombinase isothermal amplification;
(2) the colloidal gold developed color.
2. The rapid detection technique for highly sensitive pathogenic nucleic acid for non-diagnostic and therapeutic purposes as claimed in claim 1, wherein the recombinase isothermal amplification process amplifies the ORF gene and S gene separately.
3. The rapid detection technique for highly sensitive pathogenic nucleic acid for non-diagnostic and therapeutic purposes as claimed in claim 2, wherein the forward primer of ORF gene and the forward primer of S gene used in the recombinase isothermal amplification process both contain biotin label, and the reverse primer of ORF gene and the reverse primer of S gene both contain FAM label or digoxigenin label.
4. The rapid detection technique for highly sensitive pathogenic nucleic acid for non-diagnostic and therapeutic purposes as claimed in claim 1, wherein the recombinase isothermal amplification time is 15-25 min.
5. The rapid detection technique for highly sensitive pathogenic nucleic acid for non-diagnostic and therapeutic purposes as claimed in claim 1, wherein the temperature of the recombinase isothermal amplification is 40-44 ℃.
6. The rapid detection technique for highly sensitive pathogenic nucleic acids for non-diagnostic and therapeutic purposes as claimed in claim 1, wherein the colloidal gold is developed using PCRD colloidal gold test paper.
7. The rapid detection technique for highly sensitive pathogenic nucleic acid for non-diagnostic and therapeutic purposes as claimed in claim 2, wherein the amplified product of ORF gene and the amplified product of S gene are simultaneously detected on the same colloidal gold strip during the development of colloidal gold.
8. The rapid detection technique for highly sensitive pathogenic nucleic acid for non-diagnostic and therapeutic purposes according to any of claims 1 to 7, further comprising PCR of recombinase amplification products between the steps (1) and (2).
9. The primer pair of the ORF gene of the novel coronavirus is characterized by comprising an ORF gene forward primer and an ORF gene reverse primer, wherein the sequence of the ORF gene forward primer is shown as SEQ ID NO: 1, the reverse primer sequence of the ORF gene is shown as SEQ ID NO: 2, respectively.
10. A primer pair of S gene of new coronavirus is characterized by comprising an S gene forward primer and an S gene reverse primer, wherein the sequence of the S gene forward primer is shown as SEQ ID NO: 3, the sequence of the S gene reverse primer is shown as SEQ ID NO: 4, respectively.
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Cited By (2)
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| CN112779354A (en) * | 2021-01-06 | 2021-05-11 | 常州市疾病预防控制中心 | Amplification system for rapidly detecting novel coronavirus at normal temperature and application thereof |
| CN114075611A (en) * | 2020-08-10 | 2022-02-22 | 华南理工大学 | Double-target SARS-CoV-2 virus nucleic acid detection primer group, application and fluorescent kit |
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| CN111074007A (en) * | 2020-02-15 | 2020-04-28 | 上海迪飞医学检验实验室有限公司 | Isothermal amplification kit and primer probe set for detecting SARS-COV-2 virus |
| CN111257555A (en) * | 2020-04-03 | 2020-06-09 | 安徽省疾病预防控制中心（省健康教育所） | Rapid detection method and test strip for lateral chromatography colloidal gold of new coronavirus nucleic acid recombinase mediated isothermal amplification |
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| CN111074007A (en) * | 2020-02-15 | 2020-04-28 | 上海迪飞医学检验实验室有限公司 | Isothermal amplification kit and primer probe set for detecting SARS-COV-2 virus |
| CN111257555A (en) * | 2020-04-03 | 2020-06-09 | 安徽省疾病预防控制中心（省健康教育所） | Rapid detection method and test strip for lateral chromatography colloidal gold of new coronavirus nucleic acid recombinase mediated isothermal amplification |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114075611A (en) * | 2020-08-10 | 2022-02-22 | 华南理工大学 | Double-target SARS-CoV-2 virus nucleic acid detection primer group, application and fluorescent kit |
| CN112779354A (en) * | 2021-01-06 | 2021-05-11 | 常州市疾病预防控制中心 | Amplification system for rapidly detecting novel coronavirus at normal temperature and application thereof |
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