CN111560475A - Redondvirus virus nucleic acid detection kit - Google Patents
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a Redondorvus virus nucleic acid detection kit, which comprises the following components of a Redondorvus virus PCR amplification reaction solution, a positive control and a negative control; the invention firstly establishes a way for diagnosing the nucleic acid detection of Redondorvus virus infection, the kit has very high specificity and sensitivity, the technical operation is not complex, and the kit can be beneficial to early diagnosis, diagnosis of asymptomatic patients and tracking of disease course; the detection of the kit is a totally-enclosed operation, and a tube cover can not be opened after a sample is added, so that the probability of pollution is reduced; the kit has strong repeatability and rapid and objective detection result; has great application prospect in the fields of clinical diagnosis and disease prevention and detection.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a Redondovus virus nucleic acid detection kit.
Background
Redondrovir was found to be present in humans in 2019, colonizes the respiratory system of humans, and is associated with a variety of diseases in humans. Redondrovir encodes two single-stranded DNA viruses from the circular, Rep-encoded, proteins similar to capsid and Rep proteins, and a highly conserved open reading frame, with no homology to any known protein family. Redondroviruses are the second pandemic virus in respiratory specimens, second only to rubella virus.
The development of the monitoring of the Redondorvus virus is of great significance to epidemiological research and the development of lung transplantation. At present, no method for diagnosing Redondorvus virus infection is established in hospitals, but virus detection methods mainly comprise virus isolation culture, immunological diagnosis and nucleic acid detection methods from the virology perspective. The virus isolation culture detection operation is complex, the time is long, the result can be reported in several weeks, and the sensitivity is low; the immunological diagnosis mainly takes a gold standard method and EIA as main materials, has the advantages of rapidness, simplicity, convenience and the like, but has poorer specificity and sensitivity; the nucleic acid detection method is the latest detection method which is just emerging, has rapid development, very high specificity and sensitivity and uncomplicated technical operation, and can be beneficial to early diagnosis, asymptomatic patient diagnosis and disease course tracking.
The most widely used real-time fluorescence PCR technology based on fluorescence labeling probe is currently applied to nucleic acid diagnosis in China. The detection probe is an oligonucleotide comprising a 5 'end reporter group and a 3' end quenching group. When the probe is intact, the fluorescence emitted by the reporter is greatly reduced due to the proximity of the quencher to the reporter. When the primer is extended, the probe bound to the template is cleaved by Taq enzyme (5 '→ 3' exonuclease activity), and the reporter group is separated from the quencher group, thereby generating a fluorescent signal. In each PCR cycle, a new reporter group is cleaved, and thus the increase in fluorescence signal intensity is proportional to the amount of amplification product. For fluorescent PCR detection of viruses, the design of primers is crucial, which directly affects the specificity of detection. If the specificity of the primer is insufficient, false positives may occur.
The double fluorescence quantitative PCR technology is to add two probes with different fluorescence labels into the same reaction system. For example, the probe for target gene 1 is labeled FAM and the probe for target gene 2 is labeled VIC. In the PCR reaction process, if a sample to be detected contains a target gene 1, a probe for marking FAM generates a fluorescence signal; if the sample to be detected contains the target gene 2, the VIC-labeled probe generates a fluorescent signal. Thus, the same tube reaction can identify two target genes.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a method for tracing stem cells of cynomolgus monkeys by fluorescence.
In order to achieve the purpose, the technical scheme of the invention is as follows: a Rendoviras virus nucleic acid detection kit is designed, and comprises the following components of a Rendoviras virus PCR amplification reaction solution, a positive control and a negative control.
Preferably, the PCR amplification reaction solution for the Redondovir viruses comprises the following components:
a component (1): a pair of primers for detecting a Redondovirus virus; wherein, the sequences of the two primers are respectively SEQ ID No.1 and SEQ ID No.2, and the nucleotide sequences of the primers SEQ ID No.1 and SEQ ID No.2 are shown in Table 1:
TABLE 1 primer of Redonovarus virus and probe sequence thereof
A component (2): the probe for detecting the Redondovirus virus is shown in SEQ ID No.3, the nucleotide sequence of SEQ ID No.3 is shown in Table 1, the 5 'end of the probe is marked with a fluorescent reporter group, and the 3' end of the probe is marked with a fluorescent quenching group.
Preferably, the molar ratio of the primer for detecting the Redondorvus virus to the probe for detecting the Redondorvus virus is as follows: SEQ ID No. 1: SEQ ID No. 2: SEQ ID No.3 is 1:1: 2.
Preferably, the fluorescence reporter group in the component (2) is FAM, and the fluorescence quencher group is BHQ 1.
Preferably, the Redondovir virus PCR amplification reaction solution further comprises an internal standard, an internal standard primer and an internal standard probe for detecting the internal standard, wherein the internal standard is human glyceraldehyde-3-phosphate dehydrogenase housekeeping gene GAPDH, the internal standard primer and the internal standard probe are respectively SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, the molar ratio is 1:1:2, and the nucleotide sequences of SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 are shown in Table 2:
TABLE 2 internal standard primers and internal standard probes
| Name (R) | Sequence of |
| SEQ ID No.4 | 5'-TCAAGAAGGTGGTGAAGCAGG-3' |
| SEQ ID No.5 | 5'-CAGCGTCAAAGGTGGAGGAGT-3' |
| SEQ ID No.6 | 5'-VIC-CCTCAAGGGCATCCTGGGCTACACT-BHQ1-3' |
Preferably, the positive control is inactivated respiratory tract Redondorovirus virus culture solution, and the negative control is double distilled water.
Preferably, the PCR amplification reaction solution for the Redondovir viruses further comprises DNA polymerase, dNTPs and PCRbuffer.
Preferably, the DNA polymerase is Taq DNA polymerase.
Preferably, the PCR buffer comprises: mg (magnesium)2+，Tris-Cl、KCL。
Preferably, any one of the Redondorvus virus nucleic acid detection kit is applied to preparation of a reagent for detecting Redondorvus virus.
Compared with the prior art, the beneficial effect of this application is:
1. the invention establishes a way for diagnosing the nucleic acid detection of Redondorvus virus infection for the first time, the kit has very high specificity and sensitivity, the technical operation is not complex, and the kit can be beneficial to early diagnosis, diagnosis of asymptomatic patients and tracking of disease course;
2. the detection of the kit is a totally-enclosed operation, and a tube cover can not be opened after a sample is added, so that the probability of pollution is reduced;
3. the kit has strong repeatability and rapid and objective detection result; has great application prospect in the fields of clinical diagnosis and disease prevention and detection.
Drawings
FIG. 1 is a graph showing a test sample of the kit of the present invention;
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The reagents used in the examples are all commercially available;
example 1
A Redondorvus virus nucleic acid detection kit comprises the following components of a Redondorvus virus PCR amplification reaction solution, a positive control and a negative control.
Preferably, the PCR amplification reaction solution for the Redondovir viruses comprises the following components:
a component (1): a pair of primers for detecting a Redondovirus virus; wherein, the sequences of the two primers are SEQ ID No.1 and SEQ ID No.2 respectively;
a component (2): the probe for detecting the Redondovirus virus is shown in SEQ ID No.3, the nucleotide sequence of SEQ ID No.3 is shown in Table 1, the 5 'end of the probe is marked with a fluorescent reporter group, and the 3' end of the probe is marked with a fluorescent quenching group.
Preferably, the molar ratio of the primer for detecting the Redondorvus virus to the probe for detecting the Redondorvus virus is as follows: SEQ ID No. 1: SEQ ID No. 2: SEQ ID No.3 is 1:1: 2.
Preferably, the fluorescence reporter group in the component (2) is FAM, and the fluorescence quencher group is BHQ 1.
Preferably, the Redondovir virus PCR amplification reaction solution further comprises an internal standard, an internal standard primer and an internal standard probe for detecting the internal standard, wherein the internal standard is human glyceraldehyde-3-phosphate dehydrogenase housekeeping gene GAPDH, the internal standard primer and the internal standard probe are respectively SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, and the molar ratio is 1:1: 2;
preferably, the positive control is inactivated respiratory tract Redondorovirus virus culture solution, and the negative control is double distilled water.
Preferably, the PCR amplification reaction solution for the Redondovir viruses comprises DNA polymerase, dNTPs and PCRbuffer.
Preferably, the DNA polymerase is Taq DNA polymerase.
Preferably, the PCR buffer comprises: mg (magnesium)2+，Tris-Cl、KCL。
Preferably, any one of the Redondorvus virus nucleic acid detection kit is applied to preparation of a reagent for detecting Redondorvus virus.
Example 2
Preparing a Redondorvus virus nucleic acid detection kit;
the kit comprises a PCR amplification reaction solution of a Redondovus virus, a positive control and a negative control.
The Redondorvus virus PCR amplification reaction solution comprises the following components:
a component (1): a pair of primers for detecting a Redondovirus virus; wherein, the sequences of the two primers are respectively SEQ ID No.1 and SEQ ID No.2, and the concentration of the primer for detecting the Redondovirus is as follows: SEQ ID No.1 was 10. mu.M (. mu.mol/L) and SEQ ID No.2 was 10. mu.M.
A component (2): the probe for detecting the Redondovirus virus has a sequence of SEQ ID No.3, the concentration of the SEQ ID No.3 is 10 mu M, the 5 'end of the probe is marked with a fluorescence reporter group, and the 3' end of the probe is marked with a fluorescence quenching group.
Wherein the molar ratio of the primer for detecting the Redondorvus virus to the probe for detecting the Redondorvus virus is as follows: SEQ ID No. 1: SEQ ID No. 2: SEQ ID No.3 is 1:1: 2; the fluorescence reporter group in the component (2) is FAM, and the fluorescence quenching group is BHQ 1.
The Redondovirus PCR amplification reaction solution also comprises an internal standard primer and an internal standard probe for detecting an internal standard, wherein the internal standard is human glyceraldehyde-3-phosphate dehydrogenase housekeeping gene GAPDH, the internal standard primer and the internal standard probe are respectively SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, the molar ratio is 1:1:2, and the concentrations of the three are 10 mu M; the probe is marked with a fluorescence reporter group VIC at the 5 'end and a fluorescence quenching group BHQ1 at the 3' end.
Wherein the positive control is inactivated respiratory Redondorovirus virus culture solution, and the negative control is double distilled water.
The Redondovirus PCR amplification reaction solution also comprises DNA polymerase, dNTPs and PCR buffer, wherein the concentration of the dNTPs is 0.2 mmol/L/reaction.
Wherein the DNA polymerase is Taq DNA polymerase, and the concentration of the Taq DNA polymerase is 5U/reaction.
Wherein, the PCR buffer comprises: mg (magnesium)2+The concentration was 1.5mmol/L, the concentration of Tris-Cl was 10mmol/L, KCL and the concentration was 50 mmol/L.
1. Extraction of sample DNA
1.1 taking a corresponding amount of 1.5ml centrifuge tubes, marking the specimen, negative control and positive control, and adding 600. mu.l of nucleic acid extraction solution 1(SDS, Triton-100, GuScN and magnetic beads) into each tube;
1.2 adding 200 mul specimen, negative control and positive control into each tube, mixing, and isolating instantly;
1.3 adding 100 μ l of nucleic acid extraction solution 2(HEPES, NaCl) into each tube, mixing, and standing at room temperature for 30 min;
1.4 placing the centrifugal tube on a magnetic frame, adsorbing for 3min, and slowly sucking out the solution;
1.5 adding 600 mul of nucleic acid extraction solution 3(Triton-100, NaCl) and 200 mul of nucleic acid extraction solution 4 (mineral oil) into each tube, mixing uniformly, placing the centrifugal tube on a magnetic frame after instantaneous separation, adsorbing for 3min, inserting a suction head into the bottom of the centrifugal tube, slowly and completely sucking out and discarding the liquid from the bottom, and completely sucking out and discarding the residual liquid at the bottom of the centrifugal tube after instantaneous separation;
1.6 adding 30 mul of nucleic acid eluent, eluting the magnetic beads on the wall of the centrifugal tube to the bottom of the tube, sucking and uniformly mixing for 3-4 times, standing for 10min at room temperature, placing the centrifugal tube on a magnetic frame again, and obtaining the solution which is the purified DNA.
2. A Redondorvus virus detection kit (prepared in example 2) having the components shown in Table 3 below and a DNA sample were placed in each PCR reaction tube;
TABLE 3
| Reaction solution Components | Adding (mu l)/1 part of the mixture |
| PCR amplification reaction solution for Redondvirus virus | 14.5 |
| Negative control (double distilled water) | 6.5 |
| RNA samples | 4 |
| Total | 25 |
3. PCR amplification was performed according to the following procedure;
95 ℃ for 20 seconds
4. Judging whether the Redonvirous virus is infected or not according to the fluorescence curve, wherein the amplification curve is shown in figure 1;
5. and (4) judging a result:
5.1, firstly, analyzing whether an amplification curve exists in the VIC channel by the internal standard, wherein Ct is less than or equal to 40, if yes, the detection is effective, and the subsequent analysis can be continued;
5.1.1 if the FAM channel detects a typical S-type amplification curve and Ct is less than or equal to 40, indicating that the Redondorvus virus is positive;
5.1.2 if the FAM channel does not detect a typical S-type amplification curve (NoCt) or Ct > 40, this indicates that the Redondovirus virus is negative.
5.2 if the internal standard does not detect Ct or Ct is more than 40 in the VIC channel, the result shows that the concentration of the detected sample is too low or the reaction is inhibited by the interfering substances, and the experiment needs to be repeated.
5.3 for positive samples, the internal standard detection result does not make requirements; for a negative detection sample, the internal standard detection is positive, if the internal standard detection is negative, the detection result of the sample is invalid, the reason should be searched and eliminated, the sample is re-sampled, and the repeated experiment is carried out.
5.4 judging the gray scale region result: if the fluorescence signal of a certain sample in the FAM channel is obviously amplified, but the Ct value is more than 40, the sample is in a gray area and needs to be rechecked. If the rechecking result is still in the gray scale area, the result is judged to be positive.
Sequence listing
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Claims (10)
1. A Redondorvus virus nucleic acid detection kit is characterized by comprising a PCR amplification reaction solution of the Redondorvus virus, a positive control and a negative control.
2. The Redondorus virus nucleic acid detection kit as claimed in claim 1, wherein the Redondorus virus PCR amplification reaction solution comprises the following components:
a component (1): a pair of primers for detecting a Redondovirus virus; wherein, the sequences of the two primers are respectively shown as SEQ ID No.1 and SEQ ID No. 2;
a component (2): the probe for detecting the Redondovirus virus has a sequence shown in SEQ ID No.3, and is marked with a fluorescent reporter group at the 5 'end and a fluorescent quenching group at the 3' end.
3. The Redondorus virus nucleic acid detection kit as claimed in claim 2, wherein the molar ratio of the primer for detecting the Redondorus virus to the probe for detecting the Redondorus virus is as follows: SEQ ID No. 1: SEQ ID No. 2: SEQ ID No.3 is 1:1: 2.
4. The Redondovir virus nucleic acid detection kit as claimed in claim 2, wherein the component (2) comprises FAM as a fluorescence reporter group and BHQ1 as a fluorescence quencher group.
5. The Redondovir virus nucleic acid detection kit according to claim 1, wherein the Redondovir virus PCR amplification reaction solution further comprises an internal standard, an internal standard primer and an internal standard probe for detecting the internal standard, the internal standard is human glyceraldehyde-3-phosphate dehydrogenase housekeeping gene GAPDH, the internal standard primer and the internal standard probe are respectively SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, and the molar ratio is 1:1: 2.
6. The Redondorvus virus nucleic acid detection kit according to claim 1, wherein the positive control is an inactivated respiratory Redondorvus virus culture solution, and the negative control is double distilled water.
7. The Rendovir virus nucleic acid detection kit as claimed in claim 1, wherein the Rendovir virus PCR amplification reaction solution further comprises DNA polymerase, dNTPs and PCR buffer.
8. The Redondovir virus nucleic acid detection kit as claimed in claim 6, wherein the DNA polymerase is Taq DNA polymerase.
9. The Redondovir viral nucleic acid detection kit according to claim 6, wherein the PCR buffer comprises: mg (magnesium)2+，Tris-Cl、KCL。
10. Use of a Redondorvus virus nucleic acid detection kit according to any one of claims 1 to 7 in the preparation of a reagent for detecting a Redondorvus virus.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012002594A1 (en) * | 2010-07-01 | 2012-01-05 | (주)바이오니아 | Diagnostic primer for the hepatitis c virus, probe, kit including same, and method for diagnosing the hepatitis c virus using the kit |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012002594A1 (en) * | 2010-07-01 | 2012-01-05 | (주)바이오니아 | Diagnostic primer for the hepatitis c virus, probe, kit including same, and method for diagnosing the hepatitis c virus using the kit |
Non-Patent Citations (3)
| Title |
|---|
| ARWA A ABBAS等: "Redondoviridae, a Family of Small, Circular DNA Viruses of the Human Oro-Respiratory Tract Associated with Periodontitis and Critical Illness" * |
| KRISTIN NOELL等: "Further Defining the Human Virome using NGS: Identification of Redondoviridae" * |
| ZHILIANG H等: "Clinical characteristics of 24 asymptomatic infections with COVID-19 screened among close contacts in Nanjing, China" * |
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