CN116334315A - Primer for detecting human respiratory tract related circovirus HRAPLV and application thereof - Google Patents
Primer for detecting human respiratory tract related circovirus HRAPLV and application thereof Download PDFInfo
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- 108020004707 nucleic acids Proteins 0.000 claims abstract description 20
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- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 11
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- 239000013641 positive control Substances 0.000 claims description 5
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- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000012165 high-throughput sequencing Methods 0.000 abstract description 2
- 238000003908 quality control method Methods 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 230000003321 amplification Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
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- 102000004169 proteins and genes Human genes 0.000 description 3
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- 102000053602 DNA Human genes 0.000 description 1
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- 230000001681 protective effect Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a primer for detecting human respiratory tract related circovirus HRAPLV and application thereof, belonging to the technical field of molecular biology. The primer sequence for detecting the human respiratory tract related cyclic virus HRAPLV is as follows: upstream primer-F: 5'-GTGGGTGCAGTTTCTTCACT-3', downstream primer-R: 5'-TGCACGTTTGCACGGATAAA-3'; the specific primer for detecting the HRAPLV virus has higher sensitivity and specificity, can give qualitative detection results within 2 hours after receiving a HRAPLV virus nucleic acid sample, does not need complex operations such as high-throughput sequencing, saves detection cost and time, has low requirements on personnel and equipment, and is an effective tool for detecting the HRAPLV.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a primer for detecting human respiratory tract related cyclic virus HRAPLV and application thereof.
Background
Human respiratory related circovirus (Human respiratory-associated PSCV-5-like virus, HRAPLV) is a human circovirus newly found in respiratory tract samples of national port entry personnel in China in recent years. HRAPLV is isolated and identified from a human upper respiratory tract sample, and has a genome structure which has higher consistency with porcine feces-related circovirus (porcine stop-associated circular virus 5 isolate CP3, poSCV 5) and a relatively similar evolutionary relationship. The HRAPLV virus genome is single-stranded circular DAN (single-strand DNA, ssDNA), the genome is 3018 bp in full length, comprising two major open reading frames ORFs (open reading frames, ORFs), encoding two major proteins, namely, envelope and replicase proteins (Rep), respectively. The Cap protein and the Rep protein of the HRAPLV virus have the amino acid sequence identity of 53% and 48.9% respectively with PoSCV 5. In addition, there is a neck ring structure inside the HRAPLV virus genome.
Tissue tropism, pathogenicity, immunological properties, epidemic characteristics, etc. of HRAPLV are currently unknown and related biological characteristics are still under investigation. Because the HRAPLV virus belongs to the newly discovered human respiratory tract related cyclic virus, the related detection technical researches of molecular biology, serology and the like are not reported so far, the research on the accurate, rapid, sensitive and specific molecular detection technical method aiming at the virus is urgently needed to be developed, and the technical support effect is provided for early discovery of early treatment. Early prevention and control are preconditions for preventing infectious diseases from spreading, guaranteeing public health safety and maintaining human life health, and developing a rapid, sensitive and specific molecular detection technical method is a key for early prevention and control, and provides important basis for guiding clinic, reasonably selecting antiviral drugs and the like.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide a primer for detecting the human respiratory tract related annular virus HRAPLV. The invention aims to provide an application of a primer for detecting the human respiratory tract related annular virus HRAPLV, which is used for rapidly, sensitively and specifically detecting the HRAPLV virus.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a primer for detecting human respiratory-related cyclic virus HRAPLV, the primer sequence being:
upstream primer-F: 5'-GTGGGTGCAGTTTCTTCACT-3' the number of the individual pieces of the plastic,
downstream primer-R: 5'-TGCACGTTTGCACGGATAAA-3'.
Application of primer for detecting human respiratory tract related circovirus (HRAPLV) in detecting HRAPLV virus nucleic acid sample.
Further, the specific steps include:
1) Extracting nucleic acid from the sample;
2) And carrying out PCR pathogen detection by taking the extracted nucleic acid as a template.
Further, the PCR reaction system comprises: 2.5. Mu.L of 10 Xbuffer, 1.5. Mu.L of dNTPs, 1.5. Mu.L of MgCl 2 0.5 mu L of Taq enzyme, 1 mu L of each of the upstream and downstream primers, 0.5 mu L of the fluorescent probe, 2 mu L of the template DNA, and the volume of the sterile double distilled water is up to 25 mu L.
Further, the concentration of the upstream and downstream primers was 50 pmol/. Mu.L.
Further, the concentration of the fluorescent probe was 25 pmol/. Mu.L.
Further, the fluorescent probe sequence is as follows:
fluorescent probe Pro:5'-FAM-CCTGCAAACTCCTTAACGCTAAGCGC-3' MGB.
The kit for detecting the human respiratory tract-related cyclic virus HRAPLV comprises a primer reagent for detecting the human respiratory tract-related cyclic virus HRAPLV, wherein the primer sequence is as follows:
upstream primer-F: 5'-GTGGGTGCAGTTTCTTCACT-3' the number of the individual pieces of the plastic,
downstream primer-R: 5'-TGCACGTTTGCACGGATAAA-3'.
Further, the kit also comprises a positive control sequence shown as SEQ ID NO.1 and a fluorescent probe, wherein the sequence of the fluorescent probe is as follows:
fluorescent probe Pro:5'-FAM-CCTGCAAACTCCTTAACGCTAAGCGC-3' MGB.
The kit for detecting the human respiratory tract-related circovirus HRAPLV is applied to detection of a HRAPLV virus nucleic acid sample.
Compared with the prior art, the invention has the beneficial effects that:
1) The specific primer for detecting the HRAPLV virus has higher sensitivity and specificity, can give a qualitative detection result within 2 hours after receiving a HRAPLV virus nucleic acid sample, does not need complex operations such as high-throughput sequencing, saves detection cost and time, has low requirements on personnel and equipment, and is an effective tool for detecting the HRAPLV.
2) The specific primer for detecting the HRAPLV virus can conveniently, rapidly and accurately detect the human respiratory tract related annular virus (HRAPLV), and provides a molecular detection means for related researches such as molecular epidemiology, molecular biology, bioinformatics and etiology of the HRAPLV.
Drawings
FIG. 1 is a diagram of the detection results of a specificity experiment (1: positive quality control; 2: HRAPLV nucleic acid template; 3: negative control; 4: other respiratory viruses such as influenza virus);
FIG. 2 is a graph showing the results of sensitivity test (HRAPLV nucleic acid template concentrations 1-8 are 10 ng/. Mu.L, 1 ng/. Mu.L, 10, respectively) -1 ng/μL、10 -2 ng/μL、10 -3 ng/μL、10 -4 ng/μL、10 -5 ng/μL、10 -6 ng/μL）；
FIG. 3 is a graph of the fluorescent quantitative PCR detection result of group A samples (1 is a quality control product; 2 is a positive detection sample);
FIG. 4 is a graph of the fluorescent quantitative PCR detection result of the group B sample (1 is a quality control product);
FIG. 5 is a graph of the results of fluorescent quantitative PCR detection of group C samples (1 is a quality control product; 2 is a positive detection sample).
Description of the embodiments
The present invention will be further described with reference to specific embodiments for the purpose of making the objects, technical solutions and advantages of the present invention more apparent. Unless otherwise indicated, all technical means used in the following examples are conventional means well known to those skilled in the art.
The instrument used in this experiment:
full automatic nucleic acid extractor (DB-48, jiangsu Bernard Biotechnology development Co., ltd., su Ningxie, 20200112).
Fluorescent quantitative PCR instrument (Quantum 5, life Technologyies, national institute of technology, 20183400252).
Biological safety cabinets, ultra-clean benches, low temperature high speed centrifuges, ultraviolet spectrophotometers, metal baths, and the like are all purchased from zemoer femtoo (ThermoFisher Scientific).
Examples
1. Referring to the whole genome sequence information (GenBank No. KY 052047) of human respiratory tract-related circovirus (Human respiratory-associated PSCV-5-like virus), a pair of specific primers, a specific fluorescent probe and a synthetic gene fragment (quality control product) are designed, and the sequence information of the primers, the probe and the quality control product are shown in Table 1:
2. the nucleic acid detection is carried out by taking RNA reverse transcription products of influenza virus, parainfluenza virus, respiratory syncytial virus, human coronavirus and rhinovirus as templates, taking adenovirus genome and bocavirus genome as templates, taking water as a negative control and taking a quality control as a positive control.
The 25. Mu.L reaction system comprises: 2.5. Mu.L of 10 Xbuffer, 1.5. Mu.L of dNTPs, 1.5. Mu.L of MgCl2, 0.5. Mu.L of Taq enzyme, 1. Mu.L of each of the upstream and downstream primers (concentration: 50 pmol/. Mu.L), 0.5. Mu.L of fluorescent probe (concentration: 25 pmol/. Mu.L), 2. Mu.L of template DNA, and a volume of sterile double distilled water to 25. Mu.L. Sterile double distilled water is used as a template and a quality control product is used as a positive control. The PCR reaction conditions were set as follows: 42 ℃ for 10min; pre-denaturation at 95 ℃ for 5min;94℃for 15s and 58℃for 45s (fluorescence signal was collected), 40 cycles.
The result is shown in figure 1, the negative control has no amplification curve (3), the positive quality control (1) has an S-shaped amplification curve, the detection sample added with the HRAPLV nucleic acid template (2) has the S-shaped amplification curve, the Ct value is less than or equal to 38, and other respiratory tract virus templates (4) such as influenza virus have no S-shaped amplification curve, so that the detection method has good specificity for the human respiratory tract related annular virus HRAPLV.
3. Quantifying HRAPLV nucleic acid template with spectrophotometer, adjusting concentration to 10 ng/. Mu.L, and diluting to 10 times -6 ng/. Mu.L, corresponding to the indication of line 8 in FIG. 2, a total of 8 concentrations, determining a lower detection limit of 10 -5 ng/. Mu.L, experimental operations were performed according to the primer, probe and reagent configuration procedure and the PCR reaction procedure in the present invention, and it was determined that the detection limit was 10 -5 ng/. Mu.L, the results are shown in FIG. 2 when diluted to 10 in sequential doubling ratios -5 The standard S-shaped curve can be amplified at ng/. Mu.L, and the standard S-shaped curve can be diluted to 10 -6 No S-shaped curve is amplified at ng/. Mu.L, which shows that the detection limit of the method is 10 -5 ng/. Mu.L, has good detection sensitivity.
Examples
1. Collecting 1200 parts of throat swab samples of patients entering the mouth of Xinjiang from 3 months 2021 to 2 months 2023, and averagely dividing the throat swab samples into A, B, C groups of 400 parts each, and freezing at-80 ℃.
Extracting nucleic acid from collected throat swab sample of inbound personnel by using a full-automatic nucleic acid extractor (DB-48), and freezing the extracted nucleic acid at-80 ℃ for later use.
2. PCR pathogen detection
The extracted nucleic acid is used as a template, and PCR pathogen detection is carried out by using the primer and the probe in the invention.
The 25. Mu.L reaction system comprises: 2.5. Mu.L of 10 Xbuffer, 1.5. Mu.L of dNTPs, 1.5. Mu.L of MgCl 2 0.5. Mu.L of Taq enzyme, 1. Mu.L of each of the upstream and downstream primers (For, rev) (concentration: 50 pmol/. Mu.L), 0.5. Mu.L of the fluorescent probe (Pro) (concentration: 25 pmol/. Mu.L), 2. Mu.L of the template DNA, and the volume of the sterile double distilled water was made up to 25. Mu.L. Sterile double distilled water is used as a template and a quality control product is used as a positive control.
Setting the PCR reaction condition at 42 ℃ for 10min; pre-denaturation at 95 ℃ for 5min; the sample was subjected to 40 cycles at 94℃for 15s and 58℃for 45s (fluorescence signal was collected).
3. Results are shown in fig. 3-5, by using the primer, probe, quality control product and detection method of the present invention to detect each 1 of the HRAPLV from the group a and group C entry throat swab samples, rapid, sensitive and specific detection of the HRAPLV virus in the respiratory tract sample can be achieved, and a kit for HRAPLV virus detection can be prepared.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as protective scope of the present invention.
Claims (10)
1. A primer for detecting human respiratory-related cyclic virus HRAPLV, the primer sequence being:
upstream primer-F: 5'-GTGGGTGCAGTTTCTTCACT-3' the number of the individual pieces of the plastic,
downstream primer-R: 5'-TGCACGTTTGCACGGATAAA-3'.
2. Use of the primer for detecting human respiratory-related cyclic virus HRAPLV according to claim 1 in detecting a nucleic acid sample of HRAPLV virus.
3. The use of a primer for detecting human respiratory-related cyclic virus HRAPLV according to claim 2, in detecting a nucleic acid sample of HRAPLV virus, comprising the specific steps of:
1) Extracting nucleic acid from the sample;
2) And carrying out PCR pathogen detection by taking the extracted nucleic acid as a template.
4. The use of a primer for detecting human respiratory-related cyclic virus HRAPLV according to claim 3, wherein the PCR reaction system comprises: 2.5. Mu.L of 10 Xbuffer, 1.5. Mu.L of dNTPs, 1.5. Mu.L of MgCl 2 0.5 mu L of Taq enzyme, 1 mu L of each of the upstream and downstream primers, 0.5 mu L of the fluorescent probe, 2 mu L of the template DNA, and the volume of the sterile double distilled water is up to 25 mu L.
5. The use of the primer for detecting human respiratory-related circovirus HRAPLV according to claim 4, wherein the concentration of the upstream and downstream primer is 50 pmol/. Mu.l.
6. The use of the primer for detecting human respiratory-related circovirus HRAPLV according to claim 4, wherein the concentration of the fluorescent probe is 25 pmol/. Mu.l.
7. The use of the primer for detecting human respiratory-related cyclic virus HRAPLV according to claim 6, wherein the fluorescent probe sequence is:
fluorescent probe Pro:5'-FAM-CCTGCAAACTCCTTAACGCTAAGCGC-3' MGB.
8. The kit for detecting the human respiratory tract-related cyclic virus HRAPLV is characterized by comprising a primer reagent for detecting the human respiratory tract-related cyclic virus HRAPLV, wherein the primer sequence is as follows:
upstream primer-F: 5'-GTGGGTGCAGTTTCTTCACT-3' the number of the individual pieces of the plastic,
downstream primer-R: 5'-TGCACGTTTGCACGGATAAA-3'.
9. The kit for detecting human respiratory-related cyclic virus HRAPLV according to claim 8, further comprising a positive control sequence shown in SEQ ID No.1 and a fluorescent probe having the sequence:
fluorescent probe Pro:5'-FAM-CCTGCAAACTCCTTAACGCTAAGCGC-3' MGB.
10. Use of a kit for detecting the human respiratory-related cyclic virus HRAPLV according to any one of claims 8-9 for the detection of HRAPLV virus.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206715A (en) * | 2011-04-22 | 2011-10-05 | 上海市动物疫病预防控制中心 | Kit and method for detecting porcine circovirus type-2 (PCV-2) and subtypes thereof |
| US20120100168A1 (en) * | 2010-01-25 | 2012-04-26 | Blood Systems, Inc. | Cyclovirus and methods of use |
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- 2023-04-13 CN CN202310392288.0A patent/CN116334315A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120100168A1 (en) * | 2010-01-25 | 2012-04-26 | Blood Systems, Inc. | Cyclovirus and methods of use |
| CN102206715A (en) * | 2011-04-22 | 2011-10-05 | 上海市动物疫病预防控制中心 | Kit and method for detecting porcine circovirus type-2 (PCV-2) and subtypes thereof |
Non-Patent Citations (1)
| Title |
|---|
| LUNBIAO CUI 等: "Identification and genetic characterization of a novel circular single‑stranded DNA virus in a human upper respiratory tract sample", ARCH VIROL, vol. 162, pages 3305, XP036338669, DOI: 10.1007/s00705-017-3481-3 * |
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