CN113981147B - Composition for detecting various respiratory viruses, kit and application thereof - Google Patents
Composition for detecting various respiratory viruses, kit and application thereof Download PDFInfo
- Publication number
- CN113981147B CN113981147B CN202111362220.5A CN202111362220A CN113981147B CN 113981147 B CN113981147 B CN 113981147B CN 202111362220 A CN202111362220 A CN 202111362220A CN 113981147 B CN113981147 B CN 113981147B
- Authority
- CN
- China
- Prior art keywords
- seq
- composition
- virus
- viruses
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- C12Q1/70—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
-
- 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
- C12Q1/6851—Quantitative amplification
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a composition for detecting various respiratory viruses, a kit and application thereof, wherein the kit can simultaneously and rapidly detect and identify 6 viruses causing acute respiratory infection of human and 2 viruses possibly causing respiratory infection of animals which are commonly suffered by human and livestock, has extremely high detection sensitivity and specificity, accurate and reliable detection results, can effectively distinguish the pathogens, realizes symptomatic drug delivery, is beneficial to epidemic prevention and control, and is an ideal choice for detecting and identifying acute respiratory infection.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a composition and application thereof in virus detection, and in particular relates to a composition for detecting various respiratory viruses, a kit and application thereof.
Background
Acute respiratory infections are a significant cause of morbidity and mortality in patients of all ages, especially young children, elderly and immunocompromised persons. Most of these infections are caused by influenza B (IFB), adenovirus (AdV), human Respiratory Syncytial Virus (HRSV), rhinovirus (HRV) and parainfluenza virus (HPIV). The novel coronavirus (SARS-CoV-2) is similar to the clinical symptoms and epidemic characteristics of these common respiratory viruses, and it is difficult to identify the actual pathogen only by clinical symptoms. The treatment methods, the curative effects and the disease courses of infections caused by different pathogens are different, and the pathogens cannot be effectively distinguished, so that symptomatic drug delivery is difficult to achieve, and epidemic disease prevention and control are not facilitated. Meanwhile, various infectious diseases are increasingly in the trend of 'zoonosis', and the zoonosis needs to be emphasized, so that the acute respiratory tract infection possibly happened in the zoonosis is necessary to be prevented and controlled. In the pig raising industry, where a large number of people are in operation, management personnel, breeders and the like in a pig farm are in close contact with a pig farm every day, if the pig farm has 'zoonotic' acute respiratory tract infection, the safety of relevant workers is jeopardized, and if the prevention and control are not in place, the population infection event can be caused in human beings. The mutation speed of viruses, especially RNA viruses, is high, under the existing immune pressure, there is a risk of cross-species transmission, and in order to prevent large-scale infection of people after mutation, public safety is jeopardized, and respiratory tract infection of livestock is necessary to be brought into a prevention and control range.
For respiratory viruses with huge harm and outstanding transmission capacity, timely and accurate laboratory diagnosis is an important link in many aspects such as determining individual infection state, properly carrying out patient treatment and management, investigating virus transmission path, controlling further transmission and the like. At present, the nucleic acid detection kit for SARS-CoV-2 is not available in the market, but the mixed infection of other pathogens is ignored, and the viruses have the characteristics of strong infectivity, quick transmission, high mixed infection rate and the like, so that new challenges are brought to the prevention and control of SARS-CoV-2 epidemic situation, and meanwhile, the existing products in the market do not bring the respiratory tract infection of livestock into the prevention and control range, and the detection is not carried out, so that the group infection risk possibly causing 'zoonotic' exists.
There is therefore a need to establish a rapid detection and identification method that can detect acute respiratory infections in humans and animal respiratory infections that may lead to "zoonotic".
Disclosure of Invention
In view of the above, the invention provides a composition for detecting multiple respiratory viruses, a kit and application thereof, and the kit can simultaneously and rapidly detect and identify 6 viruses causing acute respiratory infection of human and 2 viruses possibly causing respiratory infection of animals with 'human and animal co-disease', including rhinovirus, influenza B virus, adenovirus, parainfluenza virus, novel coronavirus, respiratory syncytial virus porcine epidemic diarrhea virus and porcine acute diarrhea syndrome coronavirus, and has extremely high detection sensitivity and specificity, and accurate and reliable detection result.
To achieve the above object, the present invention provides in a first aspect a composition for detecting a plurality of respiratory viruses, comprising the nucleic acid sequence combinations shown in the following 1) to 8):
1) A rhinovirus upstream primer shown as SEQ ID NO. 1, a rhinovirus downstream primer shown as SEQ ID NO. 2, and a rhinovirus probe shown as SEQ ID NO. 3;
2) An upstream primer of the porcine epidemic diarrhea virus shown as SEQ ID NO. 4, a downstream primer of the porcine epidemic diarrhea virus shown as SEQ ID NO. 5, and a porcine epidemic diarrhea virus probe shown as SEQ ID NO. 6;
3) An influenza B virus upstream primer shown as SEQ ID NO. 7, an influenza B virus downstream primer shown as SEQ ID NO. 8, and an influenza B virus probe shown as SEQ ID NO. 9;
4) An adenovirus upstream primer shown as SEQ ID NO. 10, an adenovirus downstream primer shown as SEQ ID NO. 11, and an adenovirus probe shown as SEQ ID NO. 12;
5) A parainfluenza virus upstream primer shown as SEQ ID NO. 13, a parainfluenza virus downstream primer shown as SEQ ID NO. 14, and a parainfluenza virus probe shown as SEQ ID NO. 15;
6) A novel coronavirus upstream primer shown as SEQ ID NO. 16, a novel coronavirus downstream primer shown as SEQ ID NO. 17, and a novel coronavirus probe shown as SEQ ID NO. 18;
7) An upstream primer of the respiratory syncytial virus shown as SEQ ID NO. 19, a downstream primer of the respiratory syncytial virus shown as SEQ ID NO. 20, and a probe of the respiratory syncytial virus shown as SEQ ID NO. 21;
8) An upstream primer of the porcine acute diarrhea syndrome coronavirus shown as SEQ ID NO. 22, a downstream primer of the porcine acute diarrhea syndrome coronavirus shown as SEQ ID NO. 23, and a porcine acute diarrhea syndrome coronavirus probe shown as SEQ ID NO. 24.
In one embodiment of the present invention, the composition for detecting a plurality of respiratory viruses comprises composition 1 and composition 2, wherein composition 1 comprises the nucleic acid sequence combination of the first aspect 1) to 4) of the present invention, and composition 2 comprises the nucleic acid sequence combination of the first aspect 5) to 8) of the present invention.
In a preferred embodiment of the present invention, the 5' end of the probe in composition 1 comprises fluorescent reporter groups, which are different from each other and do not interfere with each other, including but not limited to any of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY, CY5, texas red.
In a further preferred embodiment of the invention, in composition 1, the fluorophore of the rhinovirus probe is FAM, the fluorophore of the porcine epidemic diarrhea virus probe is HEX, the fluorophore of the influenza B virus probe is Texas red, and the fluorophore of the adenovirus probe is CY5.
In a preferred embodiment of the present invention, the 5' end of the probe in composition 2 comprises fluorescent reporter groups, which are different from each other and do not interfere with each other, including but not limited to any of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY, CY5, texas red.
In a preferred embodiment of the present invention, in the composition 2, the fluorescent group of the respiratory syncytial virus probe is FAM, the fluorescent group of the novel coronavirus probe is HEX, the fluorescent group of the parainfluenza virus probe is Texas red, and the fluorescent group of the porcine acute diarrhea syndrome coronavirus probe is CY5.
In a second aspect the invention provides a kit for detecting a plurality of respiratory viruses, the kit comprising a composition for detecting a plurality of respiratory viruses according to the first aspect of the invention.
In one embodiment of the present invention, the reaction system of the kit includes: 50-500 nM PCR primer and 50-250 nM probe.
In a preferred embodiment of the present invention, the bladder cancer gene methylation detection kit reaction system comprises: 2X ChamQ SYBR qPCR Master Mix. Mu.L, 10. Mu.M each of the rhinovirus upstream/downstream primers 0.4. Mu.L, 10. Mu.M of the rhinovirus probe 0.2. Mu.L; 10 mu M of upper/lower primers of porcine epidemic diarrhea virus and 0.4 mu L of porcine epidemic diarrhea virus probes respectively, and 10 mu M of probes of porcine epidemic diarrhea virus and 0.2 mu L of probes; influenza B virus up/down primers 10. Mu.M each 0.4. Mu.L, influenza B virus probe 10. Mu.M 0.2. Mu.L; adenovirus up/down primer 10. Mu.M each 0.4. Mu.L, adenovirus probe 10. Mu.M 0.2. Mu.L; the templates were 2. Mu.L, respectively. Sterile deionized water was supplemented to a final volume of 20 μl. Composition 2: the four-fold real-time fluorescent quantitative PCR amplification conditions are as follows: 2X ChamQ SYBR qPCR Master Mix. Mu.L of parainfluenza virus upstream/downstream primer 10. Mu.M each 0.3. Mu.L, parainfluenza virus probe 10. Mu.M 0.2. Mu.L; 10. Mu.M each of the novel coronavirus upstream/downstream primers was 0.3. Mu.L, and 10. Mu.M each of the novel coronavirus downstream primers was 0.2. Mu.L; 10. Mu.M of respiratory syncytial virus upstream/downstream primer each 0.4. Mu.L, 10. Mu.M of respiratory syncytial virus probe 0.2. Mu.L; 10. Mu.M of respiratory syncytial virus upstream/downstream primer each 0.4. Mu.L, 10. Mu.M of respiratory syncytial virus probe 0.2. Mu.L; porcine acute diarrhea syndrome coronavirus upstream/downstream primer 10. Mu.M each 0.4. Mu.L, porcine acute diarrhea syndrome coronavirus probe 10. Mu.M 0.2. Mu.L; the plates were 2. Mu.L each. Sterile deionized water was supplemented to a final volume of 20 μl.
In one embodiment of the invention, the reaction procedure of the kit comprises: digesting for 2min at 37 ℃ and pre-denaturing for 5min at 95 ℃; denaturation at 95℃for 10s, annealing at 55-65℃for 30s, fluorescence collection for 35-55 cycles.
In a preferred embodiment of the present invention, the reaction procedure of the kit comprises: digesting for 2min at 37 ℃ and pre-denaturing for 5min at 95 ℃; denaturation at 95℃for 10s, annealing at 60℃and extension for 30s, fluorescence was collected for 45 cycles.
In a third aspect, the invention provides a composition for detecting a plurality of respiratory viruses according to the first aspect of the invention, and the use of a kit for detecting a plurality of respiratory viruses according to the second aspect of the invention in the preparation of a kit for detecting respiratory viruses.
The invention has the following beneficial effects:
1) The virus of respiratory tract infection can be detected and identified rapidly, 6 human respiratory tract infection viruses are detected and identified simultaneously, 2 porcine respiratory tract infection viruses possibly causing 'zoonosis' are detected and identified simultaneously, the virus types of respiratory tract infection are effectively distinguished, and meanwhile, the prevention of respiratory tract infection possibly causing 'zoonosis' is also carried out.
2) Multiplex gene methylation detection: and multiple gene methylation detection site joint inspection is established, so that reagent consumption is reduced, consumable cost is reduced, and meanwhile, operation steps of experimenters are reduced, and labor cost is reduced.
3) The accuracy is high: the kit for detecting various respiratory viruses provided by the invention has the advantages that in the composition 1, the detection lower limit of rhinovirus (HRV), porcine Epidemic Diarrhea Virus (PEDV), influenza B virus (IFB) and adenovirus (AdV) can reach 2.7 copies/mu L, and in the composition 2, the detection lower limit of Human Respiratory Syncytial Virus (HRSV), novel coronavirus (SARS-CoV-2), parainfluenza virus (HPIV) and porcine acute diarrhea syndrome coronavirus (SADS-CoV) can reach 2.5 copies/mu L, so that the kit has good detection sensitivity; meanwhile, the kit also shows good detection specificity in detecting other common respiratory viruses including mumps virus, rubella virus, human metapneumovirus, measles virus and the like.
Drawings
FIG. 1 is a graph of quadruple fluorescent quantitative PCR amplification of composition 1 in a plurality of respiratory virus detection kits provided by embodiments of the present invention;
FIG. 2 is a standard graph of a quadruple fluorescence quantitative PCR for composition 1 in a plurality of respiratory virus detection kits provided by the embodiments of the present invention;
FIG. 3 is a graph showing the results of a quadruple fluorescence quantitative PCR specificity test of composition 1 in a plurality of respiratory virus detection kits provided by the embodiment of the invention;
FIG. 4 is a graph showing the results of a quadruple fluorescence quantitative PCR sensitivity test of composition 1 in a plurality of respiratory virus detection kits provided by the embodiments of the present invention;
FIG. 5 is a graph of quadruple fluorescent quantitative PCR amplification of composition 2 in various respiratory virus detection kits provided by embodiments of the present invention;
FIG. 6 is a standard graph of a quadruple fluorescence quantitative PCR for composition 2 in a plurality of respiratory virus detection kits provided by embodiments of the present invention;
FIG. 7 is a graph showing the results of a quadruple fluorescence quantitative PCR specificity test of composition 2 in a plurality of respiratory virus detection kits provided by the examples of the present invention;
FIG. 8 is a graph showing the results of a quadruple fluorescence quantitative PCR sensitivity test of composition 2 in a plurality of respiratory virus detection kits provided by the embodiment of the invention.
Detailed Description
The present invention is described in detail below by way of specific examples to enable those skilled in the art to readily practice the invention in light of the present disclosure. The embodiments described below are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Unless specifically stated otherwise, reagents, methods and apparatus employed in the present invention are those conventional in the art, and experimental methods without specifying specific conditions are generally carried out under conventional conditions or conditions suggested by the manufacturer.
The invention adopts a multiplex fluorescence PCR technology to synchronously and rapidly detect and identify 6 viruses causing acute respiratory tract infection of human and 2 viruses possibly causing respiratory tract infection of animals which are co-suffering from human and livestock, including rhinovirus, influenza B virus, adenovirus, parainfluenza virus, novel coronavirus, respiratory syncytial virus porcine epidemic diarrhea virus and porcine acute diarrhea syndrome coronavirus, and has extremely high detection sensitivity and specificity and accurate and reliable detection result.
The invention provides a composition for detecting various respiratory viruses, which comprises the following nucleic acid sequence combinations shown in 1) -8):
1) A rhinovirus upstream primer shown as SEQ ID NO. 1, a rhinovirus downstream primer shown as SEQ ID NO. 2, and a rhinovirus probe shown as SEQ ID NO. 3;
2) An upstream primer of the porcine epidemic diarrhea virus shown as SEQ ID NO. 4, a downstream primer of the porcine epidemic diarrhea virus shown as SEQ ID NO. 5, and a porcine epidemic diarrhea virus probe shown as SEQ ID NO. 6;
3) An influenza B virus upstream primer shown as SEQ ID NO. 7, an influenza B virus downstream primer shown as SEQ ID NO. 8, and an influenza B virus probe shown as SEQ ID NO. 9;
4) An adenovirus upstream primer shown as SEQ ID NO. 10, an adenovirus downstream primer shown as SEQ ID NO. 11, and an adenovirus probe shown as SEQ ID NO. 12;
5) A parainfluenza virus upstream primer shown as SEQ ID NO. 13, a parainfluenza virus downstream primer shown as SEQ ID NO. 14, and a parainfluenza virus probe shown as SEQ ID NO. 15;
6) A novel coronavirus upstream primer shown as SEQ ID NO. 16, a novel coronavirus downstream primer shown as SEQ ID NO. 17, and a novel coronavirus probe shown as SEQ ID NO. 18;
7) An upstream primer of the respiratory syncytial virus shown as SEQ ID NO. 19, a downstream primer of the respiratory syncytial virus shown as SEQ ID NO. 20, and a probe of the respiratory syncytial virus shown as SEQ ID NO. 21;
8) An upstream primer of the porcine acute diarrhea syndrome coronavirus shown as SEQ ID NO. 22, a downstream primer of the porcine acute diarrhea syndrome coronavirus shown as SEQ ID NO. 23, and a porcine acute diarrhea syndrome coronavirus probe shown as SEQ ID NO. 24.
Preferably, the composition for detecting a plurality of respiratory viruses comprises a composition 1 and a composition 2, wherein the composition 1 comprises the nucleic acid sequence combination shown in the 1) to 4), and the composition 2 comprises the nucleic acid sequence combination shown in the 5) to 8).
Preferably, the 5' end of the probe in composition 1 comprises fluorescent reporter groups, which are different from each other and do not interfere with each other, including but not limited to any of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY, CY5, texas red.
Further preferably, in composition 1, the fluorescent group of the rhinovirus probe is FAM, the fluorescent group of the porcine epidemic diarrhea virus probe is HEX, the fluorescent group of the influenza b virus probe is Texas red, and the fluorescent group of the adenovirus probe is CY5.
Preferably, the 5' end of the probe in composition 2 comprises fluorescent reporter groups that are different from each other and do not interfere with each other, including but not limited to any of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY, CY5, texas red.
Further preferably, in composition 2, the fluorescent group of the rhinovirus probe is FAM, the fluorescent group of the porcine epidemic diarrhea virus probe is HEX, the fluorescent group of the influenza b virus probe is Texas red, and the fluorescent group of the adenovirus probe is CY5.
In order to more clearly demonstrate the technical solution of the present invention, the present invention will be further described below with reference to specific examples.
Example 1: target selection, primer and probe screening and optimization
1. Primer and probe design and synthesis
The complete gene sequences of novel coronaviruses (SARS-CoV-2), porcine Epidemic Diarrhea Virus (PEDV), influenza B virus (IFB), adenovirus (AdV), human Respiratory Syncytial Virus (HRSV), rhinovirus (HRV), parainfluenza virus (HPIV) and porcine acute diarrhea syndrome coronavirus (SADS-CoV) were downloaded in the national center for Biotechnology information (National Center for Biotechnology Information, NCBI), screened for conserved sequences using Mega7.0, primers and probes were designed and screened using Primer 6.0 and Oliogo7, and the final Primer probe sequences screened were as follows:
2. construction of plasmid Standard
The conserved sequences are screened out by MEGA7.0 homology alignment and tree analysis, and the plasmid standards based on pUC19 are respectively synthesized by the virus conserved sequences of the composition 1 and the composition 2 in order to homogenize the plasmid concentration during the subsequent optimization of three viruses. Plasmids were synthesized by Huada Gene Co., ltd. The copy number of each plasmid standard was calculated according to the formula (concentration ng/. Mu.L. Times.6.02X1023X 10-9)/(DNA length. Times.660) and diluted to the appropriate copy number.
3. Optimization of reaction conditions
By optimizing the concentration of different primers, the concentration of different probes, the different annealing temperatures and the total reaction volume of 8 viruses of the composition 1 and the composition 2, the optimal system with high amplification efficiency and small fluorescence signal strength Ct value is selected, and the composition 1: the four-fold real-time fluorescent quantitative PCR amplification conditions are as follows: 2X ChamQ SYBR qPCR Master Mix. Mu.L, 10. Mu.M each of the rhinovirus upstream/downstream primers 0.4. Mu.L, 10. Mu.M of the rhinovirus probe 0.2. Mu.L; 10 mu M of upper/lower primers of porcine epidemic diarrhea virus and 0.4 mu L of porcine epidemic diarrhea virus probes respectively, and 10 mu M of probes of porcine epidemic diarrhea virus and 0.2 mu L of probes; influenza B virus up/down primers 10. Mu.M each 0.4. Mu.L, influenza B virus probe 10. Mu.M 0.2. Mu.L; adenovirus up/down primer 10. Mu.M each 0.4. Mu.L, adenovirus probe 10. Mu.M 0.2. Mu.L; the templates were 2. Mu.L, respectively. Sterile deionized water was supplemented to a final volume of 20 μl. Composition 2: the four-fold real-time fluorescent quantitative PCR amplification conditions are as follows: 2X ChamQ SYBR qPCR Master Mix. Mu.L of parainfluenza virus upstream/downstream primer 10. Mu.M each 0.3. Mu.L, parainfluenza virus probe 10. Mu.M 0.2. Mu.L; 10. Mu.M each of the novel coronavirus upstream/downstream primers was 0.3. Mu.L, and 10. Mu.M each of the novel coronavirus probes was 0.2. Mu.L; 10. Mu.M of respiratory syncytial virus upstream/downstream primer each 0.4. Mu.L, 10. Mu.M of respiratory syncytial virus probe 0.2. Mu.L; porcine acute diarrhea syndrome coronavirus upstream/downstream primer 10. Mu.M each 0.4. Mu.L, porcine acute diarrhea syndrome coronavirus probe 10. Mu.M 0.2. Mu.L; the plates were 2. Mu.L each. Sterile deionized water was supplemented to a final volume of 20 μl.
The reaction procedure is: digesting for 2min at 37 ℃ and pre-denaturing for 5min at 95 ℃; denaturation at 95℃for 10s, annealing at 60℃for 30s and extension for 45 cycles.
And (3) amplifying by using the optimized reaction conditions to obtain an amplification kinetic curve. And (3) deriving a standard linear regression equation (standard curve) by taking the common logarithmic number (lgC) of the initial copy number of the standard product as an abscissa and taking a cycle threshold (Ct value) as an ordinate, so as to obtain the sensitivity test data.
And (3) result judgment: if the negative control has no Ct value and the amplification curve is irregular, the Ct value of the positive control is smaller than 35.0; the Ct value of the sample to be detected is smaller than 35.0, and a typical amplification curve appears, and the sample to be detected is judged to be positive; the Ct value of the sample to be detected is larger than 35.0, the amplification curve is accordant, the sample is judged to be suspicious and needs to be re-detected, if the detection result is accordant with the previous detection result, the sample to be detected is judged to be positive, and if the detection result is not accordant with the previous detection result, the sample to be detected is judged to be negative; and judging that the sample to be detected has no Ct value or is larger than 35 and has a random amplification curve as negative.
Example 2: sensitivity test
The recombinant standard plasmid (pUC 19 vector) dry powder synthesized in Huada gene was diluted to 100 ng/. Mu.L with proper sterile water, the copy number of each returned plasmid was calculated according to the formula (concentration ng/. Mu.L. Times.6.02X103X 10-9)/(plasmid length. Times.660) and diluted in a gradient (diluted in a 10-fold gradient), and the diluted standard plasmid was used as a template for sensitivity test. Quadruple real-time fluorescent quantitative PCR detection was performed using the reaction system and reaction procedure in composition 1. The results are shown in FIG. 4, where the lower detection limits for HRV, PEDV, IFB and AdV are all: 2.7 copies/. Mu.L, corresponding respectively to the number of cycles: 37.01, 36.64, 37.22 and 35.29; the reaction system and reaction procedure of composition 2 were subjected to a triple real-time fluorescent quantitative PCR assay. The results are shown in FIG. 8, where the lower detection limits for HRSV, 2019-nCoV, HPIV and SADS-CoV are all: 2.5 copies/. Mu.L, corresponding respectively to the number of cycles: 35.83, 35.53, 34.74 and 34.65. The established method is proved to have good sensitivity.
Example 3: standard Curve establishment
Composition 1: the standard plasmid is used as a template, and diluted according to a 10-time gradient: 2.744×10 6 copies/μL、2.744×10 5 copies/μL、2.744×10 4 copies/μL、2.744×10 3 copies/μL、2.744×10 2 copies/μL、2.744×10 1 The copies/. Mu.L, and then sub-packaging and preserving at-20 ℃; the diluted plasmid was amplified by using the reaction system in composition 1, the amplification result is shown in FIG. 1, the template copy number and cycle number (Ct value) were obtained, after the detection, the concentration lg value (X axis) of each standard was plotted against the corresponding Ct value (Y axis), and the standard curve was drawn, and the result is shown in FIG. 2.
Composition 2: the standard plasmid is used as a template, and diluted according to a 10-time gradient: 2.544 ×10 6 copies/μL、2.544×10 5 copies/μL、2.544×10 4 copies/μL、2.544×10 3 copies/μL、2.544×10 2 copies/μL、2.544×10 1 The copies/. Mu.L, and then sub-packaging and preserving at-20 ℃; the diluted plasmid was amplified by using the reaction system in composition 2, the amplification result is shown in FIG. 5, the template copy number and cycle number (Ct value) were obtained, after the detection, the concentration lg value (X axis) of each standard was plotted against the corresponding Ct value (Y axis), and the standard curve was drawn, and the result is shown in FIG. 6.
According to the formula: amplification efficiency e=10-1/slope-1, as shown in fig. 2 and 6, composition 1: HRV, PEDV, IFB and AdV slopes are respectively: -3.295, -3.372, -3.390 and-3.271, the calculated amplification efficiencies of HRV, PEDV, IFB and AdV are respectively: 101.14%, 97.95%, 97.24% and 102.17%, and the constructed quadruple fluorescent quantitative PCR has high amplification efficiency. Composition 2: the slopes of HRSV, 2019-nCoV, HPIV and SADS-CoV are respectively: -3.200, -3.310, -3.250 and-3.387, the calculated amplification efficiencies of HRSV, 2019-nCoV, HPIV and SADS-CoV are respectively: 105.35%, 100.50%, 103.09% and 97.36%. Plasmid standards of composition 1 and composition 2 (calibrated copy number 106 copies/. Mu.L) were diluted to 101 copies/. Mu.L in a double ratio, and then subjected to fluorescent quantitative PCR amplification with virus concentration on the abscissa and CT value on the ordinate, and a standard curve was established.
Example 4: repeatability test
Quantitative PCR standard plasmid diluted with 10-fold gradient was used as template, composition 1: diluted in 10-fold gradient: 2.744×10 6 copies/μL、2.744×10 5 copies/μL、2.744×10 4 copies/μL、2.744×10 3 copies/μL、2.744×10 2 copies/μL、2.744×10 1 The samples/. Mu.L was subjected to quadruple real-time fluorescent quantitative PCR detection using the reaction system and the reaction procedure of example 1, three repetitions each, and the cycle number average, standard deviation and coefficient of variation were calculated respectively; the results are shown in Table 1, and the variation coefficients in the groups and among the groups are less than 0.76%, which proves that the established method has good repeatability. Composition 2: the standard plasmid is used as a template, and diluted according to a 10-time gradient: 2.544 ×10 6 copies/μL、2.544×10 5 copies/μL、2.544×10 4 copies/μL、2.544×10 3 copies/μL、2.544×10 2 copies/μL、2.544×10 1 The samples/. Mu.L was subjected to quadruple real-time fluorescent quantitative PCR detection using the reaction system and the reaction procedure of example 1, three repetitions each, and the cycle number average, standard deviation and coefficient of variation were calculated respectively; the results are shown in Table 2, and the variation coefficients in the groups and among the groups are less than 0.94%, which proves that the established method has good repeatability.
Table 1 results of the repeatability test of the composition 1 quadruple fluorescent quantitative PCR
Table 2 results of the repeatability test of composition 2 quadruple fluorescent quantitative PCR
Example 5: specific detection
The composition of the invention is used for detecting other common respiratory viruses including mumps virus, rubella virus, human metapneumovirus, measles virus and the like, and the experimental results are shown in fig. 3 and 7. (the amplification curve presented in the figure is the plasmid standard amplification curve).
The detection results of the composition 1 and the composition 2 show that the detection results of respiratory viruses such as mumps virus, rubella virus, human metapneumovirus, measles virus and the like are negative, and the plasmid standard products are detected normally, so that the kit has good specificity.
Example 6: clinical specimen detection and patent contrast test
Using the compositions 1 and 2 of the present invention, 200 samples were tested according to an optimized test method, with 100 parts of throat test paper from healthy people with low risk areas and normal body temperature, 10 parts of porcine epidemic diarrhea virus positive sample, 10 parts of porcine acute diarrhea syndrome coronavirus positive sample, 25 parts of influenza B virus positive sample, 10 parts of adenovirus positive sample, 15 parts of respiratory syncytial virus positive sample, 15 parts of parainfluenza virus positive sample and 15 parts of rhinovirus positive sample. Patent 1 (composition, kit, method and use for detecting and typing pathogens causing respiratory tract infections, patent application number 202010302240.2), patent 2 (triple FQ-PCR detection method for porcine epidemic diarrhea virus, porcine delta coronavirus and porcine acute diarrhea syndrome coronavirus, patent application number 202010531586. X), patent 3 (kit for detecting rhinovirus, respiratory syncytial virus and parainfluenza virus and methods for using the same, patent application number 201810458223.0) and the present patent were compared with commercial kits (Shanghai river biotechnology Co., ltd.) and the results are shown in Table 3 below.
As a result, the coincidence rate of the sample detected by the method is 96.5-98.5%, which is better than 95.5-96.5% of patent 1, 96.5-97.0% of patent 2 and 94.0-96.0% of patent 3. The invention has the highest coincidence rate with the detection result of commercial reagents basically and has higher specificity. The invention is different from the gene detection sites of patent 1, patent 2 and patent, the primer and probe sequences have no similar or coincident sites, and the invention selects a conserved site sequence which does not conflict with other patents. Meanwhile, the sensitivity of the invention is different from the sensitivity data statistics mode of the patent 1, the patent 1 uses a scatter diagram as a sensitivity experiment result diagram, the scatter diagram is relatively disordered, only related, distributed and aggregated information can be basically seen, and other information can not be well displayed; the sensitivity of the invention is close to that of the patent 2, and a single copy of the virus can be detected, but compared with a patent comparison test, the specificity of the invention is slightly better than that of the patent 2, and the virus detectable by the invention is also richer than that of the patent 2; the sensitivity data of the present invention has higher sensitivity than that of patent 3, and limit detection can detect 2.5 copies/uL. Therefore, the invention not only has higher sensitivity, but also has higher accuracy of sample detection.
TABLE 3 comparison of patent negative and positive results
/>
Note that: coincidence rate: (true positive + true negative)/(true positive + true negative + false positive + false negative)
In conclusion, the kit for detecting various respiratory viruses provided by the invention has extremely high detection sensitivity and specificity, can simultaneously and rapidly detect and identify 6 viruses causing acute respiratory infection of human and 2 viruses possibly causing respiratory infection of animals suffering from both human and livestock, realizes effective differentiation of the pathogens, achieves symptomatic drug delivery, is beneficial to epidemic prevention and control, and is an ideal choice for detecting and identifying acute respiratory infection.
While the foregoing description illustrates and describes several preferred embodiments of the invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of use in various other combinations, modifications and environments and is capable of changes or modifications within the spirit of the invention described herein, either as a result of the foregoing teachings or as a result of the knowledge or skill of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Sequence listing
<110> agricultural university of south China
<120> composition for detecting various respiratory viruses, kit and use thereof
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gcgacagtgt tctagcctgc gt 22
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tgtgtgctct agctgcaggg t 21
<210> 3
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
tcctccggcc cctgaatgtg gctaacctt 29
<210> 4
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
ctcttagggt tactaatgac aagc 24
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
cagcgaattt gctcattcca gt 22
<210> 6
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
caacgctgta cccactaaca aggggaa 27
<210> 7
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
attggattrt gcaaacgcat c 21
<210> 8
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
taygtccact cyggttctgg g 21
<210> 9
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
aggctgtgaa ccgttctgca aca 23
<210> 10
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
ctctctgcag ctaacatgct t 21
<210> 11
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
tgtttctgca catcataatt agg 23
<210> 12
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
caccatccaa cggagcacag gagacagc 28
<210> 13
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
ctcaacaaag atcaacttct gt 22
<210> 14
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
ttcggtaaag attcgagtaa ggaac 25
<210> 15
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
tgtttgggtt aataaatgga gttcg 25
<210> 16
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
acaaataggc gtggtaagag a 21
<210> 17
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
gtagtcccaa aatctttgag gc 22
<210> 18
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
ccttacacgt aaccctgctt ggag 24
<210> 19
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
gatctacaag ttggcacagc a 21
<210> 20
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
tgcatcagtg tatactcccg tt 22
<210> 21
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
tggcataatg tgctatcaag accagga 27
<210> 22
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
ctttctgtga cgtctaaagt gcat 24
<210> 23
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
tgcgggtaga aagtggcaac ct 22
<210> 24
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
ttgttgattg taaggcttgg cgctgga 27
Claims (9)
1. A composition for detecting a plurality of respiratory viruses comprising the nucleic acid sequence set forth in 1) -8) below:
1) A rhinovirus upstream primer shown as SEQ ID NO. 1, a rhinovirus downstream primer shown as SEQ ID NO. 2, and a rhinovirus probe shown as SEQ ID NO. 3;
2) An upstream primer of the porcine epidemic diarrhea virus shown as SEQ ID NO. 4, a downstream primer of the porcine epidemic diarrhea virus shown as SEQ ID NO. 5, and a porcine epidemic diarrhea virus probe shown as SEQ ID NO. 6;
3) An influenza B virus upstream primer shown as SEQ ID NO. 7, an influenza B virus downstream primer shown as SEQ ID NO. 8, and an influenza B virus probe shown as SEQ ID NO. 9;
4) An adenovirus upstream primer shown as SEQ ID NO. 10, an adenovirus downstream primer shown as SEQ ID NO. 11, and an adenovirus probe shown as SEQ ID NO. 12;
5) A parainfluenza virus upstream primer shown as SEQ ID NO. 13, a parainfluenza virus downstream primer shown as SEQ ID NO. 14, and a parainfluenza virus probe shown as SEQ ID NO. 15;
6) A novel coronavirus upstream primer shown as SEQ ID NO. 16, a novel coronavirus downstream primer shown as SEQ ID NO. 17, and a novel coronavirus probe shown as SEQ ID NO. 18;
7) An upstream primer of the respiratory syncytial virus shown as SEQ ID NO. 19, a downstream primer of the respiratory syncytial virus shown as SEQ ID NO. 20, and a probe of the respiratory syncytial virus shown as SEQ ID NO. 21;
8) An upstream primer of the porcine acute diarrhea syndrome coronavirus shown as SEQ ID NO. 22, a downstream primer of the porcine acute diarrhea syndrome coronavirus shown as SEQ ID NO. 23, and a porcine acute diarrhea syndrome coronavirus probe shown as SEQ ID NO. 24.
2. The composition for detecting multiple respiratory viruses of claim 1, wherein the composition for detecting multiple respiratory viruses comprises composition 1 and composition 2, wherein composition 1 comprises the combination of nucleic acid sequences as set forth in claims 1-4), and wherein composition 2 comprises the combination of nucleic acid sequences as set forth in claims 1-5).
3. The composition for detecting multiple respiratory viruses of claim 2, wherein the combination of nucleic acid sequences shown in 1) -4) in composition 1) comprises fluorescent reporter groups at the 5' end of the probe, wherein the fluorescent reporter groups are different from each other and do not interfere with each other, and are selected from any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY, CY5 and Texas red.
4. The composition for detecting multiple respiratory viruses of claim 2, wherein the 5' end of the probe comprises a fluorescent reporter group which is different from each other and does not interfere with each other and is selected from any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY, CY5 and Texas red, in combination with the nucleic acid sequences shown in 5) -8) of the composition 2.
5. A kit for detecting a plurality of respiratory viruses, comprising a composition for detecting a plurality of respiratory viruses as claimed in any one of claims 1 to 4.
6. The kit for detecting multiple respiratory viruses of claim 5, wherein the reaction system of the kit comprises: 50-500 nM PCR primer and 50-250 nM probe.
7. The kit for detecting multiple respiratory viruses of claim 5, wherein the minimum detection of composition 1 in said composition is as low as 2.7 copies/. Mu.L and the minimum detection of composition 2 is as low as 2.5 copies/. Mu.L.
8. The kit for detecting multiple respiratory viruses of claim 5, wherein the kit is capable of specifically detecting and identifying one or more of rhinoviruses, porcine epidemic diarrhea viruses, influenza b viruses, adenoviruses, human respiratory syncytial viruses, novel coronaviruses, parainfluenza viruses, porcine acute diarrhea syndrome coronaviruses, and is incapable of detecting one or more of mumps, rubella viruses, human metapneumoviruses, measles viruses.
9. Use of a composition for detecting a plurality of respiratory viruses as claimed in claim 1 in the preparation of a kit for detecting respiratory viruses.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111362220.5A CN113981147B (en) | 2021-11-17 | 2021-11-17 | Composition for detecting various respiratory viruses, kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111362220.5A CN113981147B (en) | 2021-11-17 | 2021-11-17 | Composition for detecting various respiratory viruses, kit and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113981147A CN113981147A (en) | 2022-01-28 |
| CN113981147B true CN113981147B (en) | 2023-08-01 |
Family
ID=79749064
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111362220.5A Active CN113981147B (en) | 2021-11-17 | 2021-11-17 | Composition for detecting various respiratory viruses, kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113981147B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1802440A (en) * | 2003-07-14 | 2006-07-12 | 北京博奥生物芯片有限责任公司 | Methods and compositions for detecting SARS virus and other infectious agents |
| CN109371148A (en) * | 2018-12-10 | 2019-02-22 | 山东省农业科学院畜牧兽医研究所 | Identify the fluorescent PCR kit and quantitative detecting method of three kinds of porcine respiratory bacteriums |
| CN109609665A (en) * | 2018-12-24 | 2019-04-12 | 西安博睿康宁生物科技有限公司 | Detect the multiple PCR primer probe and kit of pet source property Amphixenosis |
| CN110982937A (en) * | 2019-12-13 | 2020-04-10 | 华南农业大学 | LAMP primer composition for detecting porcine acute diarrhea syndrome coronavirus by adopting microfluidic chip technology and kit thereof |
| CN111876524A (en) * | 2020-06-22 | 2020-11-03 | 江苏康为世纪生物科技有限公司 | Primer, probe combination and kit for detecting 34 respiratory pathogenic microorganisms based on multiple PCR-time-of-flight mass spectrometry |
| CN112501356A (en) * | 2020-12-16 | 2021-03-16 | 山东省动物疫病预防与控制中心 | Probe primer group, kit and detection method for rapidly identifying and detecting mink coronavirus and novel coronavirus |
| CN112813196A (en) * | 2020-12-31 | 2021-05-18 | 广州微远基因科技有限公司 | Capture probe set, method and kit for detecting pathogenic microorganisms and application |
| CN113151590A (en) * | 2021-02-24 | 2021-07-23 | 陈清森 | Novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit and preparation and detection methods thereof |
| WO2021198228A1 (en) * | 2020-03-30 | 2021-10-07 | Original G B.V. | Diagnostic peptide for use in a method of diagnosis of viral infection, kit and system |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210311054A1 (en) * | 2020-04-01 | 2021-10-07 | Institut Pasteur | Severe acute respiratory syndrome (sars) - associated coronavirus diagnostics |
| US11308612B2 (en) * | 2020-04-17 | 2022-04-19 | Qure.Ai Technologies Private Limited | Systems and methods for detection of infectious respiratory diseases |
-
2021
- 2021-11-17 CN CN202111362220.5A patent/CN113981147B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1802440A (en) * | 2003-07-14 | 2006-07-12 | 北京博奥生物芯片有限责任公司 | Methods and compositions for detecting SARS virus and other infectious agents |
| CN109371148A (en) * | 2018-12-10 | 2019-02-22 | 山东省农业科学院畜牧兽医研究所 | Identify the fluorescent PCR kit and quantitative detecting method of three kinds of porcine respiratory bacteriums |
| CN109609665A (en) * | 2018-12-24 | 2019-04-12 | 西安博睿康宁生物科技有限公司 | Detect the multiple PCR primer probe and kit of pet source property Amphixenosis |
| CN110982937A (en) * | 2019-12-13 | 2020-04-10 | 华南农业大学 | LAMP primer composition for detecting porcine acute diarrhea syndrome coronavirus by adopting microfluidic chip technology and kit thereof |
| WO2021198228A1 (en) * | 2020-03-30 | 2021-10-07 | Original G B.V. | Diagnostic peptide for use in a method of diagnosis of viral infection, kit and system |
| CN111876524A (en) * | 2020-06-22 | 2020-11-03 | 江苏康为世纪生物科技有限公司 | Primer, probe combination and kit for detecting 34 respiratory pathogenic microorganisms based on multiple PCR-time-of-flight mass spectrometry |
| CN112501356A (en) * | 2020-12-16 | 2021-03-16 | 山东省动物疫病预防与控制中心 | Probe primer group, kit and detection method for rapidly identifying and detecting mink coronavirus and novel coronavirus |
| CN112813196A (en) * | 2020-12-31 | 2021-05-18 | 广州微远基因科技有限公司 | Capture probe set, method and kit for detecting pathogenic microorganisms and application |
| CN113151590A (en) * | 2021-02-24 | 2021-07-23 | 陈清森 | Novel coronavirus 2019-nCoVORF1ab and N, E gene detection kit and preparation and detection methods thereof |
Non-Patent Citations (4)
| Title |
|---|
| 22种呼吸道病原体核酸多重联检试剂盒检测效能评估;罗琴 等;疾病监测;第37卷(第01期);第132-138页 * |
| A RT-PCR assay for the detection of coronaviruses from four genera;Leshan Xiu 等;Journal of Clinical Virology;第128卷;第1-7页 * |
| Development of a TaqMan-probe -vased multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses;Zhongzhou Pan 等;Virulence;第11卷(第1期);第707-718页 * |
| 广东省部分猪场猪圆环病毒、猪呼吸道冠状病毒、猪流感病毒的血清学抗体调查情况初报;鱼海琼 等;中国畜牧兽医学会2009学术年会论文集（下册）;第400页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113981147A (en) | 2022-01-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112725537A (en) | Multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) kit and method for detecting 2019 novel coronavirus and primer probe composition | |
| CN110760620A (en) | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method | |
| CN110453011A (en) | A kind of method and application based on CRISPR/Cas12a fast accurate detection African swine fever virus | |
| CN108504778B (en) | Kit that is a kind of while detecting porcine circovirus 2 type and porcine pseudorabies virus and application | |
| CN107034309B (en) | Real-time fluorescent RPA kit and test strip RPA kit for rapidly detecting porcine pseudorabies virus and application thereof | |
| CN102140543B (en) | Multiple real-time quantitative PCR primer, probe and detection method for identifying viral pathogens relevant to fever with eruption syndrome as infection diseases | |
| CN103160615B (en) | Multiple PCR primer used for simultaneously detecting infectious Bovine Rhinotracheitis virus and akabane virus as well as its design method | |
| CN105603123A (en) | Real-time fluorescence RPA reagent kit and test strip RPA reagent kit for rapidly detecting porcine parvovirus and application of reagent kits | |
| WO2022179494A1 (en) | Salmonella typhi detection kit, preparation method therefor and application thereof | |
| CN110373485A (en) | A kind of ureaplasma urealyticum, three joint inspection kit of chlamydia trachomatis and gonococcus | |
| CN115478120A (en) | Method for simultaneously detecting nodavirus and decapod iridovirus 1 of macrobrachium rosenbergii | |
| CN108531627A (en) | One kind is for detecting the streptococcic RPA fluorescent quantitations primer pair of B races, probe, kit and detection method | |
| CN106148565A (en) | A kind of Koi herpesvirus CPA detection primer and application | |
| CN117551812A (en) | Composition for detecting PaBV type 2, paBV type 4 and PaBV type 5, kit and application thereof | |
| CN113981147B (en) | Composition for detecting various respiratory viruses, kit and application thereof | |
| CN107686865A (en) | Detect primer pair, probe, kit and the method for I type bovine herpes virus | |
| CN108411014A (en) | Differentiate the primer and probe and detection method of A types and the dual real-time fluorescence quantitative PCR of Type B ox pasteurella multocida | |
| CN103805717B (en) | A kind of dual Eva Green real-time fluorescence quantitative PCR detection kit for detecting I type and IV type EHV and application thereof | |
| KR102267326B1 (en) | Method and kit for detecting coronavirus | |
| CN102312013A (en) | Primers and probes for detecting 89K pathogenicity island genes of Streptococcus suis serotype 2, real-time fluorescence quantitative PCR method and kit thereof | |
| JP5215726B2 (en) | Detection method of mouse Pneumocystis carini | |
| KR20210073220A (en) | Primer and probe sets for simultaneous detecting severe fever with thrombocytopenia syndrome and orientia tsutsugamushi | |
| CN113943834B (en) | Primer probe combination for isothermal amplification rapid nucleic acid detection of novel coronavirus 2019-nCoV and application thereof | |
| RU2778855C1 (en) | Set of oligonucleotide primers and fluorescently labeled probes for the identification of human coronavirus sars-cov-2 rna by isothermal pcr with real-time hybridization-fluorescence detection | |
| CN116042918B (en) | Five virus joint inspection compositions, kit, method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |