CN116135995A - Dual fluorescence PCR detection kit for simultaneously detecting middle east respiratory syndrome coronavirus and west nile virus - Google Patents

Dual fluorescence PCR detection kit for simultaneously detecting middle east respiratory syndrome coronavirus and west nile virus Download PDF

Info

Publication number
CN116135995A
CN116135995A CN202111368753.4A CN202111368753A CN116135995A CN 116135995 A CN116135995 A CN 116135995A CN 202111368753 A CN202111368753 A CN 202111368753A CN 116135995 A CN116135995 A CN 116135995A
Authority
CN
China
Prior art keywords
primer
west nile
nile virus
respiratory syndrome
middle east
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.)
Pending
Application number
CN202111368753.4A
Other languages
Chinese (zh)
Inventor
丁小静
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Uninovo Biological Technology Co ltd
Original Assignee
Jiangsu Uninovo Biological Technology Co ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Jiangsu Uninovo Biological Technology Co ltd filed Critical Jiangsu Uninovo Biological Technology Co ltd
Priority to CN202111368753.4A priority Critical patent/CN116135995A/en
Publication of CN116135995A publication Critical patent/CN116135995A/en
Pending legal-status Critical Current

Links

Images

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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a double fluorescence PCR detection method for simultaneously detecting middle east respiratory syndrome coronavirus and west nile virus and application thereof. The invention provides specific primers and probes for detecting the middle east respiratory syndrome coronavirus and the west nile virus, and establishes a double fluorescence PCR detection method for the middle east respiratory syndrome coronavirus and the west nile virus. The specific primer probe and the detection method have strong specificity, high sensitivity and good repeatability and stability, and provide a rapid, sensitive and specific clinical detection method for the differential diagnosis of the middle east respiratory syndrome coronavirus and the west nile virus.

Description

Dual fluorescence PCR detection kit for simultaneously detecting middle east respiratory syndrome coronavirus and west nile virus
Technical Field
The invention relates to a double fluorescence PCR detection kit for simultaneously detecting middle east respiratory syndrome coronavirus and west nile virus, provides a specific primer and a probe for detecting middle east respiratory syndrome coronavirus and west nile virus, and establishes a double fluorescence PCR detection method for middle east respiratory syndrome coronavirus and west nile virus. The specific primer probe and the detection method have strong specificity, high sensitivity and good repeatability and stability, provide a rapid, sensitive and specific clinical detection method for differential diagnosis of the middle east respiratory syndrome coronavirus and the west nile virus, and belong to the field of in vitro diagnosis.
Background
The middle east respiratory syndrome coronavirus (Respiratory Syndrome Coronavirus in the Middle East, MERS) is a novel coronavirus that has been named middle east respiratory syndrome coronavirus, most MERS virus infection cases occurring in sauter. Coronaviruses in humans were isolated in the uk the earliest 1960 s, and the viruses were named for their surface crown-like projections. It may be associated with respiratory infections in humans, pigs, cats, dogs, mice and chickens. SARS virus appearing in china in 2003 belongs to coronavirus. MERS is thus the sixth known human coronavirus, and is the third one isolated over the last 10 years.
West Nile Virus (West Nile Virus) belongs to the genus flaviviridae, and is the same genus as viruses such as encephalitis b, yellow fever, and hepatitis c. The membrane has a capsule membrane, and the nucleic acid is single-stranded RNA and has infectivity. West Nile virus is an encephalitis virus, birds are used as main storage hosts, horses, mosquitoes and people can be infectious hosts, and the disease time of people is about 33 days later than the infection time of birds. All people who are not contacted with West Nile virus are susceptible, and the old and the people with weak immunity are easy to develop and have high disease death rate. The incubation period for west nile virus infection is typically 3-12 days. Most (80%) are recessive infections, no symptoms appear, few people appear as west nile fever, and patients have common cold symptoms such as fever, headache, muscle pain, nausea, vomiting, rash, lymphadenectasis and the like for 3-6 days. Few (1%) showed west nile virus encephalitis, meningoepithymitis, and meningitis after infection.
At present, a nucleic acid diagnosis method based on a real-time fluorescence quantitative PCR technology is one of the most widely used methods in China, and the principle of the method is mainly based on marking Taq-Man fluorescent probes with different colors. Most of the existing nucleic acid detection kits adopt a single probe method to realize rapid diagnosis of target genes. The invention adopts a double fluorescence quantitative PCR technology, and 2 fluorescence probes marked by different colors are added into the same reaction system, so that the aim of simultaneously detecting 2 target genes in one tube of reaction can be fulfilled, and the coronavirus and the West Nile virus of the middle east respiratory syndrome can be simultaneously detected, and the flux and the detection speed are high. Has important application prospect in the aspects of disease diagnosis, prevention and treatment, etc.
Disclosure of Invention
The invention aims to provide a double fluorescence PCR detection kit for simultaneously detecting middle east respiratory syndrome coronavirus and west nile virus and a use method thereof, and the kit can be used for rapidly and accurately detecting whether the middle east respiratory syndrome coronavirus and the west nile virus exist in a sample and specifically detecting the middle east respiratory syndrome coronavirus and the west nile virus.
The components comprise PCR reaction liquid, enzyme mixed liquid, negative quality control product and positive quality control product. Wherein the PCR reaction liquid mainly comprises the primers and probes of the 2 viruses, a reaction buffer solution and Mg 2+ And dNTP, wherein the enzyme mixed solution mainly contains reverse transcriptase and hot start Taq enzyme, the negative quality control is RNase-free water and DNase-free water, and the positive quality control is recombinant plasmid containing detection target sequences of middle east respiratory syndrome coronavirus and west nile virus.
The primers used for nucleic acid amplification in the PCR reaction liquid are P1 and P2, wherein P1 and P2 are specific primers designed aiming at specific conserved sequences of the coronavirus of the middle east respiratory syndrome and the gene group of the west nile virus and screened out by a pre-experiment; the oligonucleotide Probe used for fluorescent signal monitoring in the PCR reaction liquid is Probe1, and the Probe1 is a specific primer sequence of a specific Probe designed and screened by a pre-experiment aiming at specific conserved sequences of the coronavirus of the middle east respiratory syndrome and the gene group of the west nile virus (see table 1).
TABLE 1 primer and probe sequences
Figure 468502DEST_PATH_IMAGE002
The PCR reaction system selected by the kit is 20 mu L, and comprises 10 mu L of 2X PCR buffer solution, 1.0 mu L of 10mmol/L dNTP, 0.8 mu L of mixed solution of reverse transcriptase and hot start Taq enzyme, 0.35 mu L of mixed primer, 2 mu L of sample RNA and 20 mu L of sterilizing water to the final system.
PCR reaction cycle of the kit, reverse transcription stage: 42 ℃ for 10min; a pre-denaturation stage: 94 ℃,10sec; pre-amplification stage: denaturation at 94 ℃,5sec; annealing at 50 ℃ for 20sec; extending at 72 ℃ for 20sec; for a total of 5 cycles. Note that no fluorescent signal was acquired at this stage. And (3) detection: denaturation at 94 ℃,5sec; annealing at 56 ℃ for 50sec; extending at 72 ℃ for 15sec; for a total of 40 cycles. The fluorescent signal is detected during the annealing step.
And (3) quality control: negative and positive controls are set up in each experiment, the negative control has no Ct value (or Ct value is 0), the Ct value of the positive control is less than or equal to 30, otherwise, the experimental result is not established.
Interpretation of the results:
positive: an S-shaped amplification curve appears, and the Ct value is less than or equal to 35; suspicious: an "S" type amplification curve occurs, but Ct values > 35; negative: the "S" type amplification curve does not appear, or the curve is not "S" type although exceeding the threshold; for suspicious results, the experiment should be repeated, and if the repeated experiment or the S-shaped amplification curve appears, the negative control has no pollution, and can be judged to be positive.
Sample requirements: clinical sample types include pharyngeal swabs, sputum, and bacterial cultures; after clinical samples are collected, the samples are transported with ice and stored at the temperature of minus 20 ℃ and can not be repeatedly frozen and thawed; the RNA is extracted from clinical samples, commercial kits with stable and reliable performance are recommended to be adopted, the specific method is referred to the instruction book of the corresponding commercial kit, the extracted RNA is detected immediately, otherwise, the RNA is stored at-80 ℃ to-20 ℃ after split charging.
The kit provided by the invention has good specificity, can detect the coronavirus of the middle east respiratory syndrome and the West Nile virus, but can not detect the nucleic acid of the coronavirus of the non-middle east respiratory syndrome and the West Nile virus; the lower limit of detection of the nucleic acid of the middle east respiratory syndrome coronavirus and the west nile virus is 10 copies per reaction system; the detection can be completed within 2 hours, and experimental basis can be provided for disease monitoring and clinical diagnosis of the middle east respiratory syndrome coronavirus and the west nile virus.
Drawings
Fig. 1 is an amplification curve diagram of a real-time fluorescence PCR detection of a coronavirus positive standard of middle east respiratory syndrome, and fig. 2 is an amplification curve diagram of a real-time fluorescence PCR detection of a west nile virus positive standard. The corresponding concentration of each group of curves from left to right is 2×10 in turn 6 -2×10 2 The detection limit of the kit for the middle east respiratory syndrome coronavirus and the west nile virus is 10 copies per reaction system. The abscissa is the number of reaction cycles, and the ordinate is the fluorescence detection signals of different cycle numbersIs a ΔRn value of (1).
FIG. 3 is a graph showing amplification of 10 viral genomes using a multiplex real-time fluorescent PCR detection system. The 10 viruses include: middle east respiratory syndrome coronavirus, west nile virus and 8 negative control microorganisms. The west nile virus and middle eastern respiratory syndrome coronavirus present an S-type amplification curve, whereas the other 8 pathogenic microorganisms did not present an S-type amplification curve.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to specific examples. It should be noted that the examples set forth herein are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way. The reagents used therein, such as kits, buffers, etc., are only those specifically selected in this particular example, and it is understood that those skilled in the art can select corresponding reagents of other companies as needed to achieve the object of the present invention.
1. Design and synthesis of primers and TaqMan probes
The genome sequences of the middle east respiratory syndrome coronavirus and the west nile virus in Genbank and domestic and foreign documents are analyzed by using Blast tools, stable conserved regions of the genome sequences are respectively selected as detection target sequences, and primers and probes are designed and synthesized aiming at the detection target sequences (see table 1). Both primers and probes were synthesized by TaKaRa Dalianbao Biochemical company, japan.
2. Preparation for detecting bacterial species
The middle east respiratory syndrome coronavirus and west nile virus used in this example and other negative control strains (rabies, mumps, adenovirus type 3, enterovirus, measles, rubella, herpes simplex, polio) were purchased from the chinese pharmaceutical biologicals institute.
3. Extraction of bacterial Strain RNA
The RNA of the strain was extracted using a nucleic acid extraction or purification reagent QIAamp DSP Virus Spin Kit (national institute 20140008 QIAGEN GmbH) produced by QIAGEN, germany. Specific steps refer to the kit operating instructions.
4. Screening of primers and probes
The genome RNA of the extracted middle east respiratory syndrome coronavirus, west nile virus and negative control strain is detected by adopting the designed primers and probes, and the primer probe combination with the best sensitivity, specificity and repeatability is screened out through repeated experiments. (see sequence Listing 1)
5. Construction and preparation of standards
The mideast respiratory syndrome coronavirus specific gene fragment was amplified by RT-PCR using P1 and P2 primers, respectively, and cloned into a plasmid containing a T7 promoter sequence (e.g., pGEM-T easy vector), RNA was synthesized using RNA Polymerase from dalbergia biology, and rnase-free pancreatic dnase I was added to terminate the in vitro transcription reaction by using phenol: and extracting and purifying RNA by chloroform. Absorbance at wavelengths of 260nm, 280nm was measured using uv-vis spectrophotometry, respectively, and then RNA concentration and purity were calculated and then diluted 10-fold gradient to 200 copies per microliter.
6. Optimization of reaction conditions
The elements of the primer, the probe, the enzyme and the like are optimized one by one, and a determined reaction system is as follows: 2X PCR buffer solution 10 [ mu ] L, 10mmol/L dNTP 1.0 [ mu ] L, 25 [ mu ] mol/L, primer mixture 0.35 [ mu ] L, 10 [ mu ] mol/L probe 0.2 [ mu ] L, enzyme mixture 0.8 [ mu ] L, template 2ul, and sterilizing water to a final system 20 [ mu ] L.
According to the length of the amplified fragment, the annealing temperature of the primer and the probe and the enzyme characteristics, the reaction annealing temperature and the extension time are mainly optimized, and finally, the determined circulation parameters are as follows: reverse transcription stage: 42 ℃ for 10min; a pre-denaturation stage: 94 ℃,10sec; pre-amplification stage: denaturation at 94 ℃,5sec; annealing at 50 ℃ for 20sec; extending at 72 ℃ for 20sec; for a total of 5 cycles. Note that no fluorescent signal was acquired at this stage. And (3) detection: denaturation at 94 ℃,5sec; annealing at 56 ℃ for 50sec; extending at 72 ℃ for 15sec; for a total of 40 cycles. The fluorescent signal is detected during the annealing step. After amplification, the data were analyzed under the same conditions to determine Ct values for each sample.
7. Evaluation of detection Limit
The positive standard in the above 5 is used for evaluating the detection limit of the kit provided by the invention, and the concentration of the positive standard is as follows: 2X 10 6 copies/μl、2×10 5 copies/μl、2×10 4 copies/μl、2×10 3 copies/μl、2×10 2 The detection lower limit of the kit provided by the invention on the nucleic acid of the coronavirus of the middle east respiratory syndrome and the West Nile virus is 10 copies per reaction system.
8. Evaluation of detection specificity
The specificity of the kit was evaluated using the RNA of the strain as a template. Clear amplification curves can be seen when the RNA of the middle east respiratory syndrome coronavirus and the West Nile virus are detected, positive amplification curves are not generated when the RNA of the 8 other common pathogenic microorganisms are detected, and the fact that the used probes and primers do not have cross reaction with other selected virus strains is shown.
Although certain embodiments of the present invention have been described above by way of example in a preferred manner, it will be understood by those skilled in the art that the present invention is not limited to the embodiments disclosed above, but may be modified in light of the knowledge of those skilled in the art to which the present invention pertains without exceeding the scope of the claimed invention. For example, the fluorescent real-time PCR used in the present invention may also employ, as required, a labeling substance other than the fluorescent reporter group and the fluorescent quencher group indicated in examples listed in the specification, such as a label of Tet, HEX, TAMRA, cy3, txRd, JOE or the like; or other labeling systems than Taqman technology, such as fluorescent probe labeling technologies, e.g., molecular beacon MB probes, scorpion probes, fluorescent double hybridization probes, etc.; or using dye-embedding method such as SYBR Green I and the like unsaturated dye and LC Green and the like saturated dye, and qualitatively or quantitatively detecting the existence of the target gene only by using the specific primer sequence and the primer ratio, thereby rapidly and specifically detecting the existence of the middle east respiratory syndrome coronavirus and the West Nile virus by the same reaction system. Therefore, such modifications and alternatives as would be apparent to one skilled in the art are intended to fall within the scope of the present invention. The scope of the invention should be defined by the appended claims.
Sequence listing
SEQUENCE LISTING
<110> Jiangsu and creative biotechnology Co., ltd
<120> Dual fluorescence PCR assay for simultaneous detection of middle east respiratory syndrome coronavirus and West Nile Virus
Kit for detecting a substance in a sample
<130> 2021111104
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> RNA
<213> artificial sequence
<400> 1
tgctaagaat agagctcgca ctgtt 25
<210> 2
<211> 23
<212> RNA
<213> artificial sequence
<400> 2
tagtaccaat gacgcaagtc gct 23
<210> 3
<211> 16
<212> RNA
<213> artificial sequence
<400> 3
tcgccagtac catcag 16
<210> 4
<211> 24
<212> RNA
<213> artificial sequence
<400> 4
cctgtgtgag ctgacaaact tagt 24
<210> 5
<211> 22
<212> RNA
<213> artificial sequence
<400> 5
gcgttttagc atattgacag cc 22
<210> 6
<211> 30
<212> RNA
<213> artificial sequence
<400> 6
cctggtttct tagacatcga gatcttcgtg 30
Sequence listing
<110> Jiangsu and creative biotechnology Co., ltd
<120> Dual fluorescence PCR detection kit for simultaneously detecting middle east respiratory syndrome coronavirus and West Nile virus
<130> 2021111104
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence (respiratory syncytial virus))
<400> 1
tgctaagaat agagctcgca ctgtt 25
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence (respiratory syncytial virus))
<400> 2
tagtaccaat gacgcaagtc gct 23
<210> 3
<211> 16
<212> DNA
<213> Artificial sequence (artificial sequence (respiratory syncytial virus))
<400> 3
tcgccagtac catcag 16
<210> 4
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
cctgtgtgag ctgacaaact tagt 24
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
gcgttttagc atattgacag cc 22
<210> 6
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
cctggtttct tagacatcga gatcttcgtg 30

Claims (8)

1. The primer probe combination for simultaneously detecting the middle east respiratory syndrome coronavirus and the west nile virus is characterized by comprising detection primer probes for the middle east respiratory syndrome coronavirus and the west nile virus, wherein the specific primer probe sequences are as follows:
middle east respiratory syndrome coronavirus:
SEQ NO.1 upstream primer P1: 5' -TGCTAAGAATAGAGCTCGCACTGTT-3
SEQ NO.2 downstream primer P2: 5' -TAGTACCAATGACGCAAGTCGCT-3
SEQ NO.3 Probe 1: 5' -FAM-TCGCCAGTACCATCAG-MGB-3
West nile virus:
SEQ NO.4 upstream primer P1: 5' -CCTGTGTGAGCTGACAAACTTAGT-3
SEQ NO.5 downstream primer P2: 5' -GCGTTTTAGCATATTGACAGCC-3
SEQ NO.6 Probe 1:5 '-CY 5-CCTGGTTTCTTAGACATCG-MGB-3'.
2. A dual fluorescent PCR method for simultaneously detecting middle east respiratory syndrome coronavirus and west nile virus, wherein the primer probe mixture of claim 1 is used to simultaneously detect middle east respiratory syndrome coronavirus and west nile virus; the mixture ratio of the middle east respiratory syndrome coronavirus and west nile virus upstream primer, downstream primer and probe MIX, middle east respiratory syndrome coronavirus primer probe MIX, upstream primer P1: downstream primer P2: probe probe1=0.032 to 0.128: 0.032 to 0.128: 0.012 to 0.08; west nile virus primer probe MIX, upstream primer P1: downstream primer P2: probe probe1=0.032 to 0.128: 0.032 to 0.128: 0.012 to 0.08; 0.012 to 0.08.
3. The dual fluorescence PCR detection method according to claim 2, wherein the upstream primer, the downstream primer and the probe of the middle east respiratory syndrome coronavirus and the west nile virus are proportioned, the middle east respiratory syndrome coronavirus primer probe MIX, the upstream primer P1: downstream primer P2: probe probe1=0.048 to 0.08: 0.08 to 0.112: 0.02 to 0.06; west nile virus primer probe MIX, upstream primer P1: downstream primer P2: probe probe1=0.08 to 0.112: 0.08 to 0.112: 0.02 to 0.06.
4. The method for detecting double fluorescence PCR according to claim 2, wherein the reaction system of the double fluorescence PCR is: 2×PCR buffer 10 [ mu ] L, 10mmol/L dNTP 1.0 [ mu ] L, reverse transcriptase and hot start Taq enzyme mixed solution 0.8 [ mu ] L, mixed primer 0.35 [ mu ] L, sample RNA 2 [ mu ] L, and adding sterilized water to a final system 20 [ mu ] L.
5. The method for detecting double fluorescence PCR according to claim 2, wherein the amplification procedure of the double fluorescence PCR is as follows: 42 ℃ for 10min; a pre-denaturation stage: 94 ℃,10sec; pre-amplification stage: denaturation at 94 ℃,5sec; annealing at 50 ℃ for 20sec; extending at 72 ℃ for 20sec; a total of 5 cycles; note that no fluorescent signal was collected at this stage; and (3) detection: denaturation at 94 ℃,5sec; annealing at 56 ℃ for 50sec; extending at 72 ℃ for 15sec; a total of 40 cycles; the fluorescent signal is detected during the annealing step.
6. Use of the primer mixture of claim 1 in the preparation of a kit for detection of middle east respiratory syndrome coronavirus and west nile virus.
7. A kit according to claims 6 and 3, wherein the mixture of primer probes comprises the middle east respiratory syndrome coronavirus and west nile virus primer probe MIX: west nile virus primer probe mix=0.05-0.25: 0.05 to 0.25.
8. The kit of claim 7, wherein the primer mixture comprises a mixture of middle east respiratory syndrome coronavirus and west nile virus primer probe MIX, wherein the middle east respiratory syndrome coronavirus primer probe MIX: west nile virus primer probe mix=0.05-0.15: 0.10 to 0.20.
CN202111368753.4A 2021-11-18 2021-11-18 Dual fluorescence PCR detection kit for simultaneously detecting middle east respiratory syndrome coronavirus and west nile virus Pending CN116135995A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111368753.4A CN116135995A (en) 2021-11-18 2021-11-18 Dual fluorescence PCR detection kit for simultaneously detecting middle east respiratory syndrome coronavirus and west nile virus

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111368753.4A CN116135995A (en) 2021-11-18 2021-11-18 Dual fluorescence PCR detection kit for simultaneously detecting middle east respiratory syndrome coronavirus and west nile virus

Publications (1)

Publication Number Publication Date
CN116135995A true CN116135995A (en) 2023-05-19

Family

ID=86333353

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111368753.4A Pending CN116135995A (en) 2021-11-18 2021-11-18 Dual fluorescence PCR detection kit for simultaneously detecting middle east respiratory syndrome coronavirus and west nile virus

Country Status (1)

Country Link
CN (1) CN116135995A (en)

Similar Documents

Publication Publication Date Title
CN110760620A (en) Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method
CN111334609A (en) Primer group and kit for detecting mRNA corresponding to ORF1ab gene and N gene of 2019 novel coronavirus
CN111440897A (en) Probe and primer composition for rapidly detecting seven coronaviruses and other respiratory pathogens
CN109234456B (en) Kit capable of simultaneously detecting 6 respiratory pathogens and application thereof
WO2021190633A1 (en) Primer for detecting sars-cov-2 novel coronavirus, and test kit, detection method, and application thereof
CN110724769A (en) PCR primer group, kit and detection method for detecting African swine fever virus MGF360-505R gene
CN103045754A (en) One-step process real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method and kit for detecting Z/S subtype ebola viruses
CN113652505A (en) Method and kit for detecting novel coronavirus and VOC-202012/01 mutant strain thereof
EP2658994B1 (en) Oligonucleotide probe for the detection of adenovirus
CN106471135B (en) Molecular detection of enteroviruses and echoviruses
WO2006132601A1 (en) Diagnostic primers and method for detecting avian influenza virus subtype h5 and h5n1
CN116135995A (en) Dual fluorescence PCR detection kit for simultaneously detecting middle east respiratory syndrome coronavirus and west nile virus
TWI723675B (en) Methods and kits for detecting dengue virus
US20050239055A1 (en) Enterovirus primers and probes
US20150140548A1 (en) Extraction control for rna
CN112266978A (en) Primer-probe combination, detection kit and application thereof
CN113337638A (en) Method and kit for detecting novel coronavirus (SARS-CoV-2)
CN107988429B (en) Reagent for detecting rabies virus and application thereof
CN116144832A (en) Rapid fluorescence PCR detection kit for enterovirus EV71 type and CoxA16 type
CN110714097A (en) Method for simultaneously detecting A, B, C three groups of rotaviruses
KR102632702B1 (en) Method for detection and quantification of hepatitis a virus
CN112126714B (en) Coronavirus detection product and application thereof
US20240209462A1 (en) A method for detection of target nucleic acid using isothermal amplification
CN101748218A (en) Target sequence for detection of parainfluenza virus type I and kit
CN117757990A (en) Respiratory tract adenovirus typing nucleic acid detection primer combination, kit and detection method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication