CN113106170B - Triple TaqMan fluorescent quantitative PCR detection reagent, kit, detection method and application - Google Patents
Triple TaqMan fluorescent quantitative PCR detection reagent, kit, detection method and application Download PDFInfo
- Publication number
- CN113106170B CN113106170B CN202110281112.9A CN202110281112A CN113106170B CN 113106170 B CN113106170 B CN 113106170B CN 202110281112 A CN202110281112 A CN 202110281112A CN 113106170 B CN113106170 B CN 113106170B
- Authority
- CN
- China
- Prior art keywords
- probe
- fcv
- iav
- taqman
- detection
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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 triple TaqMan fluorescent quantitative PCR detection reagent, a kit, a detection method and application. The detection reagent comprises specific qPCR detection primers and corresponding TaqMan probes of novel coronaviruses (SARS-CoV-2), feline caliciviruses (Feline Calicivirus, FCV) and influenza A (Influenza A virus, IAV). The detection method mainly designs 3 pairs of specific primers and TaqMan probes for SARS-CoV-2, FCV and IAV, and realizes simultaneous detection of three cat respiratory viruses in one PCR reaction tube, and the detection sensitivity can reach 1×10 1 The copies/. Mu.L, has excellent specificity and repeatability, and can be applied to the clinical detection of pets.
Description
Technical Field
The invention belongs to the technical field of pathogen detection, and particularly relates to a triple TaqMan fluorescent quantitative PCR detection reagent, a kit, a detection method and application.
Background
Cats, a companion animal, are closely related to human daily life and studies have been presented to show that SARS-CoV-2 can infect cats and can spread among cats, although there is no evidence that SARS-CoV-2 can spread from cat to human, as mentioned previously, cross-host spread is visible in coronavirus and is necessary for proper control of COVID-19, timely monitoring of SARS-CoV-2 in cats and isolation of positively infected cats.
Influenza a virus, a common influenza virus, has high pathogenicity to humans and has caused multiple world pandemics. The pandemic H1N1 of type a caused about 2000 tens of thousands of deaths from 1918 to 1920. Influenza a has also exploded in cats, causing about 500 cats to become infected, and clinical symptoms are mainly manifested as cough, sneeze, runny nose and other respiratory symptoms. In addition, cats are susceptible to highly pathogenic avian influenza a H5N1 and exhibit fever, depression, dyspnea, and neurological symptoms, and inter-transmission between cats is also possible. Feline calicivirus, a common causative agent of respiratory symptoms in cats, is an infectious agent that commonly causes mild, self-limiting respiratory disease. High virulent FCV strains are also present and are associated with serious systemic infections. From the clinical aspect alone, it is difficult to clearly distinguish the three viral infections, so developing a rapid, accurate, simple to operate, highly specific diagnostic method to distinguish the novel coronavirus (SARS-CoV-2), influenza A (influenza A virus, IAV), feline calicivirus (feline calicivirus, FCV) infections is of great importance.
The triple qPCR detection method can detect three pathogens of SARS-CoV-2, IAV and FCV on cats at the same time, but considering that the triple qPCR requires adding three pairs of primers and three probes in a system, the primers and the probes need to have high specificity in order to ensure that the primers and the probes do not influence each other, and the combination or the non-specific amplification between the primer probes is avoided. Furthermore, it is contemplated that the situation of nucleic acids in the template is more complex and that the annealing temperatures required for the different primers may be different. Therefore, the establishment of the qPCR detection method by the triple TaqMan probe method has high research and clinical practical values, but is quite difficult to realize.
Disclosure of Invention
In order to solve the problem that three pathogens of SARS-CoV-2, IAV and FCV can not be distinguished through clinical symptoms in the clinic of pets, and hope to develop a detection method capable of diagnosing the three pathogens at one time, the invention provides a triple TaqMan fluorescent quantitative PCR detection reagent, a kit, a detection method and application.
The invention is realized by the following technical scheme:
a triple TaqMan fluorescent quantitative PCR detection reagent comprises a specific qPCR detection primer of SARS-CoV-2, FCV and IAV and a corresponding TaqMan probe, and is specifically as follows:
the primer sequence and TaqMan probe sequence for detecting SARS-CoV-2 are as follows: upstream primer N-112F, downstream primer N-112R, taqMan probe N-probe;
the N-112F has a sequence shown as SEQ ID No.1
The N-112R has a sequence shown as SEQ ID No. 2;
the N-probe has a sequence shown as SEQ ID No. 3;
the report fluorescent dye marked at the 5 'end of the probe N-probe is HEX, and the fluorescent quenching group marked at the 3' end is BHQ1;
the primer sequence for detecting FCV and the TaqMan probe sequence are as follows: an upstream primer FCV-106F, a downstream primer FCV-106R, taqMan probe FCV-probe;
the FCV-106F has a sequence shown as SEQ ID No. 4;
the FCV-106R has a sequence shown as SEQ ID No. 5;
the FCV-probe has a sequence shown as SEQ ID No. 6;
the report fluorescent dye marked at the 5 'end of the probe FCV-probe is Texas Red, and the fluorescent quenching group marked at the 3' end is BHQ2;
the primer sequence for detecting IAV and the TaqMan probe sequence are as follows: an upstream primer IAV-149F, a downstream primer IAV-149R, taqMan probe IAV-probe;
the IAV-149F has a sequence shown in SEQ ID No. 7;
the IAV-149R has a sequence shown in SEQ ID No. 8;
the IAV-probe has a sequence shown as SEQ ID No. 9;
the reporter fluorescent dye marked at the 5 'end of the probe IAV-probe is FAM, and the fluorescence quenching group marked at the 3' end is MGB.
The detection method based on the triple TaqMan fluorescent quantitative PCR detection reagent comprises the following steps:
step 1) obtaining cDNA of a sample to be detected: extracting total RNA of a cat nasopharynx swab sample or a respiratory tract tissue or organ, and carrying out reverse transcription to obtain a cDNA template;
step 2) detection using three sets of qPCR primers and probes: qPCR amplification is carried out by using the cDNA obtained in the step 1) as a template and the primer and the probe according to the claim 1, and fluorescence signals are collected;
and 3) judging the result: judging whether SARS-CoV-2, FCV and IAV are contained in the sample according to the fluorescence signal collected in step 2) and Cq value, and judging as negative when Cq value is more than 35 or the system is judged as negative.
Preferably, the reverse transcription in step 1) is performed using HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wind), the total reaction system is 20. Mu.L containing 1. Mu. L Random hexamers, 6. Mu.L RNase-free Water, 5. Mu.L total RNA, heating for 5min at 65℃after mixing, rapidly quenching on ice, and continuing to add 4 XgDNA wind Mix after standing for 2min on ice, gently pipetting with a pipette after 4. Mu.L, mixing at 42℃for 2min; finally, adding 2 mu L of 10 xRT Mix and 2 mu L HiScript II Enzyme Mix, mixing uniformly, and obtaining the product at 25 ℃ for 5min,50 ℃ for 45min and 85 ℃ for 2 min.
Preferably, the qPCR amplification reaction system of step 2) is 10. Mu.L 2X AceQ qPCR Probe Master Mix, the initial concentration of 10. Mu.M N-112F, N-112R, FCV-106F, FCV-106R, IAV-149F, IAV-149R primers is 0.6. Mu.L each, the initial concentration of 10. Mu.M N-probe, FCV-probe and IAV-probe is 0.2. Mu.L each, and the template and ddH 2 The volume of O was 5.8. Mu.L, and the total volume of the system was 20. Mu.L.
Preferably, the reaction procedure of the qPCR amplification of step 2) is: the fluorescence channel is set to be temperature controlled, and the program is set to be 600s at 95 ℃; the 40 cycles included 95℃15s,60℃30s; the qPCR instrument fluorescence channel was set to: channel 1: FAM, channel 2: HEX, channel 3: texas Red; fluorescence signals were collected simultaneously with qPCR instrument.
The detection method based on triple TaqMan fluorescent quantitative PCR detection reagent is applied to detection of SARS-CoV-2, FCV and IAV.
A triple TaqMan fluorescent quantitative PCR detection kit comprises the triple TaqMan fluorescent quantitative PCR detection reagent.
The application of a triple TaqMan fluorescent quantitative PCR detection kit in detecting SARS-CoV-2, FCV and IAV.
The beneficial effects of the invention are as follows:
1. the invention can detect SARS-CoV-2, IAV and FCV in one reaction tube, and provides a simple, quick, high-efficiency and low-cost method for detecting cat respiratory tract pathogens.
2. The detection sensitivity of the triple TaqMan probe method qPCR detection method established by the invention to the detection result of each target virus positive sample is higher than that of the single RT-PCR, and the positive result is confirmed by sequencing, so that the feasibility of the method is further proved. And the verification proves that the detection sensitivity of the invention to each pathogen can reach 1 multiplied by 10 1 The copies/. Mu.L has better specificity and repeatability.
3. The establishment of the detection method provides a reliable basis for prevention and control of the diseases, thereby greatly reducing the workload of single qPCR detection and greatly improving the working efficiency.
4. The detection method established in the invention has better specificity and repeatability, higher sensitivity, can be directly used for clinical detection, and can diagnose and monitor SARS-CoV-2, IAV and FCV in cats at the same time by rapidly and efficiently detecting the SARS-CoV-2, IAV and FCV and timely taking prevention and control measures.
Drawings
In fig. 1: A-C is the standard detection result of the triple Taqman qPCR detection method in example 3; D-F is the standard curve of the triple Taqman qPCR detection method in example 3;
in fig. 2: a is the most suitable reaction system exploration test result of the triple Taqman qPCR detection method in the embodiment 3; b is a sensitivity test result of detecting a single pathogen by the triple Taqman qPCR detection method in the embodiment 3; c is the result of the specificity test in example 3;
FIG. 3 is a graph showing the results of a co-infection simulation test at the same concentration of any two or three pathogens in example 3: A-C are each 1X 10 1 copiThe detection condition of the co-infection of three combinations of FCV+SARS-CoV-2, FCV+IAV and IAV+SARS-CoV-2 with the concentration of es/uL; D-F is 1X 10 respectively 2 The cocies/uL concentration FCV+SARS-CoV-2, FCV+IAV, IAV+SARS-CoV-2; G-I is FCV+SARS-CoV-2+IAV at a concentration of 1×10, respectively 1 copies/uL、1×10 2 copies/uL、1× 10 7 Detection results of co-infection at copies/uL;
FIG. 4 is a graph showing the results of a simulation of co-infection at three different concentrations of pathogens in example 3: IAV in A is 1X 10 7 The copies/uL are 1X 10 in each case 1 cobies/uL; SARS-CoV-2 in B is 1X 10 7 The copies/uL are 1X 10 in each case 1 cobies/uL; FCV in C is 1×10 7 The copies/uL are 1X 10 in each case 1 cobies/uL; IAV in D is 1X 10 7 The copies/uL are 1X 10 in each case 2 cobies/uL; SARS-CoV-2 in E is 1X 10 7 The copies/uL are 1X 10 in each case 2 cobies/uL; FCV in F is 1×10 7 The copies/uL are 1X 10 in each case 2 copies/uL；
Detailed Description
The technical scheme of the present invention will be further described in detail below by means of the accompanying drawings and specific examples, but the present invention is not limited to these examples.
Example 1
A triple TaqMan fluorescent quantitative PCR detection reagent comprises a novel coronavirus (SARS-CoV-2), a feline calicivirus (feline calicivirus, FCV), a specific qPCR detection primer of influenza A (influenza A virus, IAV) and a corresponding TaqMan probe, and is specifically as follows:
(1) The primer sequence and TaqMan probe sequence for detecting SARS-CoV-2 are as follows: upstream primer N-112F, downstream primer N-112R, taqMan probe N-probe;
the N-112F has a sequence shown as SEQ ID No.1
The N-112R has a sequence shown as SEQ ID No. 2;
the N-probe has a sequence shown as SEQ ID No. 3;
the report fluorescent dye marked at the 5 'end of the probe N-probe is HEX, and the fluorescent quenching group marked at the 3' end is BHQ1:5'-HEX-TTCACCGCTCTCACTCAACAT-BHQ1-3'.
(2) The primer sequence for detecting FCV and the TaqMan probe sequence are as follows: an upstream primer FCV-106F, a downstream primer FCV-106R, taqMan probe FCV-probe;
the FCV-106F has a sequence shown as SEQ ID No. 4;
the FCV-106R has a sequence shown as SEQ ID No. 5;
the FCV-probe has a sequence shown as SEQ ID No. 6;
the reported fluorescent dye marked at the 5 'end of the probe FCV-probe is Texas Red, and the fluorescent quenching group marked at the 3' end is BHQ2:5'-Texas Red-TTGATTTGGCCTGGGCTCT-BHQ2-3'.
(3) The primer sequence for detecting IAV and the TaqMan probe sequence are as follows: an upstream primer IAV-149F, a downstream primer IAV-149R, taqMan probe IAV-probe;
the IAV-149F has a sequence shown in SEQ ID No. 7;
the IAV-149R has a sequence shown in SEQ ID No. 8;
the IAV-probe has a sequence shown as SEQ ID No. 9;
the 5 '-end marked report fluorescent dye of the probe IAV-probe is FAM, and the 3' -end marked fluorescent quenching group is MGB:5'-FAM-CTCAAAGCCGAGATCGCGCA-MGB-3'.
TABLE 1 specific qPCR detection primers for SARS-CoV-2, FCV and IAV and corresponding TaqMan probe sequence Listing
Example 2
The method for detecting the triple fluorescence quantitative qPCR based on the detection reagent in the embodiment 1 comprises the following specific steps:
(1) Obtaining cDNA of a sample to be detected: total RNA of a cat nasopharyngeal swab sample or a respiratory tract tissue or organ is extracted, and a cDNA template is obtained by reverse transcription.
The reverse transcription was performed using HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wind), the total reaction system was 20. Mu.L, containing 1. Mu. L Random hexamers, 6. Mu.L RNase-free Water, 5. Mu.L total RNA, and after mixing, heating at 65℃for 5min, rapidly quenching on ice, and after standing on ice for 2min, continuing to add 4 XgDNA wind Mix, after 4. Mu.L, gently pipetting with a pipette, mixing at 42℃for 2min; finally adding 2 μl of 10×RT Mix,2 μ L HiScript II Enzyme Mix, mixing, and standing at 25deg.C for 5min, 50deg.C for 45min, and 85deg.C for 2 min.
(2) Detection was performed using three sets of qPCR primers and probes: qPCR amplification was performed using the cDNA obtained in (1) as a template, using the primers and probes described in example 1, and fluorescence signals were collected.
The qPCR amplification reaction system is 10 mu L of 2X AceQ qPCR Probe Master Mix, the initial concentration of N-112F, N-112R, FCV-106F, FCV-106R, IAV-149F, IAV-149R primer is 0.6 mu L each, the initial concentration of N-probe, FCV-probe and IAV-probe is 0.2 mu L each, the template and ddH are 10 mu M 2 The volume of O was 5.8. Mu.L, and the total volume of the system was 20. Mu.L.
The reaction program of qPCR amplification is as follows: the fluorescence channel is set to be temperature controlled, and the program is set to be 600s at 95 ℃; the 40 cycles included 95℃15s,60℃30s; the qPCR instrument fluorescence channel was set to: channel 1: FAM, channel 2: HEX, channel 3: texas Red; fluorescence signals were collected simultaneously with qPCR instrument.
(3) And (3) result judgment: judging whether SARS-CoV-2, FCV and IAV are contained in the sample according to the fluorescence signal collected in (2) and Cq value, and judging as negative when Cq value is more than 35 or the system is judged as negative.
Example 3
The reagents described in example 1 and the detection method described in example 2 were constructed and validated as follows:
1. establishment of a Standard Curve
(1) Primer and probe design: all probes and primers in this experiment were designed using the primer design software Oligo 7 and were commissioned for synthesis by Shanghai Biotechnology Co.
(2) Preparation of plasmid standard: the primers N-112F (F/R), FCV-106F (F/R) and IAV-149F (F/R) designed by the test are adopted to amplify the target fragment by a high-fidelity PCR enzyme Phanta Max Super-Fidelity DNA Polymerase (Nanjinouzan Biotechnology Co., ltd.), and then the target fragment is connected to a pMD18-T vector (Bob Biotechnology Co., ltd.) vector to construct a recombinant plasmid. The reaction system and experimental procedure for PCR amplification were set up in accordance with the Phanta Max Super-Fidelity DNA Polymerase reagent instructions, and the reaction was carried out in an Eppendorf PCR apparatus (Ai Bende, china Co., ltd.). Ligation reactions were performed with reference to the reagent instructions and the ligation products were sent to general biosystems (Anhui) Inc. for sequencing to ensure ligation was correct, ultimately obtaining three correctly ligated plasmids.
Respectively using three plasmid standard substancesLibrary Dilution Buffer (Beijing full gold) is subjected to gradient dilution to obtain 1×10 concentrations 7 cobies/uL to 1X 10 1 Recombinant plasmid standard at seven concentrations of copies/uL.
(3) qPCR reaction system: the enzyme used for this reaction was AceQ qPCR Probe Master Mix (Nanjinouzan Biotechnology Co., ltd.); the reaction system is as follows: 10. Mu.L of qPCR Supermix, 0.4. Mu.L of upstream and downstream primers, 0.2. Mu.L of probe (initial concentration: 10. Mu.M); 2. Mu.L of DNA template; 7. Mu.L of enzyme-free water.
(4) qPCR reaction procedure: carrying out fluorescence quantitative reaction on plasmid standard substances with various concentrations of each pathogen by adopting a single qPCR reaction system, wherein the reaction is carried out in Roche96 instruments (roche diagnostics products (Shanghai)). The reaction procedure was set at 95℃for 600s; the 40 cycles included 95℃15s,60℃30s; the qPCR instrument fluorescence channel was set to: the channel was FAM when IAV was amplified, HEX when SARS-CoV-2 was amplified, and Texas Red when FCV was amplified. Meanwhile, fluorescence signals are collected by a fluorescence quantitative PCR instrument. Cq value greater than 35 is determined to be negative.
(5) Obtaining a fluorescent amplification curve and a standard curve of SARS-CoV-2, IAV and FCV plasmid standard substance gradient dilution.
As shown in FIG. 1, FIG. 1A, FIG. 1B, FIG. 1C show amplification curves of plasmid standards of FCV, IAV and SARS-CoV-2, respectively, in gradient dilution (in FIGS. 1A-C, the curves are 10 in order from left to right 7 -10 1 ) FIGS. 1D, 1E, and 1F are standard curves for standard dilution of FCV, IAV, and SARS-CoV-2, respectively. As can be seen from FIG. 1, the plasmid standard used to test the assay has been constructed successfully, thus demonstrating the reliability of the data of the assay.
2. Optimum reaction System exploration
The three qPCR primers N-112F (F/R), FCV-106F (F/R), IAV-149F (F/R) were added to the same reaction system as the three probes N-probe, FCV-probe and IAV-probe. Different primer concentrations (150 nM to 300nM per primer, 50nM increment) and probe (50 nM to 200nM per probe, 50nM increment) were set, 1X 10 per pathogen plasmid standard concentration 3 The optimal reaction conditions for this multiplex reaction were explored by the copies/. Mu.L, as shown in Table 2 below.
TABLE 2 triple Taqman qPCR detection method most suitable for reaction system exploration test results
As can be seen from Table 2 and FIG. 2A, the multiplex qPCR reaction has lower Cq values and stronger fluorescence intensities for all three templates at a total primer concentration of 1800nM and a total probe concentration of 300 nM. The optimal reaction system of the triple Taqman qPCR detection method is shown in the following table 3.
Table 3 triple Taqman qPCR detection method most suitable for reaction system
3. Sensitivity test
Using the optimal reaction system (Table 3) as explored above, 1X 10 was observed for the three pathogens FCV, IAV and SARS-CoV-2 7 COPIES/. Mu.L to 1X 10 1 Seven concentrations of the plasmid standard were tested for copies/. Mu.L, and the sensitivity of the test method was investigated. At the same time, in order to confirm the reliability of the sensitivity, the detection method is used for 1×10 3 copies/μL、1×10 2 copies/μL、1×10 1 Three concentrations of copies/. Mu.L were tested and 21 replicates were set for each concentration. And finally, taking the minimum concentration which accords with the detection rate of 95% as the sensitivity of the detection method according to the experimental result. The procedure of the reaction and the enzymes used in the reaction are as described in steps 1- (3) and 1- (4).
TABLE 4A triple Taqman qPCR detection method sensitivity test results for detecting a single pathogen (one)
TABLE 4 triple Taqman qPCR detection method sensitivity test results (two) for detecting a single pathogen
In Table 4A, each plasmid standard of FCV, SARS-CoV-2 and IAV was obtained from the highest concentration of 1X 10 7 The results of the copes/. Mu.L 10-fold gradient assay were Cq values. Table 4B shows the results of the repeated detection of low concentration standard for a single pathogen by this method and comparing the detection rate with the 95% positive detection rate. FIG. 2B shows that each plasmid standard of FCV, SARS-CoV-2 and IAV is purified from 1X 10 at the highest concentration 7 Amplification curve of the copies/. Mu.L 10-fold gradient assay (10 in order from left to right in FIG. 2B) 7 -10 1 ). As can be seen from tables 4A, 4B and 2B, the multiplex detection method of the present invention is used for detecting a single pathogenThe sensitivity can reach 10 1 copies/μL。
4. Co-infection simulation test
Co-infection simulators include triple co-infection simulators and double co-infection simulators. Triple co-infection assay was performed using the multiplex qPCR system optimized in Table 1 to detect a mixture of three pathogens FCV, SARS-CoV-2 and IAV, the mixture comprising the same concentration of mixture and different concentrations of mixture. Mixing at the same concentration comprises mixing three pathogens of FCV, SARS-CoV-2 and IAV at the same concentration, wherein the concentrations are respectively 1×10 7 copies/μL，1×10 2 COPIES/. Mu.L and 1X 10 1 COPies/. Mu.L. The mixing of different concentrations is to simulate the actual co-infection in clinical situations, so that one of three pathogens is set as high concentration, and the other two pathogens are low concentration, namely one of FCV, SARS-CoV-2 and IAV is set as high concentration 1×10 7 The other two pathogens were set to low concentrations, 1X 10 each at the same time 2 COPIES/. Mu.L or 1X 10 1 COPies/. Mu.L. The double co-infection simulation test was performed by using the multiple system optimized in Table 1 to detect three combinations of FCV+SARS-CoV-2, FCV+IAV and IAV+SARS-CoV-2, at a concentration of 1X 10 2 COPIES/. Mu.L and 1X 10 1 copies/μL。
The procedure of the reaction and the enzymes used in the reaction are as described in steps 1- (3) and 1- (4).
FIG. 3 shows the results of a co-infection simulation test at the same concentration. FIGS. 3A, 3B and 3C are 1×10, respectively 1 Coinfection detection of FCV+SARS-CoV-2, FCV+IAV and IAV+SARS-CoV-2 at concentration of copies/. Mu.L. FIGS. 3D, 3E and 3F are 1×10, respectively 2 Coinfection detection of FCV+SARS-CoV-2, FCV+IAV and IAV+SARS-CoV-2 at concentration of copies/. Mu.L. FIGS. 3G, 3H, and 3I show FCV+SARS-CoV-2+IAV at 1X 10 concentrations, respectively 1 copies/μL、 1×10 2 COPIES/. Mu.L and 1X 10 7 Detection of co-infection at copies/. Mu.L.
FIG. 4 is a graph showing co-infection simulation at different concentrations. IAV of 1X 10 in FIG. 4A 7 The copies/. Mu.L, the other two are 1X 10 1 cobies/. Mu.L; SARS-CoV-2 in FIG. 4B is 1X 10 7 The copies/. Mu.L, the other two are 1X 10 1 cobies/. Mu.L; FCV in FIG. 4C is 1×10 7 The copies/. Mu.L, the other two are 1X 10 1 COPies/. Mu.L. IAV of 1X 10 in FIG. 4D 7 The copies/. Mu.L, the other two are 1X 10 2 cobies/. Mu.L; FIG. 4E shows SARS-CoV-2 as 1X 10 7 The copies/. Mu.L, the other two are 1X 10 2 cobies/. Mu.L; FCV in FIG. 4F is 1×10 7 The copies/. Mu.L, the other two are 1X 10 2 copies/μL。
As can be seen from fig. 3 and 4, the multiplex detection method can detect double and triple infections at the lowest concentration, and can detect both high concentration and low concentration templates normally when the high concentration and low concentration are mixed, which indicates that the method can be used in co-infection detection.
5. Specificity test
The clinical positive samples or positive plasmids of FCV, IAV, SARS-CoV-2, feline herpesvirus type 1 (feline herpesvirus type, FHV-1), feline chlamydia (Chlamydophila felis, C.felis), feline Mycoplasma (M.felis), feline parvovirus (Feline parvovirus, FPV) and feline coronavirus (Feline coronavirus, FCoV) were detected by the triple fluorescence quantitative qPCR detection method of example 2, respectively, and the procedure and enzymes used for the reactions of the assays were as described in steps 1- (3), 1- (4).
As can be seen from FIG. 2C, the detection method is used for simultaneously detecting a plurality of cat-related pathogens, only the target pathogen is detected normally, and other cat-related pathogens are not detected, so that the detection method has good specificity.
6. Stability test
The standard plasmids of FCV, SARS-CoV-2 and IAV were simultaneously and separately detected by the triple fluorescence quantitative qPCR detection method of example 2 at a concentration of 1X 10 7 COPIES/. Mu.L to 1X 10 1 The copies/. Mu.L was tested in 3 groups (group 1, group 2 and group 3) at different times, 3 replicates (replicates 1, 2 and 3) were tested in each group, at 3 day intervals. CV% values were then calculated for the groups and for the groups, respectively, and the reproducibility of the method was examined based on CV%. Reaction procedure for this testThe enzymes used in the reaction are as described in steps 1- (3), 1- (4).
TABLE 5 Simultaneous detection of FCV, SARS-CoV-2 and IAV by the triple Taqman qPCR detection method, respectively
The stability of the detection method is determined by comparing the CV% values between and within groups of the detection of the single pathogen by the multiplex detection method. As shown in Table 5, the variation coefficients between groups and within groups of the concentration gradients were mostly smaller than 1%, and the variation coefficients were 1% to 5%. The results show that the detection method has excellent stability.
Sequence listing
<110> Nanjing agricultural university
<120> triple TaqMan fluorescent quantitative PCR detection reagent, kit, detection method and application
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ccaaggttta cccaataata c 21
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
actgctattg gtgttaattg 20
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
ttcaccgctc tcactcaaca t 21
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
caacatgtgg taacygttaa 20
<210> 5
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
gcacatcata tgcggctct 19
<210> 6
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
ttgatttggc ctgggctct 19
<210> 7
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atgagtcttc taaccgargt cga 23
<210> 8
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
ggtcttgtct ttagccaytc cat 23
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
ctcaaagccg agatcgcgca 20
Claims (2)
1. The triple TaqMan fluorescent quantitative PCR detection reagent is characterized by comprising a specific qPCR detection primer of SARS-CoV-2, FCV and IAV and a corresponding TaqMan probe, and specifically comprises the following steps:
the primer sequence and TaqMan probe sequence for detecting SARS-CoV-2 are as follows: upstream primer N-112F, downstream primer N-112R, taqMan probe N-probe;
the N-112F has a sequence shown as SEQ ID No.1
The N-112R has a sequence shown as SEQ ID No. 2;
the N-probe has a sequence shown as SEQ ID No. 3;
the report fluorescent dye marked at the 5 'end of the probe N-probe is HEX, and the fluorescent quenching group marked at the 3' end is BHQ1;
the primer sequence for detecting FCV and the TaqMan probe sequence are as follows: an upstream primer FCV-106F, a downstream primer FCV-106R, taqMan probe FCV-probe;
the FCV-106F has a sequence shown as SEQ ID No. 4;
the FCV-106R has a sequence shown as SEQ ID No. 5;
the FCV-probe has a sequence shown as SEQ ID No. 6;
the report fluorescent dye marked at the 5 'end of the probe FCV-probe is Texas Red, and the fluorescent quenching group marked at the 3' end is BHQ2;
the primer sequence for detecting IAV and the TaqMan probe sequence are as follows: an upstream primer IAV-149F, a downstream primer IAV-149R, taqMan probe IAV-probe;
the IAV-149F has a sequence shown in SEQ ID No. 7;
the IAV-149R has a sequence shown in SEQ ID No. 8;
the IAV-probe has a sequence shown as SEQ ID No. 9;
the reporter fluorescent dye marked at the 5 'end of the probe IAV-probe is FAM, and the fluorescence quenching group marked at the 3' end is MGB.
2. A triple TaqMan fluorescent quantitative PCR detection kit is characterized by comprising the triple TaqMan fluorescent quantitative PCR detection reagent according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110281112.9A CN113106170B (en) | 2021-03-16 | 2021-03-16 | Triple TaqMan fluorescent quantitative PCR detection reagent, kit, detection method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110281112.9A CN113106170B (en) | 2021-03-16 | 2021-03-16 | Triple TaqMan fluorescent quantitative PCR detection reagent, kit, detection method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113106170A CN113106170A (en) | 2021-07-13 |
| CN113106170B true CN113106170B (en) | 2023-07-21 |
Family
ID=76711410
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110281112.9A Active CN113106170B (en) | 2021-03-16 | 2021-03-16 | Triple TaqMan fluorescent quantitative PCR detection reagent, kit, detection method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113106170B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019149107A1 (en) * | 2018-02-05 | 2019-08-08 | 上海赛比曼生物科技有限公司 | Reagent and method for fluorescence quantitative real-time pcr detection of rcl |
| CN110592290A (en) * | 2019-10-31 | 2019-12-20 | 上海市动物疫病预防控制中心 | Kit and method for detecting feline calicivirus |
| CN111235310A (en) * | 2020-02-20 | 2020-06-05 | 南京农业大学 | Quadruple TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method for porcine viral diarrhea pathogen |
-
2021
- 2021-03-16 CN CN202110281112.9A patent/CN113106170B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019149107A1 (en) * | 2018-02-05 | 2019-08-08 | 上海赛比曼生物科技有限公司 | Reagent and method for fluorescence quantitative real-time pcr detection of rcl |
| CN110592290A (en) * | 2019-10-31 | 2019-12-20 | 上海市动物疫病预防控制中心 | Kit and method for detecting feline calicivirus |
| CN111235310A (en) * | 2020-02-20 | 2020-06-05 | 南京农业大学 | Quadruple TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method for porcine viral diarrhea pathogen |
Non-Patent Citations (2)
| Title |
|---|
| Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2;Yohei Takeda et al;《Viruses》;1-18 * |
| 猫杯状病毒荧光定量PCR检测方法的建立及初步应用;姜雪;高玉伟;胡桂学;杨松涛;赵艳丽;刘秋燕;徐春忠;梁秀娟;夏咸柱;;吉林大学学报(理学版)(第05期);973-977 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113106170A (en) | 2021-07-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111020064B (en) | Novel coronavirus ORF1ab gene nucleic acid detection kit | |
| KR102109196B1 (en) | LAMP composition for detecting 2019 novel Coronavirus and uses thereof | |
| CN110551846B (en) | Cpf1 kit for quickly detecting African swine fever virus nucleic acid and detection method thereof | |
| Hoffmann et al. | A review of RT-PCR technologies used in veterinary virology and disease control: sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health | |
| Belák | Molecular diagnosis of viral diseases, present trends and future aspects: A view from the OIE Collaborating Centre for the Application of Polymerase Chain Reaction Methods for Diagnosis of Viral Diseases in Veterinary Medicine | |
| CN108192996B (en) | Multiple RT-RPA primer combination for detecting influenza A virus and parting H1 and H3 and application thereof | |
| CN110643745B (en) | Composition and kit for detecting feline calicivirus and feline parvovirus and application of composition and kit | |
| CN112176112A (en) | Triple fluorescent quantitative RT-PCR detection kit for avian influenza virus H5, H7 and H9 subtypes and application thereof | |
| CN111763766B (en) | Primer pair, taqMan probe and method for detecting canine diarrhea virus by one-step method and application | |
| KR20210122632A (en) | PNA Probe and Primer for Detecting SARS-CoV-2 Causing Covid-19 Using RT-LAMP and Method for Detecting SARS-CoV-2 Infection Using Thereof | |
| Wang et al. | Recombinase-aided amplification–lateral flow dipstick assay—a specific and sensitive method for visual detection of avian infectious laryngotracheitis virus | |
| JP5754100B2 (en) | Detection method and detection reagent for enterovirus 71 RNA | |
| CN111808987A (en) | 2019 novel coronavirus S protein gene isothermal color development amplification primer group, screening kit and detection method | |
| Zhao et al. | A lateral flow dipstick combined with reverse transcription recombinase polymerase amplification for rapid and visual detection of the bovine respirovirus 3 | |
| CN113481325A (en) | Method and kit for detecting novel coronavirus B.1.1.7 mutant strain | |
| CN112725539A (en) | PRA/Cas12a/IF kit of respiratory syncytial virus and detection method thereof | |
| CN110527747B (en) | Kit for detecting wild strains of classical swine fever viruses | |
| CN111235310A (en) | Quadruple TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method for porcine viral diarrhea pathogen | |
| CN113106170B (en) | Triple TaqMan fluorescent quantitative PCR detection reagent, kit, detection method and application | |
| KR20220004468A (en) | Simultaneous Diagnositic methods and diagnostic kits for SARS-CoV-2 causing COVID-19 and Sarbecovirus using PNA probe | |
| EP3885455A1 (en) | Method and kit for the detection of sars-cov-2 virus in a sample based on reverse transcription loop-mediated isothermal amplification (rt-lamp) | |
| KR102346881B1 (en) | A kit for simultaneous diagnosis of coronavirus and influenza virus | |
| CN111172242B (en) | Kit for combined detection of influenza A and B virus based on double amplification technology and application thereof | |
| CN113215317A (en) | Microdroplet digital PCR (polymerase chain reaction) detection primer, probe and kit for wild strain of bovine sarcoidosis virus and application of microdroplet digital PCR detection primer, probe and kit | |
| KR20120086028A (en) | Differential detection of West nile virus and Japanese encephalitis virus |
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 |