CN113215231B - Dual PCR detection method for SARS-CoV and COVID-19 virus - Google Patents
Dual PCR detection method for SARS-CoV and COVID-19 virus Download PDFInfo
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Abstract
The invention discloses a dual PCR detection method for SARS-CoV and COVID-19 virus, comprising the following steps: s1, preparing a virus cDNA template; s2, preparing reagent consumables; s3, designing a primer; s4, synthesizing a virus gene; s5, single PCR amplification; s6, double PCR amplification and condition optimization; s7, detecting specificity of double PCR; s8, detecting the sensitivity of the double PCR method; the invention carries out single PCR amplification and double amplification on the virus after the virus gene synthesis, carries out double optimization of primer concentration condition and annealing temperature condition on the virus during amplification, and finally uses other common respiratory system disease viruses to carry out PCR to determine the specificity of establishing the double PCR method, and the result shows that the designed primer can only specifically detect two virus plasmid templates of SARS-CoV and COVID-19, but can not detect other experimental viruses, thereby improving the effectiveness of detecting SARS-CoV and COVID-19 viruses from various viruses to a certain extent.
Description
Technical Field
The invention belongs to the technical field of laboratory pathogen detection, and particularly relates to a SARS-CoV and COVID-19 virus double PCR detection method.
Background
The novel coronavirus pneumonia COVID-19 is a disease caused by the infection of a novel coronavirus SARS-CoV, because the novel coronavirus pneumonia has extremely strong infectivity, the virus needs to be detected in an effective mode for controlling the spread of the virus, and effective control is carried out according to the detected virus, however, when the virus is detected in a laboratory, because each virus gene group contains different viruses, such as SARS-CoV, COVID-19, MERS-CoV, influenza A virus H1N1, influenza B virus and adenovirus, SARS-CoV and COVID-19 virus can not be effectively detected from a plurality of viruses during detection, and the sensitivity of the method for detecting the virus is low, therefore, a dual PCR detection method for SARS-CoV and COVID-19 virus is provided.
Disclosure of Invention
The invention aims to provide a double PCR detection method for SARS-CoV and COVID-19 viruses, which solves the problems that the prior art can not effectively detect SARS-CoV and COVID-19 viruses from various viruses and the method for detecting the viruses has low sensitivity by double PCR amplification and condition optimization and then carrying out specific detection of double PCR and sensitivity detection of the PCR method.
In order to achieve the purpose, the invention adopts the following technical scheme:
a dual PCR detection method for SARS-CoV and COVID-19 virus comprises the following steps:
s1, preparing a virus cDNA template, preparing SARS-CoV, COVID-19, MERS-CoV, influenza A virus H1N1, influenza B virus and adenovirus templates;
s2, preparing reagent consumables, and preparing a template cloning reagent, an agarose gel electrophoresis reagent and a PCR amplification reagent;
s3, designing primers, obtaining complete gene sequences of SARS-CoV and COVID-19 strains from GenBank, comparing conserved regions of virus gene sequences by BioXM2.6 software, and designing specific primers of each virus by using Primer Premier 5 software;
s4, synthesizing virus genes, synthesizing SARS-CoV, COVID-19, MERS-CoV and influenza A virus H1N1 gene fragments to be detected, cloning to a pUC57 vector, and extracting plasmids for later use after correct sequencing;
s5, single PCR amplification, wherein the total volume of each reaction system is 25. mu.L, and the reaction system contains 12.5. mu.L of 2 × Master Mix, 1. mu.L (10pmol/L) of each of the upstream and downstream primers, 1. mu.L of the synthesized viral plasmid template, and 9.5. mu.L of ddH2O, and the PCR reaction conditions are as follows: 94 ℃ for 3 min; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 30s, 30 cycles; 72 ℃ for 10 min; finally storing at 12 ℃; analyzing the PCR product by 1.5% agarose gel electrophoresis, and placing the PCR product in a gel imaging system to observe an amplification result;
s6, double PCR amplification and condition optimization, mixing two virus plasmids in equal proportion, carrying out PCR amplification according to S5 reaction conditions and a reaction system, analyzing the amplified PCR product through 1.5% agarose gel electrophoresis, and observing by using a gel imaging system, wherein the condition optimization of the double PCR amplification comprises primer concentration optimization and annealing temperature optimization, wherein the primer concentration optimization is to carry out PCR on upstream and downstream primers of SARS-CoV and COVID-19 according to different concentrations, and the combination is respectively as follows: determining the optimal primer concentration by 10pmol/L +10pmol/L, 5pmol/L +5pmol/L, 10pmol/L +5pmol/L, 5pmol/L +10pmol/L and 3pmol/L +3 pmol/L; the annealing temperature optimization is to set different PCR annealing temperatures, namely 50 ℃, 53 ℃, 56 ℃, 58 ℃ and 60 ℃, and determine the optimal PCR reaction annealing temperature;
s7, performing double PCR specific detection, performing PCR by using cDNA of other common respiratory disease viruses, namely influenza A virus H1N1, influenza B virus, human coronavirus MERS-CoV and adenovirus as a template, and observing the specificity of the primers according to an electrophoresis result;
s8, detection of sensitivity by the double PCR method, mixing plasmids from 1X 10 by 10-fold gradient dilution 8 Copy/. mu.L dilution to 1X 10 0 Copy/. mu.L, for detecting the sensitivity of the dual method.
Preferably, the template cloning reagent in step 2 comprises pUC57 vector, tryptone, yeast extract, agar powder and Amp, the agarose gel electrophoresis reagent comprises agarose, nucleic acid dye Goldview, DNA Marker (DL2000) and TBE solution, and the PCR amplification reagent comprises primer, 2 xMaster Mix and ddH 2O.
Preferably, the amplification length of SARS-CoV, COVID-19 and MERS-CoV in step 4 is set to 3001-3348bp, and the gene accession number of the influenza A virus H1N1 is DJ 399201.1.
Preferably, in the single PCR amplification in step 5 and the double PCR amplification in step 6, artificially synthesized SARS-CoV and COVID-19ORF1ab fragments are used as templates, and specific primers designed and synthesized by SARS-CoV and COVID-19 are used for single PCR amplification and double PCR amplification, and the amplification results show that the specific fragments can be amplified by single PCR amplification or double PCR amplification, and the sizes of the specific fragments are consistent with those expected, and the primers are designed to be correct after sequencing, thereby indicating that the synthesized gene fragments are correct, and the target fragments can be specifically amplified by the designed primers.
Preferably, the primer concentration optimization in step 6 is to perform PCR on the SARS-CoV and COVID-19 virus upstream and downstream primers at different concentrations, and find that the two virus detection upstream and downstream primers are respectively 5pmol/L +5pmol/L most suitable by PCR amplification and electrophoresis detection.
Preferably, the annealing temperature optimization in step 6 is to perform amplification at different PCR annealing temperatures according to an optimal PCR reaction system, and the amplification result is that the optimal annealing temperature for detecting two viruses, SARS-CoV and COVID-19, is 58 ℃.
Preferably, the specificity of the double PCR method is determined by PCR using other common respiratory disease viruses as described in step 7, and the results show that the designed primers can only specifically detect two virus plasmid templates of SARS-CoV and COVID-19, but cannot detect other viruses in the experiment.
Preferably, the sensitivity detection of the double PCR method described in step 8 mixes the two viral plasmids from 1X 10 by 10-fold gradient dilution 7 Copy/. mu.L dilution to 1X 10 0 copying/mu.L, performing PCR amplification, and displaying that the concentration of the lowest virus gene fragment plasmid which can be detected by the established PCR diagnosis method is 1 multiplied by 10 2 Copies/. mu.L.
Compared with the prior art, the dual PCR detection method for SARS-CoV and COVID-19 viruses provided by the invention has the following advantages:
1. the invention carries out single PCR amplification and double amplification on the virus after the virus gene synthesis, carries out double optimization of primer concentration condition and annealing temperature condition on the virus during amplification, finally uses other common respiratory system disease viruses to carry out PCR, determines the specificity of establishing the double PCR method, and shows that the designed primer can only specifically detect two virus plasmid templates of SARS-CoV and COVID-19, but can not detect other viruses of the experiment, and improves the effectiveness of detecting the SARS-CoV and the COVID-19 viruses from a plurality of viruses to a certain extent;
2. the invention mixes plasmid from 1X 10 through 10 times of gradient dilution 8 Copy/. mu.L dilution to 1X 10 0 Sensitivity of the copy/μ L detection dual method, the lowest virus gene fragment plasmid concentration that can be detected by the established PCR diagnostic method is 1 × 10 2 Copy/. mu.L, has better sensitivity.
Drawings
FIG. 1 is a block flow diagram of the present invention;
FIG. 2 is a schematic diagram showing the result of PCR amplification according to the present invention;
FIG. 3 is a cross-sectional view of a filtration apparatus housing of the present invention;
FIG. 4 is a schematic structural view of a sewage screen of the present invention;
FIG. 5 is a diagram showing the results of specificity detection;
FIG. 6 is a schematic diagram showing the result of PCR amplification;
in fig. 2:
a: A. single PCR amplification; m, marker2000, 892000; 1. SARS-CoV virus gene fragment; 2. a fragment of the COVID-19 viral gene; 3. negative control;
b: B. performing double PCR amplification; m, marker2000, 892000; 1. SARS-CoV + COVID-19 virus gene fragment;
in fig. 3: m, marker2000, 892000; 1. 10pmol/L +10 pmol/L; 2.5 pmol/L +5 pmol/L; 3. 10pmol/L +5 pmol/L; 4. 5pmol/L +10 pmol/L; 5. 3pmol/L +3 pmol/L; 6. negative control;
in fig. 4: m, marker2000, 892000; 1. 50 ℃; 2. at 53 ℃; 3. 56 ℃; 4. at 58 ℃; 5. 60 ℃; 6. negative control;
in fig. 5: m, marker2000, 892000; 1. SARS-CoV + COVID-19; 2. influenza a virus H1N 1; 3. Influenza B virus; 4. human coronavirus MERS-CoV; 5. an adenovirus; 6. negative control;
in fig. 6: m, marker2000, 892000; 1. 1X 10 7 Copy/. mu.L; 2. 1X 10 6 Copy/. mu.L; 3. 1X 10 5 Copy/. mu.L; 4. 1X 10 4 Copy/. mu.L; 5. 1X 10 3 Copy/. mu.L; 6. 1X 10 2 Copy/. mu.L; 7. 1X 10 1 Copy/. mu.L; 8. 1X 10 0 Copies/. mu.L.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The specific embodiments described herein are merely illustrative of the invention and do not delimit the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a SARS-CoV and COVID-19 virus double PCR detection method as shown in figure 1, comprising the following steps:
s1, preparing a virus cDNA template, preparing SARS-CoV, COVID-19, MERS-CoV, influenza A virus H1N1, influenza B virus and adenovirus templates;
s2, preparing reagent consumables, and preparing a template cloning reagent, an agarose gel electrophoresis reagent and a PCR amplification reagent;
s3, designing primers, obtaining the complete gene sequences of SARS-CoV and COVID-19 strains from GenBank, comparing the conserved regions of the virus gene sequences by BioXM2.6 software, designing specific primers of each virus by using Primer Premier 5 software, wherein the Primer information is as follows:
GenBank is a DNA sequence database established by the national center for biotechnology information, obtains sequence data from public resources, is mainly directly provided by scientific researchers or derived from a large-scale genome sequencing plan, and establishes a cooperative relationship of mutual exchange data with EMBL (European EMBL-DNA database) and DDBJ (Japanese DNA database) in order to ensure that the data are as complete as possible; BioXM2.6 software introduces that Bioxm is used for conventional analysis of DNA sequence data, including ORF searching, sequence formatting, translation, calculation of protein molecular weight and isoelectric point, restriction enzyme cutting site analysis, primer auxiliary design and the like, and also provides a batch conversion tool of sequence format files, a primer concentration calculation tool, a cutting site query tool, an automatic vector sequence removal tool, an SSR site search tool, a latest influence factor query and the like.
S4, synthesizing virus genes, synthesizing SARS-CoV, COVID-19, MERS-CoV and influenza A virus H1N1 gene fragments to be detected, cloning to a pUC57 vector, and extracting plasmids for later use after correct sequencing;
s5, single PCR amplification, wherein the total volume of each reaction system is 25. mu.L, and the reaction system contains 12.5. mu.L of 2 × Master Mix, 1. mu.L (10pmol/L) of each of the upstream and downstream primers, 1. mu.L of the synthesized viral plasmid template, and 9.5. mu.L of ddH2O, and the PCR reaction conditions are as follows: 94 ℃ for 3 min; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 30s, 30 cycles; 72 ℃ for 10 min; finally storing at 12 ℃; analyzing the PCR product by 1.5% agarose gel electrophoresis, and placing the PCR product in a gel imaging system to observe an amplification result;
s6, double PCR amplification and condition optimization, mixing two virus plasmids in equal proportion, carrying out PCR amplification according to S5 reaction conditions and a reaction system, analyzing the amplified PCR product through 1.5% agarose gel electrophoresis, and observing by using a gel imaging system, wherein the condition optimization of the double PCR amplification comprises primer concentration optimization and annealing temperature optimization, wherein the primer concentration optimization is to carry out PCR on upstream and downstream primers of SARS-CoV and COVID-19 according to different concentrations, and the combination is respectively as follows: determining the optimal primer concentration by 10pmol/L +10pmol/L, 5pmol/L +5pmol/L, 10pmol/L +5pmol/L, 5pmol/L +10pmol/L and 3pmol/L +3 pmol/L; the annealing temperature optimization is to set different PCR annealing temperatures, namely 50 ℃, 53 ℃, 56 ℃, 58 ℃ and 60 ℃, and determine the optimal PCR reaction annealing temperature;
s7, performing double PCR specific detection, performing PCR by using cDNA of other common respiratory disease viruses, namely influenza A virus H1N1, influenza B virus, human coronavirus MERS-CoV and adenovirus as a template, and observing the specificity of the primers according to an electrophoresis result;
s8, detection of sensitivity by the double PCR method, mixing plasmids from 1X 10 by 10-fold gradient dilution 8 Copy/. mu.L dilution to 1X 10 0 Copy/. mu.L, for detecting the sensitivity of the dual method;
the template cloning reagent in the step 2 comprises a pUC57 carrier, tryptone, a yeast extract, agar powder and Amp, the agarose gel electrophoresis reagent comprises agarose, a nucleic acid dye Goldview, a DNA Marker (DL2000) and a TBE solution, the PCR amplification reagent comprises a primer, 2 × Master Mix and ddH2O, and the specific experimental reagents are shown in the following table:
wherein, the amplification length of SARS-CoV, COVID-19 and MERS-CoV in step 4 is set as 3001-3348bp, and the gene accession number of influenza A virus H1N1 is DJ 399201.1;
the single PCR amplification in the step 5 and the double PCR amplification in the step 6 are carried out by taking artificially synthesized SARS-CoV and COVID-19ORF1ab fragments as templates and adopting specific primers designed and synthesized by SARS-CoV and COVID-19 to carry out single PCR amplification and double PCR amplification, wherein the amplification results show that the specific fragments can be amplified by the single PCR amplification or the double PCR amplification, the sizes of the specific fragments are consistent with the expected sizes, the sequencing is correct, the synthesized gene fragments are correct, the target fragments can be specifically amplified by the designed primers, and the PCR amplification results are shown in FIG. 2;
wherein, the concentration optimization of the primers in the step 6 is to perform PCR on the upstream and downstream primers of SARS-CoV and COVID-19 viruses according to different concentrations, and the optimal results are shown in FIG. 3 when the upstream and downstream primers for the detection of the two viruses are respectively 5pmol/L +5pmol/L through PCR amplification and electrophoresis detection;
wherein, in the step 6, the annealing temperature optimization is to perform amplification at different PCR annealing temperatures according to an optimal PCR reaction system, the amplification result is that the optimal annealing temperature for detecting two viruses, SARS-CoV and COVID-19 is 58 ℃, and the optimization result is shown in FIG. 4;
wherein, in step 7, PCR is carried out by using other common respiratory disease viruses, the specificity of establishing the dual PCR method is determined, and the result shows that the designed primer can only specifically detect two virus plasmid templates of SARS-CoV and COVID-19, but can not detect other viruses of the experiment, which indicates that the designed primer has better specificity, and the specific detection result is shown in figure 5;
wherein the detection of the sensitivity of the double PCR method in step 8 mixes the two viral plasmids from 1X 10 by 10-fold gradient dilution 7 Copy/. mu.L dilution to 1X 10 0 Copying/mu L, performing PCR amplification, and displaying that the concentration of the lowest virus gene fragment plasmid detected by the established PCR diagnosis method is 1 multiplied by 10 2 Copy/. mu.L, with better sensitivity, as shown in FIG. 6.
In the experiment, the complete gene sequences of SARS-CoV and COVID-19 strains are obtained from GenBank, the conserved region of the virus gene sequences is compared through BioXM2.6 software, specific primers of each virus are firstly designed, SARS-CoV, COVID-19, MERS-CoV and influenza A virus H1N1 are subjected to gene fragment synthesis to be detected, then cloned to a pUC57 vector, plasmids are extracted after the sequencing is correct, then single PCR amplification and double PCR amplification are carried out on the extracted plasmids, the double optimization of primer concentration conditions and annealing temperature conditions is carried out on the extracted plasmids during the amplification, finally other common respiratory system disease viruses are used for carrying out PCR to determine the specificity for establishing the double PCR method, the designed primers can only specifically detect SARS-CoV and COVID-19 two virus plasmid templates, but can not detect other viruses in the experiment, the effectiveness of detecting SARS-CoV and COVID-19 virus from a plurality of viruses is improved to a certain extent.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Sequence listing
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Claims (8)
1. A dual PCR detection method for SARS-CoV and COVID-19 virus is characterized in that: the method comprises the following steps:
s1, preparing a virus cDNA template, preparing SARS-CoV, COVID-19, MERS-CoV, influenza A virus H1N1, influenza B virus and adenovirus templates;
s2, preparing reagent consumables, and preparing a template cloning reagent, an agarose gel electrophoresis reagent and a PCR amplification reagent;
s3, designing primers, obtaining complete gene sequences of SARS-CoV and COVID-19 strains from GenBank, comparing conserved regions of virus gene sequences by BioXM2.6 software, and designing specific primers of each virus by using Primer Premier 5 software, wherein the specific primers are SARS-CoV-F, SARS-CoV-R, COVID-19-F, COVID-19-R, the sequence of SARS-CoV-F is AGGAAGAAGAGGACGATGCAGAGTGT, the sequence number of SARS-CoV-R is AGGTGTAGGTTCTGGTTCTGGCTCAA, the sequence number of COVID-19-F is TGCCACTTCTGCTGCTCTTCAACCT, and the sequence number of COVID-19-R is TCTGATTGTCCTCACTGCCGTCTTGT;
s4, synthesizing virus genes, synthesizing SARS-CoV, COVID-19, MERS-CoV and influenza A virus H1N1 gene fragments to be detected, cloning to a pUC57 vector, and extracting plasmids for later use after correct sequencing;
s5, single PCR amplification, wherein the total volume of each reaction system is 25. mu.L, the reaction system contains 12.5. mu.L of 2 xMaster Mix, 1. mu.L of each upstream primer and downstream primer of 10pmol/L, the synthesized virus plasmid template is 1. mu.L, and 9.5. mu.L of ddH2O, and the PCR reaction conditions are as follows: 94 ℃ for 3 min; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 30s, 30 cycles; 72 ℃ for 10 min; finally storing at 12 ℃; analyzing the PCR product by 1.5% agarose gel electrophoresis, and placing the PCR product in a gel imaging system to observe an amplification result;
s6, double PCR amplification and condition optimization, mixing two virus plasmids in equal proportion, carrying out PCR amplification according to S5 reaction conditions and a reaction system, analyzing the amplified PCR product through 1.5% agarose gel electrophoresis, and observing by using a gel imaging system, wherein the condition optimization of the double PCR amplification comprises primer concentration optimization and annealing temperature optimization, wherein the primer concentration optimization is to carry out PCR on upstream and downstream primers of SARS-CoV and COVID-19 according to different concentrations, and the combination is respectively as follows: determining the optimal primer concentration by 10pmol/L +10pmol/L, 5pmol/L +5pmol/L, 10pmol/L +5pmol/L, 5pmol/L +10pmol/L and 3pmol/L +3 pmol/L; the annealing temperature optimization is to set different PCR annealing temperatures, namely 50 ℃, 53 ℃, 56 ℃, 58 ℃ and 60 ℃, and determine the optimal PCR reaction annealing temperature;
s7, performing double PCR specific detection, performing PCR by using cDNA of other common respiratory disease viruses, namely influenza A virus H1N1, influenza B virus, human coronavirus MERS-CoV and adenovirus as a template, and observing the specificity of the primers according to an electrophoresis result;
s8, detection of sensitivity of the double PCR method, mixing plasmids from 1X 10 by 10-fold gradient dilution 8 Copy/. mu.L dilution to 1X 10 0 Copies/. mu.L, sensitivity for detection of the dual method of SARS-CoV and COVID-19 virus dual PCR detection for non-disease diagnostic purposes.
2. The dual PCR detection method for SARS-CoV and COVID-19 virus according to claim 1, wherein: the template cloning reagent in the step S2 comprises a pUC57 vector, tryptone, a yeast extract, agar powder and Amp, the agarose gel electrophoresis reagent comprises agarose, a nucleic acid dye Goldview, DNA MarkerDL2000 and a TBE solution, and the PCR amplification reagent comprises a primer, 2 xMaster Mix and ddH 2O.
3. The dual PCR detection method for SARS-CoV and COVID-19 virus according to claim 1, wherein: the amplification length of SARS-CoV, COVID-19 and MERS-CoV in step S4 was set to 3001-3348bp, and the gene accession number of the influenza A virus H1N1 was DJ 399201.1.
4. The dual PCR detection method for SARS-CoV and COVID-19 virus according to claim 1, wherein: the single PCR amplification in the step S5 and the double PCR amplification in the step S6 are carried out by taking artificially synthesized SARS-CoV and COVID-19ORF1ab fragments as templates and adopting specific primers designed and synthesized by SARS-CoV and COVID-19 to carry out single PCR amplification and double PCR amplification, and the amplification results show that the specific fragments can be amplified by single PCR amplification or double PCR amplification, the sizes of the specific fragments are consistent with the expected sizes, the sequencing is correct, the synthesized gene fragments are correct, and the designed primers can specifically amplify the target fragments.
5. The dual PCR detection method for SARS-CoV and COVID-19 virus according to claim 1, wherein: the primer concentration optimization in step S6 is to perform PCR on the upstream and downstream primers of SARS-CoV and COVID-19 viruses according to different concentrations, and find that the upstream and downstream primers for detection of the two viruses are respectively 5pmol/L +5pmol/L most suitable through PCR amplification and electrophoresis detection.
6. The dual PCR detection method for SARS-CoV and COVID-19 virus according to claim 1, wherein: the annealing temperature optimization in step S6 is to amplify under different PCR annealing temperatures according to the optimal PCR reaction system, and the amplification result is that the optimal annealing temperature for detecting two viruses, SARS-CoV and COVID-19, is 58 ℃.
7. The dual PCR detection method for SARS-CoV and COVID-19 virus according to claim 1, wherein: the specificity of the double PCR method is determined by using other common respiratory disease viruses to carry out PCR in the step S7, and the result shows that the designed primers can only specifically detect two virus plasmid templates of SARS-CoV and COVID-19, but can not detect other viruses in the experiment.
8. The dual PCR detection method for SARS-CoV and COVID-19 virus according to claim 7, wherein: sensitivity detection of the double PCR method described in step S8 will be performed by 10-fold gradient dilutionMixing two viral plasmids from 1X 10 7 Copy/. mu.L dilution to 1X 10 0 Copying/mu L, performing PCR amplification, and displaying that the concentration of the lowest virus gene fragment plasmid detected by the established PCR diagnosis method is 1 multiplied by 10 2 Copies/. mu.L.
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