Virus Quantification
Encyclopedia
Virus
quantification involves counting the number of viruses in a specific volume to determine the virus concentration. It is utilized in both research and development (R&D) in commercial and academic laboratories as well as production situations where the quantity of virus at various steps is an important variable. For example, the production of viral vaccines, recombinant proteins using viral vectors and viral antigens all require virus quantification to continually adapt and monitor the process in order to optimize production yields and respond to ever changing demands and applications. Examples of specific instances where known viruses need to be quantified include clone screening, multiplicity of infection
(MOI) optimization and adaptation of methods to cell culture. This page discusses various techniques currently used to quantify viruses in liquid samples. These methods are separated into two categories, traditional vs. modern methods. Traditional methods are industry-standard methods that have been used for decades but are generally slow and labor intensive. Modern methods are relatively new commercially available products and kits that greatly reduce quantification time. This is not meant to be an exhaustive review of all potential methods, but rather a representative cross-section of traditional methods and new, commercially available methods. While other published methods may exist for virus quantification, non-commercial methods are not discussed here.
assays determine the number of plaque forming units (pfu) in a virus sample, which is one measure of virus quantity. This assay is based on a microbiological method conducted in petri dishes or multi-well plates. Specifically, a confluent monolayer of host
cells is infected with the virus at varying dilutions and covered with a semi-solid medium, such as agar
or carboxymethyl cellulose
, to prevent the virus infection from spreading indiscriminately. A viral plaque is formed when a virus infects a cell within the fixed cell monolayer. The virus infected cell will lyse and spread the infection to adjacent cells where the infection-to-lysis cycle is repeated. The infected cell area will create a plaque (an area of infection surrounded by uninfected cells) which can be seen visually or with an optical microscope. Plaque formation can take 3 – 14 days, depending on the virus being analyzed. Plaques are generally counted manually and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per sample unit volume (pfu/mL). The pfu/mL result represents the number of infective particles within the sample and is based on the assumption that each plaque formed is representative of one infective virus particle.
in 50% of inoculated tissue culture cells. This assay may be more common in clinical research applications where the lethal dose of virus must be determined or if the virus does not form plaques. When used in the context of tissue culture, host cells are plated and serial dilutions of the virus are added. After incubation, the percentage of cell death (i.e. infect ed cells) is manually observed and recorded for each virus dilution, and results are used to mathematically calculate a TCID50 result. Due to distinct differences in assay methods and principles, TCID50 and pfu/mL or other infectivity assay results are not equivalent. This method can take up to a week due to cell infectivity time.
techniques with fluorescently-labeled antibodies specific for the virus antigen
to measure host cell infection before an actual plaque is formed. Like the plaque assay, host cell monolayers are infected with various dilutions of the virus sample and allowed to incubate for a relatively brief incubation period (e.g., 24–72 hours). Plates are subsequently stained with fluorescent antibodies, and fluorescence microscopy is used to count and quantify how many cells are infected. The FFA method yields results in less time than plaque or TCID50 assays but is more expensive in terms of required reagents and equipment. Assay time is also dependent on the size of area that the user is counting, the larger area will take more time but will provide a more accurate representation of the sample. Results are expressed as fluorescent focus forming units or FFU/mL.
-based virus quantification assays. In general, these methods quantify either the amount of all protein or the amount of a specific virus protein in the sample rather than the number of infected cells or virus particles. Quantification most commonly relies on fluorescence
detection. Some assay variations quantify protein directly in a sample while other variations require host cell infection and incubation to allow virus growth prior to protein quantification. The variation used depends primarily on the amount of protein (i.e. virus) in the initial sample and the sensitivity of the assay itself. If incubation and virus growth are required, cell and/or virus lysis/digestion are often conducted prior to analysis. Most protein-based methods are relatively fast and sensitive but require quality standards for accurate calibration, and quantify protein, not actual virus particle concentrations. Below are specific examples of widely used protein-based assays.
. It relies on the fact that hemagglutinin
, a surface protein of influenza viruses, agglutinates red blood cells (i.e. causes red blood cells to clump together). In this assay, dilutions of an influenza sample are incubated with a 1% erythrocyte solution for one hour and the virus dilution at which agglutination first occurs is visually determined. The assay produces a result of hemagglutination units (HAU), with typical pfu to HAU ratios in the 106 range. This assay takes ~1–2 hours to complete and results can differ widely based on the technical expertise of the operator.
The hemagglutination inhibition assay is a common variation of the HA assay used to measure flu-specific antibody levels in blood serum. In this variation, serum antibodies to the influenza virus will interfere with the virus attachment to red blood cells. Therefore hemagglutination is inhibited when antibodies are present at a sufficient concentration.
assay (BCA) is based on a simple colorimetric measurement and is the most common protein quantification assay. BCA is similar to the Lowry
or Bradford
protein assays and was first made commercially available by Pierce, which is now owned by Thermo Fisher Scientific
. In the BCA assay, a protein’s peptide bonds quantitatively reduce Cu2+ to Cu1+, which produces a light blue color. BCA chelates Cu1+ at a 2:1 ratio resulting in a more intensely colored species that absorbs at 562 nm. Absorbance of a sample at 562 nm is used to determine the bulk protein concentration in the sample. Assay results are compared with known standard curves after analysis with a spectrophotometer or plate reader. Total assay time is 30 minutes to one hour. While this assay is ubiquitous and fast, it lacks specificity since it counts all protein, the virus preparation to be quantified must contain very low levels host cell proteins.
assay (SRID), also known as the Mancini method, is a protein assay that detects the amount of specific viral antigen by immunodiffusion in a semi-solid medium (e.g. agar). The medium contains antiserum
specific to the antigen of interest and the antigen is placed in the center of the disc. As the antigen diffuses into the medium it creates a precipitate ring that grows until equilibrium is reached. Assay time can range from 10 hours to days depending on equilibration time of the antigen and antibody. The zone diameter from the ring is linearly related to the log of protein concentration and is compared to zone diameters for known protein standards for quantification. There are kits and serums commercially available for this assay (e.g. The Binding Site Inc.).
is a specialized type of microscopy that utilizes a beam of electrons focused with a magnetic field to image a sample. TEM provides imaging with 1000x greater spatial resolution than a light microscope (resolution down to 0.2 nm). An ultrathin, negatively stained
sample is required. Sample preparations involve depositing specimens onto a coated TEM grid and negative staining with an electron-opaque liquid. Tissue embedded samples can also be examined if thinly sectioned. Sample preparations vary depending on protocol and user but generally require hours to complete. TEM images can show individual virus particles and quantitative image analysis
can be used to determine virus concentrations. These high resolution images also provide particle morphology information that most other methods cannot. Quantitative TEM results will often be greater than results from other assays as all particles, regardless of infectivity, are quantified in the reported virus-like particles per mL (vlp/mL) result. Quantitative TEM generally works well for virus concentrations greater than 106 particles/mL. Because of high instrument cost and the amount of space and support facilities needed, TEM equipment is available in a limited number of facilities.
is a benchtop flow cytometer designed specifically for virus quantification by InDevR
Inc. This system is significantly more sensitive than other commercial cytometers, which enables efficient detection and accurate quantification of viruses while maintaining the speed and versatility advantages of flow cytometry. The Virus Counter assay and instrument quantify the number of intact virus particles in a sample using fluorescence to detect colocalized proteins and nucleic acids. Samples are stained with two dyes, one specific for proteins and one specific for nucleic acids, and analyzed as they flow through a laser beam. The quantity of particles producing simultaneous events on each of the two distinct fluorescence channels is determined, along with the measured sample flow rate, to calculate a concentration of virus particles (vp/mL). The results are generally similar in absolute quantity to a TEM result. The assay has a linear working range of 105-109 vp/mL and an analysis time of ~10 min with a short sample preparation time.
chemistry to amplify viral DNA
or RNA
to produce high enough concentrations for detection and quantification by fluorescence. In general, quantification by qPCR relies on serial dilutions of standards of known concentration being analyzed in parallel with the unknown samples for calibration and reference. Quantitative detection can be achieved using a wide variety of fluorescence detection strategies, including sequence specific probes or universal probes such as SYBR Green
dye. Sequence specific probes, such as TaqMan
(i.e. Applied Biosystems), Molecular Beacons, or Scorpion, bind only to the copied, or cDNA of the appropriate sequence produced during the reaction. SYBR Green dye binds to all double-stranded DNA produced during the reaction. While SYBR Green is easy to use, its lack of specificity and lower sensitivity lead most labs to use probe-based qPCR detection schemes. There are many variations of qPCR including the comparative threshold method, which allows relative quantification through comparison of Ct values (PCR cycles that show statistically significant increases in the product) from multiple samples that include an internal standard. Since PCR amplifies all target nucleic acid
, whether from an intact virion or free nucleic acids in solution, qPCR results (expressed in terms of genome copies/mL) are likely to be higher in quantity than TEM results. For viral quantification, the ratio of whole virions to copies of nucleic acid is seldom one to one due to the fact that during viral replication, viral assembly in the cytoplasm requires the production of proteins (structural and non-structural) and nucleic acid which are the precursors needed to assemble a whole virion. In the example of foot-and-mouth disease virus, the ratio of whole virions to RNA copies within an actively replicating host cell is approximately 1:1000. Commercially available products for qPCR are available through numerous companies such as Invitrogen, Roche and Qiagen just to name a few. Real-time qPCR takes around 1–4 hours and can provide quantitative results containing too few viruses to be analyzed by other methods.
is a more modern variation of a protein assay that utilizes a specific antibody linked to an enzyme to detect the presence of an unknown amount of antigen (i.e. virus) in a sample. The antibody-antigen binding event is detected and/or quantified through the enzyme’s ability to convert a reagent to a detectable signal that can be used to calculate the concentration of the antigen in the sample. Horseradish peroxidase
(HRP) is a common enzyme utilized in ELISA schemes due to its ability to amplify signal and increase assay sensitivity. There are many variations, or types of ELISA assays but they can generally be classified as either indirect, competitive, sandwich or reverse. ELISA kits are commercially available from numerous companies and quantification generally occurs via chromogenic
reporters or fluorescence (e.g. Invitrogen, Santa Cruz Biotechnology Inc.). This technique is much less labor intensive than the traditional methods and can take anywhere from 4 to 24 hours based on antibody incubation time.
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
quantification involves counting the number of viruses in a specific volume to determine the virus concentration. It is utilized in both research and development (R&D) in commercial and academic laboratories as well as production situations where the quantity of virus at various steps is an important variable. For example, the production of viral vaccines, recombinant proteins using viral vectors and viral antigens all require virus quantification to continually adapt and monitor the process in order to optimize production yields and respond to ever changing demands and applications. Examples of specific instances where known viruses need to be quantified include clone screening, multiplicity of infection
Multiplicity of infection
The multiplicity of infection or MOI is the ratio of infectious agents to infection targets . For example, when referring to a group of cells inoculated with infectious virus particles, the multiplicity of infection or MOI is the ratio of the number of infectious virus particles to the number of...
(MOI) optimization and adaptation of methods to cell culture. This page discusses various techniques currently used to quantify viruses in liquid samples. These methods are separated into two categories, traditional vs. modern methods. Traditional methods are industry-standard methods that have been used for decades but are generally slow and labor intensive. Modern methods are relatively new commercially available products and kits that greatly reduce quantification time. This is not meant to be an exhaustive review of all potential methods, but rather a representative cross-section of traditional methods and new, commercially available methods. While other published methods may exist for virus quantification, non-commercial methods are not discussed here.
Plaque assay
Plaque-based assays are the standard method used to determine virus concentration in terms of infectious dose. Viral plaqueViral plaque
A viral plaque is a visible structure formed within a cell culture, such as bacterial cultures within some nutrient medium . The bacteriophage viruses replicate and spread, thus generating regions of cell destructions known as plaques....
assays determine the number of plaque forming units (pfu) in a virus sample, which is one measure of virus quantity. This assay is based on a microbiological method conducted in petri dishes or multi-well plates. Specifically, a confluent monolayer of host
Host (biology)
In biology, a host is an organism that harbors a parasite, or a mutual or commensal symbiont, typically providing nourishment and shelter. In botany, a host plant is one that supplies food resources and substrate for certain insects or other fauna...
cells is infected with the virus at varying dilutions and covered with a semi-solid medium, such as agar
Agar
Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae. Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium...
or carboxymethyl cellulose
Carboxymethyl cellulose
Carboxymethyl cellulose or cellulose gum is a cellulose derivative with carboxymethyl groups bound to some of the hydroxyl groups of the glucopyranose monomers that make up the cellulose backbone...
, to prevent the virus infection from spreading indiscriminately. A viral plaque is formed when a virus infects a cell within the fixed cell monolayer. The virus infected cell will lyse and spread the infection to adjacent cells where the infection-to-lysis cycle is repeated. The infected cell area will create a plaque (an area of infection surrounded by uninfected cells) which can be seen visually or with an optical microscope. Plaque formation can take 3 – 14 days, depending on the virus being analyzed. Plaques are generally counted manually and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per sample unit volume (pfu/mL). The pfu/mL result represents the number of infective particles within the sample and is based on the assumption that each plaque formed is representative of one infective virus particle.
50% Tissue Culture Infective Dose (TCID50)
This endpoint dilution assay quantifies the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effectCytopathic effect
Cytopathic effect or cytopathogenic effect refers to degenerative changes in cells, especially in tissue culture, and may be associated with the multiplication of certain viruses....
in 50% of inoculated tissue culture cells. This assay may be more common in clinical research applications where the lethal dose of virus must be determined or if the virus does not form plaques. When used in the context of tissue culture, host cells are plated and serial dilutions of the virus are added. After incubation, the percentage of cell death (i.e. infect ed cells) is manually observed and recorded for each virus dilution, and results are used to mathematically calculate a TCID50 result. Due to distinct differences in assay methods and principles, TCID50 and pfu/mL or other infectivity assay results are not equivalent. This method can take up to a week due to cell infectivity time.
Fluorescent Focus Assay (FFA)
This assay is a more rapid variation of the plaque assay because it uses immunostainingImmunostaining
Immunostaining is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. The term immunostaining was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941...
techniques with fluorescently-labeled antibodies specific for the virus antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
to measure host cell infection before an actual plaque is formed. Like the plaque assay, host cell monolayers are infected with various dilutions of the virus sample and allowed to incubate for a relatively brief incubation period (e.g., 24–72 hours). Plates are subsequently stained with fluorescent antibodies, and fluorescence microscopy is used to count and quantify how many cells are infected. The FFA method yields results in less time than plaque or TCID50 assays but is more expensive in terms of required reagents and equipment. Assay time is also dependent on the size of area that the user is counting, the larger area will take more time but will provide a more accurate representation of the sample. Results are expressed as fluorescent focus forming units or FFU/mL.
Protein assays
There are several variations of proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
-based virus quantification assays. In general, these methods quantify either the amount of all protein or the amount of a specific virus protein in the sample rather than the number of infected cells or virus particles. Quantification most commonly relies on fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
detection. Some assay variations quantify protein directly in a sample while other variations require host cell infection and incubation to allow virus growth prior to protein quantification. The variation used depends primarily on the amount of protein (i.e. virus) in the initial sample and the sensitivity of the assay itself. If incubation and virus growth are required, cell and/or virus lysis/digestion are often conducted prior to analysis. Most protein-based methods are relatively fast and sensitive but require quality standards for accurate calibration, and quantify protein, not actual virus particle concentrations. Below are specific examples of widely used protein-based assays.
Hemagglutination assay
The hemagglutination assay (HA) is a common non-fluorescence protein quantification assay specific for influenzaInfluenza
Influenza, commonly referred to as the flu, is an infectious disease caused by RNA viruses of the family Orthomyxoviridae , that affects birds and mammals...
. It relies on the fact that hemagglutinin
Hemagglutinin
Influenza hemagglutinin or haemagglutinin is a type of hemagglutinin found on the surface of the influenza viruses. It is an antigenic glycoprotein. It is responsible for binding the virus to the cell that is being infected...
, a surface protein of influenza viruses, agglutinates red blood cells (i.e. causes red blood cells to clump together). In this assay, dilutions of an influenza sample are incubated with a 1% erythrocyte solution for one hour and the virus dilution at which agglutination first occurs is visually determined. The assay produces a result of hemagglutination units (HAU), with typical pfu to HAU ratios in the 106 range. This assay takes ~1–2 hours to complete and results can differ widely based on the technical expertise of the operator.
The hemagglutination inhibition assay is a common variation of the HA assay used to measure flu-specific antibody levels in blood serum. In this variation, serum antibodies to the influenza virus will interfere with the virus attachment to red blood cells. Therefore hemagglutination is inhibited when antibodies are present at a sufficient concentration.
Bicinchoninic acid assay
The bicinchoninic acidBicinchoninic acid
Bicinchoninic acid is a weak acid composed of two carboxylated quinoline rings.Bicinchoninic acid is most commonly employed by biochemists in the bicinchoninic acid assay, which is used to determine the total level of protein in a solution...
assay (BCA) is based on a simple colorimetric measurement and is the most common protein quantification assay. BCA is similar to the Lowry
Lowry protein assay
The Lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques. It is...
or Bradford
Bradford protein assay
The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. It is subjective, i.e., dependent on the amino acid composition of the measured protein. The Bradford protein assay was developed by Marion M...
protein assays and was first made commercially available by Pierce, which is now owned by Thermo Fisher Scientific
Thermo Fisher Scientific
Thermo Fisher Scientific is a large life sciences supply company that was created in 2006 by the merger of Thermo Electron and Fisher Scientific.-Predecessors and merger:...
. In the BCA assay, a protein’s peptide bonds quantitatively reduce Cu2+ to Cu1+, which produces a light blue color. BCA chelates Cu1+ at a 2:1 ratio resulting in a more intensely colored species that absorbs at 562 nm. Absorbance of a sample at 562 nm is used to determine the bulk protein concentration in the sample. Assay results are compared with known standard curves after analysis with a spectrophotometer or plate reader. Total assay time is 30 minutes to one hour. While this assay is ubiquitous and fast, it lacks specificity since it counts all protein, the virus preparation to be quantified must contain very low levels host cell proteins.
Single radial immunodiffusion assay
Single radial immunodiffusionRadial immunodiffusion
Radial immunodiffusion is an immunodiffusion technique used in immunology to determine the quantity of an antigen by measuring the diameters of circles of precipitin complexes surrounding samples of the antigen that mark the boundary between the antigen and an antibody suspended in a medium, such...
assay (SRID), also known as the Mancini method, is a protein assay that detects the amount of specific viral antigen by immunodiffusion in a semi-solid medium (e.g. agar). The medium contains antiserum
Antiserum
Antiserum is blood serum containing polyclonal antibodies. Antiserum is used to pass on passive immunity to many diseases. Passive antibody transfusion from a previous human survivor is the only known effective treatment for Ebola infection .The most common use of antiserum in humans is as...
specific to the antigen of interest and the antigen is placed in the center of the disc. As the antigen diffuses into the medium it creates a precipitate ring that grows until equilibrium is reached. Assay time can range from 10 hours to days depending on equilibration time of the antigen and antibody. The zone diameter from the ring is linearly related to the log of protein concentration and is compared to zone diameters for known protein standards for quantification. There are kits and serums commercially available for this assay (e.g. The Binding Site Inc.).
Transmission Electron Microscopy (TEM)
TEMTransmission electron microscopy
Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...
is a specialized type of microscopy that utilizes a beam of electrons focused with a magnetic field to image a sample. TEM provides imaging with 1000x greater spatial resolution than a light microscope (resolution down to 0.2 nm). An ultrathin, negatively stained
Negative stain
Negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid....
sample is required. Sample preparations involve depositing specimens onto a coated TEM grid and negative staining with an electron-opaque liquid. Tissue embedded samples can also be examined if thinly sectioned. Sample preparations vary depending on protocol and user but generally require hours to complete. TEM images can show individual virus particles and quantitative image analysis
Image analysis
Image analysis is the extraction of meaningful information from images; mainly from digital images by means of digital image processing techniques...
can be used to determine virus concentrations. These high resolution images also provide particle morphology information that most other methods cannot. Quantitative TEM results will often be greater than results from other assays as all particles, regardless of infectivity, are quantified in the reported virus-like particles per mL (vlp/mL) result. Quantitative TEM generally works well for virus concentrations greater than 106 particles/mL. Because of high instrument cost and the amount of space and support facilities needed, TEM equipment is available in a limited number of facilities.
Flow cytometry
While most flow cytometers do not have sufficient sensitivity, there are a few commercially available instruments that can be used for virus quantification. For example, the Virus CounterVirus Counter
The Virus Counter is an instrument for rapid quantification of viruses in liquid samples. It is a specialized flow cytometer that uses high-sensitivity fluorescence detection to give a direct measurement of the concentration of virus particles in a fraction of the time required for traditional...
is a benchtop flow cytometer designed specifically for virus quantification by InDevR
InDevR
InDevR is a biotechnology company that develops advanced life science instrumentation and assays for analysis of viruses and other microorganisms, with product focus on Virus Quantification and pathogen detection / identification. InDevR Inc. is a privately held, woman-owned small business located...
Inc. This system is significantly more sensitive than other commercial cytometers, which enables efficient detection and accurate quantification of viruses while maintaining the speed and versatility advantages of flow cytometry. The Virus Counter assay and instrument quantify the number of intact virus particles in a sample using fluorescence to detect colocalized proteins and nucleic acids. Samples are stained with two dyes, one specific for proteins and one specific for nucleic acids, and analyzed as they flow through a laser beam. The quantity of particles producing simultaneous events on each of the two distinct fluorescence channels is determined, along with the measured sample flow rate, to calculate a concentration of virus particles (vp/mL). The results are generally similar in absolute quantity to a TEM result. The assay has a linear working range of 105-109 vp/mL and an analysis time of ~10 min with a short sample preparation time.
Quantitative Polymerase Chain Reaction (qPCR)
Quantitative PCR utilizes polymerase chain reactionPolymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
chemistry to amplify viral DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
or RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
to produce high enough concentrations for detection and quantification by fluorescence. In general, quantification by qPCR relies on serial dilutions of standards of known concentration being analyzed in parallel with the unknown samples for calibration and reference. Quantitative detection can be achieved using a wide variety of fluorescence detection strategies, including sequence specific probes or universal probes such as SYBR Green
SYBR Green
SYBR Green I is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to DNA. The resulting DNA-dye-complex absorbs blue light and emits green light . The stain preferentially binds to double-stranded DNA, but will stain single-stranded DNA with lower...
dye. Sequence specific probes, such as TaqMan
TaqMan
TaqMan probes are hydrolysis probes that are designed to increase the specificity of real-time PCR assays. The method was first reported in 1991 by researchers at Cetus Corporation, and the technology was subsequently developed by Roche Molecular Diagnostics for diagnostic assays and by Applied...
(i.e. Applied Biosystems), Molecular Beacons, or Scorpion, bind only to the copied, or cDNA of the appropriate sequence produced during the reaction. SYBR Green dye binds to all double-stranded DNA produced during the reaction. While SYBR Green is easy to use, its lack of specificity and lower sensitivity lead most labs to use probe-based qPCR detection schemes. There are many variations of qPCR including the comparative threshold method, which allows relative quantification through comparison of Ct values (PCR cycles that show statistically significant increases in the product) from multiple samples that include an internal standard. Since PCR amplifies all target nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
, whether from an intact virion or free nucleic acids in solution, qPCR results (expressed in terms of genome copies/mL) are likely to be higher in quantity than TEM results. For viral quantification, the ratio of whole virions to copies of nucleic acid is seldom one to one due to the fact that during viral replication, viral assembly in the cytoplasm requires the production of proteins (structural and non-structural) and nucleic acid which are the precursors needed to assemble a whole virion. In the example of foot-and-mouth disease virus, the ratio of whole virions to RNA copies within an actively replicating host cell is approximately 1:1000. Commercially available products for qPCR are available through numerous companies such as Invitrogen, Roche and Qiagen just to name a few. Real-time qPCR takes around 1–4 hours and can provide quantitative results containing too few viruses to be analyzed by other methods.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISAELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
is a more modern variation of a protein assay that utilizes a specific antibody linked to an enzyme to detect the presence of an unknown amount of antigen (i.e. virus) in a sample. The antibody-antigen binding event is detected and/or quantified through the enzyme’s ability to convert a reagent to a detectable signal that can be used to calculate the concentration of the antigen in the sample. Horseradish peroxidase
Horseradish peroxidase
The enzyme horseradish peroxidase , found in horseradish, is used extensively in biochemistry applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule.-Applications:...
(HRP) is a common enzyme utilized in ELISA schemes due to its ability to amplify signal and increase assay sensitivity. There are many variations, or types of ELISA assays but they can generally be classified as either indirect, competitive, sandwich or reverse. ELISA kits are commercially available from numerous companies and quantification generally occurs via chromogenic
Chromogenic
Chromogenic refers to color photographic processes in which a traditional silver image is first formed, and then later replaced with a colored dye image.- Description :...
reporters or fluorescence (e.g. Invitrogen, Santa Cruz Biotechnology Inc.). This technique is much less labor intensive than the traditional methods and can take anywhere from 4 to 24 hours based on antibody incubation time.