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Why not use SDS-PAGE as a method to detect viruses?

Why not use SDS-PAGE as a method to detect viruses?


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Recently, I have been researching about DNA and I know the most popular method for detecting viruses is based on DNA. After learning about proteins, I wonder why we do not detect viruses based on virus proteins?

If it becomes true we can save a lot of money, instead of doing a PCR reaction, we can detect more than one disease in a single reaction.

For example, when I have the sequence of a virus from NCBI Genebank, I can know how many proteins the target organism produces and I can calculate the molecular weight of each protein. Then running it in a SDS-PAGE gel to see what the bands look like, may be that I can know the target organism is present or not?

For example, HIV produces 9 proteins and the molecular weight of each protein is different. So after doing electrophoresis, I have 9 bands. Based on the distance between each band and based on the molecular weight, it's possible to know HIV is present or not?


Put simply, the answer is that you could seek to detect viral proteins, but because these proteins would be very minor components of your sample, you would have to use an immunoblotting technique to detect a specific viral protein. While immunoblotting is quite a sensitive technique, I think its fair to say that it can be technically demanding. In contrast a PCR reaction is exquisitely sensitive and is fairly robust. PCR is also technically more staightfoward because DNA is generally easier to work with than are proteins.

Before the advent of PCR, immunoblotting would have been the method of choice.


Molecular Methods for Virus Detection

Molecular diagnostic procedures have been described in a number of recent books and articles. However, these publications have not focused on virus detection, nor have they provided practical protocols for the newer molecular methods.

Written by the inventors or principal developers of these technologies, Molecular Methods for Virus Detection provides both reviews of individual methods and instructions for detecting virus nucleic acid sequences in clinical specimens. Each procedure includes quality assurance protocols that are often ignored by other methodology books. Molecular Methods for Virus Detection provides clinically relevant procedures for many of the newer diagnostic methodologies.

Molecular diagnostic procedures have been described in a number of recent books and articles. However, these publications have not focused on virus detection, nor have they provided practical protocols for the newer molecular methods.

Written by the inventors or principal developers of these technologies, Molecular Methods for Virus Detection provides both reviews of individual methods and instructions for detecting virus nucleic acid sequences in clinical specimens. Each procedure includes quality assurance protocols that are often ignored by other methodology books. Molecular Methods for Virus Detection provides clinically relevant procedures for many of the newer diagnostic methodologies.


5.1. Laboratory quality assurance

In order to build laboratory capacity, national HIV/AIDS programmes should invest in a QA programme for all laboratories performing diagnostic testing, and should use existing available services provided by WHO and others including the Centers for Disease Control and Prevention (CDC) to support external quality assessment schemes (EQAS).

Having highly accurate tests does not necessarily guarantee reliable laboratory results. Many processes are involved from the time the specimen is collected and transported to the laboratory, tested and until the results are reported, during which errors can occur. Therefore, ongoing QA within the context of the laboratory quality system, both internally and externally, is essential. Clinicians and staff providing laboratory services need regular communication about the performance of tests to improve and ensure appropriate performance. Well-defined standard operating procedures (SOPs), following nationally defined and validated testing algorithms, are essential for optimal use of all laboratory-based testing.


The stacking gel is of no use to the analysis and it can be removed. Top of the gel refers to the top of the separating gel, that is, the point at which different polypeptides began to separate. A mix of protein standards usually consists of five to eight individual polypeptides that produce a prominent "ladder." Standards are identified from the top down. Depending on %T one or more of the lowest mass standards may not resolve.

To prepare a standard curve for molecular mass one estimates a relative mobility for each standard and plots a standard curve of molecular mass versus relative mobility on semi-log paper or log molecular mass versus relative mobiltiy on conventional graph paper. Relative mobility is determined by measuring the distance from the top of the gel to the middle of the dye front or arbitrary reference point, measuring the distance from the top of the gel to the middle of the band, and dividing the second measurement by the first. This is the Rf, which is always between 0 and 1.

Note that the relative mobility of a given protein depends on gel concentration. Any single gel has an upper and lower limit to its useful range for estimating molecular mass.


3. Cell Culture (Tissue Culture)

There are three types of tissue culture organ culture, explant culture and cell culture.

Organ cultures are mainly done for highly specialized parasites of certain organs e.g. tracheal ring culture is done for isolation of coronavirus.

Explant culture is rarely done.

Cell culture is mostly used for identification and cultivation of viruses.

  • Cell culture is the process by which cells are grown under controlled conditions.
  • Cells are grown in vitro on glass or a treated plastic surface in a suitable growth medium.
  • At first growth medium, usually balanced salt solution containing 13 amino acids, sugar, proteins, salts, calf serum, buffer, antibiotics and phenol red are taken and the host tissue or cell is inoculated.
  • On incubation the cell divide and spread out on the glass surface to form a confluent monolayer.

Methods That Detect Whole Phage Particles

Transmission electron microscopy (TEM) uses a beam of electrons to produce 1000x higher resolution compared to traditional light microscopes. Increased resolution (down to 0.2 nm) is enough to visualize even viruses. This technology can be used to quantify viral particles, albeit the sample needs to be highly concentrated (� 6 particles/mL) to produce reliable results (Mann, 2005 Goldsmith and Miller, 2009). Viral quantification using TEM is highly accurate in determining the morphotype and the total number, but it is considered time-consuming, expensive and impractical for running many samples and cannot be used for complex samples. Moreover, sample preparation is tedious and the technique requires a skilled operator on top of the sophisticated instrument (Ackermann, 2012).

Another technique to count whole particles is flow cytometry. Herein, viral particles are marked with a fluorescent dye and directed through a capillary. Small diameter of the capillary forces particles to flow in a single line, allowing detection of light scatter caused by each particle. The method is rapid and rigorous, thus employed widely (Picot et al., 2012). A seminal study showed that the fluorescent signal does not correlate with the genome size, but that different viruses in a mixed sample could be discriminated based on their fluorescence and side scatter distribution (Brussaard et al., 2000). Recently, it was shown that the fluorescent signal can be used to correlate the number of target nucleic acid molecules to the number of viral particles only if samples are handled gently, the use of surfactants is avoided, negative controls are included to determine auto-fluorescence of the medium and the instrument and assay sensitivity is estimated beforehand, using a panel of bacteriophages of various genome sizes (Dlusskaya et al., 2019).

Finally, a laser-based method developed by NanoSight Limited allows real time visualization and to enumerate viral particles in few minutes based on dynamic light scattering by laser-illuminated optical microscopy. Drawbacks are the need for relative high sample concentration (10 7 � 9 PFU/ml) and a clear liquid (Anderson et al., 2011) that is difficult to obtain from complex samples such as soil and fecal material.


Blotting

Following the separation of the protein mix the polypeptide bands are transferred to a membrane carrier. For this purpose the membrane is attached to the gel and this so-called sandwich is transferred to an electrophoresis chamber. It is possible that some of the SDS is washed out, and the protein partially re-naturates again, i.e. regains its 2D- and 3D structure. However, the applied electric charge causes the proteins to travel out of the gel vertically to the direction they traveled in on the gel, onto the membrane. The protein bands are thereby bound to the membrane. The "blotted" bands are now available to be treated further (e.g. for detection of specific proteins with specific antibodies).


Quantitation of Nucleic Acids

Estimating DNA Concentration on an Ethidium Bromide-Stained Gel

Agarose gel electrophoresis is commonly used to separate DNA fragments following restriction endonuclease digestion or PCR amplification. Fragments are detected by staining the gel with the intercalating dye, ethidium bromide, followed by visualization/photography under ultraviolet light. Ethidium bromide stains DNA in a concentration-dependent manner such that the more DNA present in a band on the gel, the more intensely it will stain. This relationship makes it possible to estimate the quantity of DNA present in a band through comparison with another band of known DNA amount. If the intensities of two bands are similar, then they contain similar amounts of DNA. Ethidium bromide stains single-stranded DNA and RNA only very poorly. These forms of nucleic acid will not give reliable quantitation by gel electrophoresis.


Sample denaturation

Various sample buffers have been used for SDS-PAGE but all use the same principles to denature samples. We obtain good denaturation by preparing a sample to a final concentration of 2 mg/ml protein with 1% SDS, 10% glycerol, 10 mM Tris-Cl, pH 6.8, 1 mM ethylene diamine tetraacetic acid (EDTA), a reducing agent such as dithiothreitol (DTT) or 2-mercaptoethanol, and a pinch of bromophenol blue to serve as a tracking dye (

We prepare a 2x concentrate of sample buffer consisting of 2% SDS, 20% glycerol, 20 mM Tris-Cl, pH 6.8, 2 mM ethylene diamine tetraacetic acid (EDTA), 160 mM dithiothreitol (DTT), and 0.1 mg/ml bromphenol blue dye. I prefer DTT to 2-mercaptoethanol because the latter has a much stronger unpleasant odor and it doesn't denature our blood fractions very well. Part of the problem is that our water baths don't reach the boiling point, and boiling may be necessary with 2-mercaptoethanol. We prepare all of our unknowns to the same concentration then mix 1 volume prepared sample to 1 volume 2x buffer.

So, what do the various components do? EDTA is a preservative that chelates divalent cations, which reduces the activity of proteolytic enzymes that require calcium and magnesium ions as cofactors. The tris acts as a buffer, which is very important since the stacking process in discontinuous electrophoresis requires a specific pH. Glycerol makes the sample more dense than the sample buffer, so the sample will remain in the bottom of a well rather than float out. The dye allows the investigator to track the progress of the electrophoresis.

SDS, DTT, and heat are responsible for the actual denaturation of the sample. SDS breaks up the two- and three-dimensional structure of the proteins by adding negative charge to the amino acids. Since like charges repel, the proteins are more-or-less straightened out, immediately rendering them functionless. Some quaternary structure may remain due to disulfide bonding (covalent) and due to covalent and noncovalent linkages to other types molecules. By the way, another name for SDS is lauryl sulfate. Your shampoo may contain lauryl sulfate - now doesn't that inspire confidence in the product?

Many proteins have significant hydrophobic properties and may be tighly associated with other molecules, such as lipids, through hydrophobic interaction. Heating the samples to at least 60 degrees C shakes up the molecules, allowing SDS to bind in the hydrophobic regions and complete the denaturation.

The amino acid cysteine contains a sulfhydryl (-SH) group that spontaneously forms a disulfide bond (-S-S-) with another sulfhydryl group under normal intracellular conditions. Disulfide bonding is covalent and is not disrupted by SDS. DTT is a strong reducing agent. Its specific role in sample denaturation is to remove the last bit of tertiary and quaternary structure by reducing disulfide bonds.

Most sample buffers do not remove covalently attached carbohydrate or phosphate groups, and some associations with other types macromolecules are difficult to disrupt. Polypeptides contain varying amounts of basic and acidic amino acids that add charge to the molecules, and individual amino acids vary in molecular weight although they may bind SDS with the same affinity. Therefore, charge to mass ratio and the relative mobility of many proteins is affected by factors other than strictly the molecular weight. SDS-PAGE is very effective in providing reproducible results, but don't count on precise values for MW determination.


High sensitivity detection of coronavirus SARS-CoV-2 using multiplex PCR and a multiplex-PCR-based metagenomic method

Many detection methods have been used or reported for the diagnosis and/or surveillance of SARS-CoV-2. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive, claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by RT-PCR, probably due to loss or degradation of virus RNA in the sampling process, or even mutation of the virus genome. Therefore, developing highly sensitive methods is imperative to ensure robust detection capabilities. With the goal of improving sensitivity and accommodate various application settings, we developed a multiplex-PCR-based method comprised of 172 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. In addition, optional sequencing can provide further confirmation as well as phylogenetic information of the identified virus(es) for specific strain discrimination, which will be of paramount importance for surveillance purposes that represent a global health imperative. Finally, we also developed in parallel a multiplex-PCR-based metagenomic method that is amenable to detect SARS-CoV-2, with the additional benefit of its potential for uncovering mutational diversity and novel pathogens at low sequencing depth.