2.3 DETECTION, IDENTIFICATION, AND CLONING OF VIRUS GENOMES USING PCR AND RT-PCR

 

As discussed above, all techniques have their advantages and limitations. For example, serological methods of virus detection are effective and quick but tell us nothing about the virus genome. Neutralization tests are simple, but are confined to viruses that can be cultivated, and are slow to give a result. This depends on the time a virus takes to kill a detectable number of cells, and this can range from several days to several weeks. Such a situation is far from ideal, and the problem was solved by the discovery of a technique which makes many, many copies of a chosen part of the virus genome. This is the polymerase chain reaction (PCR) which synthesizes DNA from a DNA template, and was devised in 1985 by Kari Mullis. If the virus of interest has an RNA genome, the region of interest has first to be converted into DNA using a primer (see below) and the retrovirus enzyme, reverse transcriptase (Section 8.3). If a unique sequence is chosen and there is a positive result, the virus present is immediately identified. The system is highly sensitive and can detect one copy of a DNA genome or around 1000 copies of an RNA genome. Thus it is a detection and identification system all in one. Normally a region of 100 bp (basepairs) or so is amplified, but with care whole genomes up to 15,000 bp can be copied. PCR has the added advantage of detecting virus in primary tissue, so that mutations associated with adaptation to cell culture are avoided. It is no more expensive than a neutralization assay. The only prerequisite for PCR is knowing the sequence of the regions flanking the portion of the genome to be detected, so that oligonucleotide primers that are complementary to a sequence on each strand of DNA can be made. PCR requires two primers, each of around 20–30 nucleotides in length, and these are chemically synthesized. The DNA is denatured by heating to around 90°C and the primers added in high molar excess together with deoxyribonucleotide triphosphates (dNTPs

cooling, the primers anneal to their respective template strands and the template is copied by the enzyme. It is convenient to use a polymerase that is not inactivated at high temperatures, such as the Taq polymerase from Thermophilus aquaticus, a bacterium that lives in natural hot springs; otherwise fresh polymerase would have to be added after each denaturation step. The mix is again denatured and cooled so that further primers can anneal, and the next round of DNA synthesis take place. The defined PCR product is now present, but around 30 rounds of synthesis are required before there is sufficient product to be analyzed. To determine if the PCR result is positive, DNA is electrophoresed to determine if an amplified fragment (amplicon) of the expected size has been synthesized. Size is measured by comparison with DNA size markers electrophoresed in a parallel track. Confirmation of the result can be obtained by isolating the DNA fragment from the gel and sequencing it. This technique rapidly gained acceptance and has very many applications. It is as widely used for diagnostic clinical virology as for research purposes. For diagnostic purposes PCR may be carried out in two phases. In the first, primers are chosen that amplify regions of the genome that are common to a whole group of viruses known to occur, say in the gut. On finding a positive, primers to a region that is unique to each virus type can then be used for exact identification. PCR can amplify complete genes or even small genomes, and these can be cloned into an appropriate vector, which can express more genomes or a protein product when transfected into animal cells in culture. In this way vaccines can be made from noncultivable viruses. Cloned DNA that expresses a virus protein can also be injected directly into animals as an experimental vaccine – this is called a DNA vaccine.

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