Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 99

The detection of viruses in cell cultures is being increasingly supplanted by molecular methods such as the polymerase chain reaction and high-throughput sequencing, especially for discovery of new viruses associated with human diseases. These methods can be used to identify viruses that cannot be propagated in cell culture, offering new ways to fulfill Koch’s postulates (Box 1.4).

Polymerase chain reaction. In this technique, specific oligonucleotides are used to amplify viral DNA sequences from infected cells or clinical specimens. Amplification is done in cycles, using a thermostable DNA polymerase (Fig. 2.16). Each cycle consists of thermal denaturation, primer annealing, and extension, carried out by automated cycler machines. The result is exponential amplification (a 2n-fold increase after n cycles of amplification) of the target sequence that is located between the two DNA primers.

Figure 2.15 Using fluorescent proteins to study virus particles and virus-infected cells. (A) Submandibular ganglia after infection of the salivary gland with three recombinant pseudorabies viruses, each expressing a different color fluorescent protein. Courtesy of Lynn Enquist, Princeton University. (B) Single-virus-particle imaging with green fluorescent protein illustrates microtubule-dependent movement of human immunodeficiency virus type 1 particles in cells. The cells were infected with virus particles that contain a fusion of green fluorescent protein with a viral protein. Rhodamine-tubulin was injected into cells to label microtubules (red). Virus particles can be seen as green dots (white arrow). Bar, 5 μm. Courtesy of David McDonald, University of Illinois.

Figure 2.16 Polymerase chain reaction. The DNA to be amplified is mixed with nucleotides, thermostable DNA polymerase, and a large excess of DNA primers. DNA polymerase initiates synthesis at the primers bound to both strands of denatured DNA, which are then copied. The product DNA strands are then separated by heating. Primer annealing, DNA synthesis steps, and DNA duplex denaturation steps are repeated multiple times, leading to geometric amplification of a specific DNA.

Clinical laboratories employ PCR assays to detect evidence for infection by a single type of virus (singleplex PCR), while screening for the presence of hundreds of different viruses can be accomplished with multiplex PCR. In contrast to conventional PCR, real-time PCR can be used to quantitate the amount of DNA or RNA in a sample. In this procedure, also called quantitative PCR, the amplified DNA is detected as the reaction progresses, not after it is completed as in conventional PCR. The product is detected either by incorporation of a dsDNA specific dye or by release of a fluorescence resonance energy transfer probe via the 5′-to-3′ exonuclease activity of DNA polymerase. The number of cycles needed to detect fluorescence above background can then be compared between standard and experimental samples. Quantitative PCR is widely used in research and clinical applications for genotyping, gene expression analysis, copy number variation assays, and pathogen detection. While PCR is often used to detect viral genomes in clinical specimens or during experimental research, it is important to recognize that the nucleic acid detected does not necessarily correspond to infectious virus (Box 2.8).