The following simplified algorithm outlines the overall working of NVRL VIROLOGY TECHNIQUES AND TERMINOLOGY A) Nucleic acid amplification Most commonly, PCR (polymerase chain reaction) is used for both DNA and RNA viruses. For RNA viruses, an initial reverse transcription step which converts RNA to DNA is used. PCR is highly sensitive and relatively rapid. Other variations of nucleic acid amplification are used such as LCR (ligase chain reaction) for Chlamydia trachomatis, NABSA (nucleic acid sequence based amplification) HIV viral load etc. HIV viral load allows for monitoring disease progression and response to treatment. Real-Time PCR Real-Time PCR analysis detects specific nucleic acid amplification products as they accumulate in real-time by detecting and quantifying a fluorescent reporter, which increases in direct proportion to the amount of PCR product in a reaction. Thus the more template molecules present at the beginning of the reaction, the fewer number of cycles it takes to reach a point in which the fluorescent signal is first recorded as statistically significant above background values. This point is termed the cycle threshold (Ct). There are several methods available allowing the quantification of amplicon in a PCR reaction: 1. DNA binding flourophores The simplest and most economical way of detecting amplicon is to use double-strand DNA-specific dyes such as SYBR® Green, which bind to double-stranded DNA, and upon excitation emits light. SYBR Green is inexpensive, easy to use, and sensitive. The disadvantage is that SYBR Green will bind to any double-stranded DNA in the reaction, including primer-dimers and other non-specific reaction products, which may result in an overestimation of the target concentration. The two most popular alternatives to SYBR Green are dual labelled probes and molecular beacons, both of which are hybridization probes relying on fluorescence resonance energy transfer (FRET) mechanisms to detect target amplification. 2. Duel Labelled probes Dual labelled probes (also known as Taqman™ probes) are oligonucleotides that contain a fluorescent dye on the 5' base, and a quenching dye located on the 3' base. When excited the flourescent dye transfers energy to the nearby quenching dye molecule rather than fluorescing, resulting in a nonfluorescent probe. Dual labelled probes are designed to hybridize to an internal region of a PCR product. During PCR, when the polymerase replicates a template on which the probe is bound, the 5' exonuclease activity of the polymerase cleaves the probe. This separates the fluorescent and quenching dyes and FRET no longer occurs, allowing detection of the signal from the reporter dye. Fluorescence increases in each cycle, proportional to the rate of probe cleavage. Figure 1: Real time PCR using dual labelled probes 3. Molecular beacons Molecular beacons also contain fluorescent and quenching dyes and are designed to adopt a hairpin structure while free in solution, bringing the fluorescent dye and quencher into close proximity. When a molecular beacon hybridizes to a target, the fluorescent dye and quencher are separated, FRET does not occur, and the fluorescent dye emits light upon irradiation. Unlike dual labelled probes, molecular beacons are designed to remain intact during the amplification reaction. Figure 2: Real time PCR using molecular beacons. 4. Self fluorescing amplicons There are now numerous methods available which use fluorescent labelled primers, which release flourescence once incorporated into the amplicon. Examples include scorpion primers™, sunrise primers™ and lux primers™. These systems claim to be as effective as probe based real time assays but are less costly as a probe is no longer required. Multiplex real time PCR Dual labelled probes and molecular beacons can be used to detect multiple DNA targets within the same sample (multiplex PCR), since fluorescent dyes with different emission spectra may be attached to different probes. These hybridization probes afford a level of discrimination impossible to obtain with SYBR Green, since they will only hybridize to true targets in a PCR and not to primer-dimers or other spurious products. Real time PCR Machines Real-time PCR requires an instrumentation platform that consists of a thermal cycler, computer, optics for fluorescence excitation and data acquisition and analysis software. These machines, available from several manufacturers, differ in sample capacity (some are 96-well standard format, others process fewer samples or require specialized glass capillary tubes), method of excitation (some use lasers, others broad spectrum light sources with tunable filters), and overall sensitivity. There are also platform-specific differences in how the software processes data. For a presentation giving an overview of real time PCR please click here To see an animation of how PCR works please click here (728k, PC only) B) Nucleic acid sequencing DNA Sequencing Nucleotide sequencing is a powerful technique, allowing the determination of the precise order of the nucleotides in a DNA (or RNA molecule following reverse transcription). The sequence can then be compared with a library of known sequences for epidemiological and clinical purposes (e.g. typing, investigating transmission routes, identifying mutations associated with antiviral resistance). Methodology The DNA to be sequenced is added to a mixture containing dNTPs (deoxynucleotides), a primer complementary to the beginning of the sequence to be investigated, a DNA polymerase and a limited amount of four dideoxynucleotides, each labelled with a "tag" that fluoresces with a different colour (ddATP, ddGTP, ddCTP, ddTTP) upon laser excitation (figure 1). Chain elongation proceeds from the primer until, by chance, the DNA polymerase inserts a dideoxynucleotide (shown as coloured letters) instead of the normal deoxynucleotide (shown as vertical lines). At this point chain elongation stops because there is no 3' OH on the dideoxynucleotide for the next nucleotide to attach to. If the ratio of normal nucleotide to the dideoxy versions is high enough, some DNA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process. At the end of the reaction, the fragments produced are separated by length from longest to shortest using gel electrophoresis. The resolution is so good that a difference of one nucleotide is enough to differentiate one strand from the next. Figure 1: The process of DNA nucleotide sequencing. Each fragment is then analysed by an automated laser scanner. Under excitation each of the four dideoxynucleotides will fluoresce a different colour, allowing the computer to determine the nucleotide composition of the chosen sequence (figure 2). Figure 2: A electropherogram showing the nucleotide composition of a particular sequence as determined by flourescence. To see an animation on how nucleic acid sequencing works please click here (1MB, PC only) C) Serology. Two main methods available for antibody detection in blood 1. ELISA (enzyme-linked immunosorbent assay) : ELISA detects antibodies (IgM, IgG and IgA). IgM is detectable immediately after the onset of illness. IgG antibodies, which appear later, indicate past infection and immunity in most instances. ELISA can be adapted to detect antigens (e.g. HBsAg). 2. CFT (complement fixation test) : Antigen is added to patient’s serum; if specific antibody is present, then a complex forms which binds complement, so complement is no longer free to lyse indicator red blood cells, added to well. The result is given as a titre which indicates the amount of antibody present. A rise in titre between an acute and convalescent serum (10-14 days after onset of illness) is required to make a diagnosis. D) Direct antigen detection by immunofluorescence Certain specimens with lots of cells such as nasopharyngeal aspirates (NPA) and bronchoalveolar lavages (BAL) can be stained directly with monoclonal antibodies prepared against specific pathogens such as influenza virus, respiratory syncytial virus (RSV), adenovirus and Pneumocystis carinii. A good quality specimen is needed! E) Electron microscopy (EM) This requires a specimen where high concentration of virus in samples can be expected, such as diarrhoeaic stool and vesicle fluids. EM has become a reference technique. F) Virus isolation Samples are inoculated onto several different types of cells (e.g. derived from kidney or lung) selected for the types of viruses expected in the clinical sample. The cultures are inspected for evidence of cytopathic effect (effect of the virus on the cell). This technique depends on the presence of live virus. So the use of correct viral transport medium, the time the sample takes to reach the lab, and correct storage of samples prior to transfer are all important factors. Not all viruses can be grown in cell cultures. Monolayer of normal uninfected cells Monolayer of cells showing viral cytopathic effect