NUCLEIC ACID TECHNIQUES
The goal of the clinical microbiology laboratory is to provide the clinician with evidence of the presence or absence of an infection agent that may be causing a particular illness. Traditionally, this diagnosis relies upon methods that detect the pathogens, either directly through culture or antigen detection or indirectly through serological methods that would enable the detection of antibodies made in response to the pathogen. The indirect methods could also involve the detection of metabolic by-products of the agents, such as in the detection of toxins.
For some infectious agents reliable culture or serologic methods are not available or these methods may require long periods of turnaround time. The introduction of nucleic acid hybridization and amplification techniques has allowed the clinical laboratory to decrease time to detection in many situations and thereby enhance the laboratory’s role in the diagnosis of these infectious diseases.
Nucleic acid hybridization:
It is the key to the molecular techniques. This process provides for the formation of stable double-stranded nucleic acid molecules form complementary single-stranded molecules. The single-stranded molecules can be RNA or DNA and the resultant hybrids formed can be DNA-DNA, RNA-RNA or DNA-RNA. A probe is a labeled single-stranded sequence of nucleic acid that is complementary to the nucleic acid sequence to be detected and can be either DNA or RNA. The targeted nucleic acid sequence is referred to as the target, and it too can be DNA or RNA. This target can be located within the specimen, or in a colony, either from an agar plate or broth culture.
The nucleic acid probe is traditionally constructed from specific nucleic acid fragments (sequence) of the organism (target), or the probe may be a synthetically produced oligonucleotide of specific sequences. In a probe assay, the target and probe must be allowed to come together and hybridize, and the conditions whereby this may occur are referred to as the hybridization format.
- Solid hybridization: Solid support is a conventional approach in which the target nucleic acid is secured to a membrane (nitrocellulose or nylon fiber filter paper). The probe is then overlaid to target, if present, and after a series of washes, detected as a dot, spot or blot, depending upon the manner in which the nucleic acid is added to the membrane.
- Sandwich hybridization: It is a type of solid support that utilizes two probes, one labeled and attached to the solid support and the other labeled and added after the target, if present, has been allowed to hybridize to the first probe.
- In-solution hybridization: It involves movement of both target and probe in a liquid environment, providing for kinetic of interaction that may 5-10 times faster than solid support hybridization.
- In-situ hybridization: It occurs in formalin-fixed or paraffin-embedded tissue, which may contain the suspected target. A positive reaction provides a localization of the target nucleic acid within the cellular and subcellular detail of the tissue and for probing certain viruses.
Probe labels: To detect the hybridization reaction that may have occurred between probe and target, a label or marker is needed.
Labels: Radioactive: 32P, 125I, 35S, Biotin-avidin, Enzyme: Alkaline phosphatase
Chemiluminescence, Fluorescent, Antibody
Probe target: Target material for probe technology can be DNA or RNA. DNA is commonly utilized as a target with DNA or RNA as a probe.
Application of probe technology:
- Genetic defect detection
- Food microbiology
- Plant pathology
- Direct detection of microbe in clinical samples
- Organisms not able to be cultivated
- Organisms with long incubation times
- Diseases with unknown etiologies
- Identification of cultural isolates
- Strain identification
- Identification of toxins, virulence factors
- Identification of resistance markers
- Identification of microbes in environmental specimens
- Identification of resistance factors
Amplification methods are recent methodologies for detection of pathogens and for their products in infectious material. They are highly sensitive. Amplification is a molecular procedure that increases the number of nucleic acid copies in a specimen to millions in a very short period of time. Its success includes identification of organism for which culture methods are long and tedious, for which there are no culture methods available, or for diseases in which the quick diagnosis is of utmost importance.
Polymerase chain reaction:
The first method of amplification is polymerase chain reaction described by Mullis (1990). It involves a repeating three-step process in which DNA target material is denatured; annealing primers (oligonucleotides) are applied to the single-stranded DNA and then enzymatically extended to synthesize complementary strands of the DNA. A typical reaction involves a total of 25 to 40 cycles. Each cycle is a sequential series of three different temperatures settings. The methodology is similar to the in-vivo mode of DNA replication.
Nucleic acid sequence information is available for most organisms of clinical importance. Careful evaluation of this information determines the target for amplification. Microorganisms typically contain unique regions in their genetic material that is characteristic of particular strain. Primers designed to recognize these unique region permit highly specific identification of a single strain or species.
A proper selection of a pair of oligonucleotide primers is essential to the ultimate success of the amplification. Primers are short pieces of single-stranded DNA. They flank the two sides of the target sequence and serve to define the ends of the DNA that has been targeted for amplification.
DNA polymerase is the enzyme that catalyzes the formation of a new strand of dsDNA identical to content to the original DNA template. The Taq polymerase is the enzyme most often utilized. It is heat stable and retains full activity after repeat denaturation steps.
Detection of amplicons:
A variety of formats are available for the detection of the product amplicon. These include for amplification: an EIA type colored format or Chemiluminescence. Radioactivity is often used for detection of PCR amplicons in research.
|1.||Denaturation||940C||Double-stranded DNA broken into single-strands|
|2.||Primer annealing||550C||Attachments of oligonucleotide primers to complementary regions on ssDNA|
|3.||Extension||720C||Synthesis of dsDNA catalyzed by Taq DNA polymerase.|
Real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR), which is used to amplify and simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, quantitative PCR enables both detection and quantification. In real-time PCR, the amount of DNA is measured after each cycle via fluorescent dyes that yield increasing fluorescent signal in direct proportion to the number of PCR product molecules (amplicons) generated.
Real- time PCR is considered to be the most powerful, sensitive, and quantitative assay for the detection of RNA levels. It is frequently used in the expression analysis of single or multiple genes, and expression patterns for identifying infections and diseases.
The advantages of real-time PCR include:
Ability to monitor the progress of the PCR reaction as it occurs in real time
Ability to precisely measure the amount of amplicon at each cycle, which allows highly accurate quantification of the amount of starting material in samples
An increased dynamic range of detection
Amplification and detection occurs in a single tube, eliminating post-PCR manipulations.