Nucleic Acid Detection Assays

Chapter 5

Nucleic Acid Detection Assays


Assays that detect nucleic acid, also known as molecular diagnostic assays, include probe-based assays such as in situ hybridization, and methods that amplify DNA, such as the polymerase chain reaction (PCR).

DNA or RNA probes are short, single-stranded nucleic acids (usually <50 base pairs) that have a sequence that hybridizes to (i.e., is complementary to) a specific segment of DNA from the target microorganism (Figure 5-1). For in situ hybridization, the probe is labeled with a fluorescent or chemical tag so that the bound probe can be detected. The probe is reacted with a tissue specimen on a microscope slide in order to determine if the organism is present in the specimen. For PCR, two specific short (approximately 20 bases) DNA sequences called primers, which are complementary to the target microorganism’s DNA, are used in conjunction with a machine called a thermocycler to amplify a specific segment of DNA from just one copy to millions of copies that can be more easily detected. In situ PCR is a combination of these two techniques.1

Nucleic acid detection methods are ideally suited to detection of organisms that are not easily found using cytology or histopathology, are slow-growing, or are difficult to culture, as well as when a rapid (<12 hours) diagnosis is required. Other applications are shown in Box 5-1.

The use of nucleic acid–based assays for diagnosis has increased dramatically in veterinary medicine over the past 10 years. Because of the exquisite sensitivity of some assays, especially those that involve DNA amplification, positive results may reflect contamination that occurs in the laboratory. Contamination problems have decreased with increased automation and the use of real-time PCR assays (see later). In human medicine, proficiency testing programs have helped to overcome quality assurance problems in laboratories that perform molecular diagnostic assays.2 Not all PCR assays for a specific pathogen are equal, because the nucleic acid primers and probes and the equipment used to run each assay may be different. So a PCR assay for Leptospira performed in one laboratory may perform differently than an assay used in another laboratory.

A plethora of nucleic acid detection methods have been developed since the advent of PCR, many of which are commercially available for diagnosis of human infectious diseases but are not yet being widely used in veterinary medicine. These are described in detail elsewhere.1,3,4 The purpose of this chapter is to outline (1) the indications for nucleic acid detection methods, (2) the best ways to collect and transport specimens for these assays, (3) the most commonly used methods in veterinary medicine, and (4) guidelines for their interpretation.

Specimen Collection and Transport

The recommendations for specimen collection and transport presented here are based on the guidelines published by the Clinical and Laboratory Standards Institute (CLSI).5 These guidelines are updated on a regular basis and ensure that detection of nucleic acids within clinical specimens is optimal. In some instances, nucleic acid may be present and detectable in a specimen even when the specimen has not been handled in a manner that is consistent with the guidelines. Many laboratories publish their own specific guidelines for specimen collection and transport. Specimens should be properly packaged and labeled (see Chapter 3).

Nucleic Acid Probe Assays

In situ hybridization is generally performed on formalin-fixed, paraffin-embedded tissue specimens. Specimens that have been archived for several years may still be adequate, even for detection of RNA, which is more labile than DNA.6,7 The optimum specimen for fluorescence in situ hybridization (see later for discussion) is tissue that has been snap frozen in liquid nitrogen after being embedded in optimal cutting temperature (OCT) medium.5

Polymerase Chain Reaction

When collecting specimens for PCR, the timing of collection relative to the course of disease and the best specimen type must be considered. For acute diseases, PCR is often most useful early in the course of disease. For chronic diseases, timing is less critical. Knowledge of organism shedding patterns can help to determine the most appropriate timing of specimen collection. For example, Leptospira organisms are primarily present in blood in the first week of acute illness, after which they may be shed in the urine.8

Because contamination can occur outside the PCR laboratory as well as within the laboratory, collection of specimens for nucleic acid testing should be performed aseptically. Gloves should be worn, and disposable instruments should be used (e.g., disposable blades, punch biopsy instruments).

In general, for detection of pathogens that contain DNA (i.e., all bacteria, fungi, some viruses), either fresh or frozen specimens should be submitted. In general, DNA is stable in tissue for up to 24 hours at 2°C to 8°C, at least 2 weeks at −20°C, and at least 2 years at −20°C or below −70°C.5 Specimens held at room temperature should be submitted immediately and reach the laboratory within 24 hours. Specimens can also be refrigerated and submitted on wet ice within 72 hours. Provided the target of the assay is not an erythrocyte pathogen, erythrocytes should be removed before storage if possible. Specimens should not be stored in frost-free freezers, as these undergo repeated freeze-thaw cycling, which can be associated with DNA degradation.9 Box 5-2 outlines recommendations based on specimen type.

RNA is highly susceptible to degradation during storage and transport. For detection of RNA viruses, some laboratories provide an RNA stabilizing solution into which specimens are collected to prevent RNA degradation. If stabilizing solution is not readily available at the time of specimen collection, swabs and body fluids should be immediately sent to the laboratory on wet ice. Tissue should ideally be snap frozen at −70°C within half an hour of collection and shipped on dry ice without being thawed in the interim. RNA is stable for at least 2 years at or below −70°C. Ribonucleases can continue to degrade RNA at −20°C.

For tissue specimens, the optimal amount is 1 to 2 g, although more or less may be required depending on the cellularity of the specimen.5 Tissues stored in formalin, especially for prolonged periods, are suboptimal for PCR because the formalin cross-links the DNA in the specimen. Tissues fixed briefly in formalin and then paraffin-embedded are preferable to tissues stored in formalin, because the formalin is removed during the embedding process. Paraffin-embedded specimens should only be used when no other specimens are available.5

Although PCR can be performed on feces, its sensitivity may be lower than with other specimens because inhibitors of PCR are particularly abundant in fecal material.10 The use of appropriate amplification controls by the laboratory facilitates detection of false negatives due to the presence of inhibitors.

Diagnostic Methods

Nucleic Acid Probes

A variety of nucleic acid probes are available commercially as kits for detection of microorganisms that infect humans. Some of these can also be used to detect the same pathogens in specimens from dogs or cats. Probes are available for use in the clinical microbiology laboratory to identify organisms that have grown in culture (e.g., Gen-Probe Inc., San Diego), and these probes are widely used in human clinical microbiology laboratories.

Probe hybridization can be performed on a nitrocellulose membrane (solid-phase hybridization); on formalin-fixed, paraffin-embedded sections mounted on a microscope slide (in situ hybridization); or in solution (liquid-phase hybridization). For solid-phase hybridization, the probe is reacted with microorganism DNA that has been immobilized on the membrane. Unbound probe is washed away, and the bound probe is detected using fluorescence, chemiluminescence, radioactivity, or color development (in the same way that bound antibody or antigen is detected in an ELISA or immunofluorescent antibody assay). For in situ hybridization, formalin-fixed, paraffin-embedded specimens are sectioned and mounted on a special slide. The sections are deparaffinized, dried, and incubated with a solution that contains the probe, so both the presence and the location of the target pathogen within tissues can be identified. In situ hybridization assays that include a fluorescent-labeled probe are referred to as fluorescence in situ hybridization (FISH) assays (see Figure 5-1).

Because liquid-phase hybridization occurs in solution, unbound probe cannot be washed away. To overcome this problem, a chemiluminescent acridinium ester label is attached to the probe. A subsequent chemical hydrolysis step selectively degrades only unbound probe. On addition of peroxides, the intact (hybridized) probe then emits light.11

Although not yet widely used for veterinary applications, peptide nucleic acid (PNA) probes are now increasingly available to detect target DNA. PNA probes are uncharged peptides that mimic DNA and bind to complementary DNA sequences just as a nucleic acid probe would.12,13 PNA probes lack the net negative charge of nucleic acid probes; therefore, the electrostatic repulsion that normally occurs when two negatively charged DNA strands hybridize does not occur. The result is a more stable and specific binding of the probe to its target, which in turn can be associated with increased assay sensitivity and specificity.

Branched DNA assays and hybrid capture assays are highly sensitive hybridization methods that include steps to intensify the signal generated from probe hybridization.3,4 They are not yet widely used in veterinary medicine.

The Polymerase Chain Reaction

PCR allows the specific amplification of DNA sequences from just one copy to millions of copies, which can be more readily detected. The DNA from a clinical specimen is extracted using a commercially available DNA extraction kit. A pair of primers, roughly 20 nucleotides long, is then used to bracket a desired DNA sequence, which is subsequently copied using a DNA polymerase enzyme (Figure 5-2).

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Jul 10, 2016 | Posted by in INTERNAL MEDICINE | Comments Off on Nucleic Acid Detection Assays
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