Nucleic Acid Detection Assays

• Assays for detection of nucleic acid such as polymerase chain reaction assays are increasingly available commercially for diagnosis of companion animal infectious diseases.

• Molecular diagnostic assays include those that involve amplification (the copying of nucleic acid so that it can be more readily detected) or probe-based assays that do not involve nucleic acid amplification.

• Specimens for these assays should be collected using sterile technique in order to minimize contamination.

• Interpretation of the results of molecular diagnostic tests requires knowledge of the problems and pitfalls of each type of assay. The results of testing at one laboratory may differ from that at another because of variations in assay design.

• With increased automation, laboratory proficiency testing, improved quality assurance, and standardization of reporting, the availability and utility of molecular diagnostic tests for companion animal infectious diseases will increase.

Introduction

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

FIGURE 5-1 Fluorescence in situ hybridization (FISH). A, A nucleic acid probe that is complementary to a segment of leptospiral DNA is tagged with a fluorescent label and incubated with a tissue section on a slide. B, Unbound probe is washed away, and the section is examined using fluorescence microscopy. (Leptospira FISH image courtesy Dr. Richard Goldstein.)

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.

BOX 5-1 Applications of Nucleic Acid Based Assays

FCV, Feline calicivirus.

^∗Anderson B, Sims K, Regnery R, et al. Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR. J Clin Microbiol 1994;32:942-948.

^†Willi B, Boretti FS, Cattori V, et al. Identification, molecular characterization, and experimental transmission of a new hemoplasma isolate from a cat with hemolytic anemia in Switzerland. J Clin Microbiol 2005;43:2581-2585.

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.

BOX 5-2 Guidelines for Collection and Transport of Specimens for Polymerase Chain Reaction Assays

^∗Beutler E, Gelbart T, Kuhl W. Interference of heparin with the polymerase chain reaction. Biotechniques 1990;9:166.

Adapted from the CLSI guidelines MM13-A.

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.