The need for improved diagnostic procedures is outlined and variations in probe technology are briefly reviewed. A discussion of the application of probe technology to the diagnosis of disease in animals and humans is presented. A comparison of probe versus nonprobe diagnostics and isotopic versus nonisotopic probes is made and the current state of sequence amplification is described. The current market status of nucleic acid probes is reviewed with respect to their diagnostic application in human and veterinary medicine. Representative product examples are described and information on probes being developed that offer promise as future products is discussed

Oberry, Phillip A.

English

NASA Technical Reports Server

N92-22699
NUCLEIC ACID PROBES IN DIAGNOSTIC MEDICINE
Phillip A. O'Berry
National Technology Transfer Coordinator for Animal Science
National Soil Tilth Laboratory, USDA, ARS, OC!
2150 Pammel Drive, Ames, Iowa 50011
ABSTRACT
The need for improved diagnostic procedures will be outlined and variations in probe technology will
briefly be reviewed. A discussion of the appfication of probe technology to the diagnosis of disease in animals
and humans will be presented. A comparison of probe vs. nonprobe diagnostics and isotopic vs. nonisotopic
probes will be made and the current state of sequence amplification will be described. The current market status
of nucleic acid probes will be reviewed with respect to their diagnostic application in human and veterinary
medicine. Representative product examples will be described and information on probes being developed that
offer promise as future products will be presented.
INTRODUCTION
New technology will be required to meet the exponentially increasing world food needs. World
population is projected to double in the four decade period from 1960-2000 to reach 6.1 billion [1]. World
livestock and poultry numbers have also increased significantly. Healthy animals contribute immeasurably to the
nutritional status, economic productivity, land resource utilization and overall well-being of our global society.
Molecular approaches to agricultural production and animal disease diagnosis provide a ray of hope that world
food needs can be met. Nucleic acid probes provide a powerful new technology that is just now being applied
to improve our ability to more rapidly detect diseases and their causative agents.
Diagnostic Teehnolo_
Diagnosis of the hundreds of infectious and noninfectious diseases that affect man and animals is not
simple. It often involves clinical examination, serologic tests, cultural or microscopic examination of a wide range
of samnles, fluids, or tissues, postmortem examination and the gathering and assessment of additional
information. Adequate diagnostic tests do not exist for many important diseases. Even in instances where
adequate tests do exist, there is a critical need for an improved test that is more rapid, more economical or more
reliable. Thus, the field of diagnostics lends itself to the application of nucleic acid probe technology.
In simplest form, a nucleic acid probe is a specific piece of single stranded nucleic acid, usually
composed of highly conserved nucleotide sequences, that contains an isotopic or nonisotopic label allowing it to
be visualized when it finds and binds (hybridizes) to another piece of nucleic acid that has complementary
nucleotide bases. It has been known since 1953 that the structure of DNA is a double helix [2]. It was later
found that two separate but complementary strands of DNA reassociate (hybridize) under specific conditions
to form a double helix [3]. These and other principles were used in the late 1960's to identify specific pieces of
DNA in the nuclei of cells fixed to a microscope slide using labelled complementary pieces of nucleic acid. Thus
probe technology was born.
Probes are made from such materials as target-specific sequences of cloned genes, complementary DNA,
natural or synthetic single-stranded oligonucleotides or ribosomal RNA. Sample material usually is solubilized,
and either its proteins are electrophoretically separated and bound to an appropriate support membrane
(Western blotting) or they are applied to nitrocellulose filters (Dot blotting) for reaction with a probe. The
probe, labeled by incorporation of a radioisotope (e.g. 32p or t25I) or enzyme, (e.g. alkaline phosphatase or
horseradish peroxidase) then searches the sample on the support system for homologous nucleotide sequences,
or "target" DNA. Probe-target DNA hybrids are then visualized or enumerated by autoradiographic or direct
scintillation counting or by enzyme-substrate color change reactions.
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Most diagnostic probes developed thusfar utilize radioisotopes as labels with resulting advantages and
disadvantages. The advantages are high sensitivity, easy signal detection and permanent record of test results.
The disadvantages are the risk of exposure to harmful levels of radiation, expensive safety protection equipment,
strict waste disposal procedures and relatively limited probe storage life. The diagnostic community has been
slow to develop, market and use these radioactive probes.
Nonisotopic probes offer some distinct advantages over their isotopic counterparts. They are more
simple to run, obviously pose no radiation hazard and require no specialized safety equipment or disposal
procedures. These probes tend to have longer storage life. The recent development and marketing of
chemiluminescent substrates for use with nouisotopic probes allow for exposure of x-ray film in the probe
reaction with the production of a hard-copy of the test results in only a few minutes. These and other recent
improvements in probe development technology have made nonisotopic probes more sensitive and practical than
ever before.
Powerful new procedures to amplify target DNA sequences in sample material offer considerable
promise in enhancing the application of probe technology to diagnostic veterinary medicine. The best known
of these is the polymerase chain reaction, (PCR) formally introduced in 1986 [4] and patented in 1987. A recent
review article [5] describes PCR in detail and discusses its application to probe technology. Target DNA is heat
denatured and specific paired primers are repeatedly annealed and extended with a specific polymerase until
amplification is sufficient to allow signal detection even in a complex mixture. The relatively simple procedure
uses a single thermostable enzyme, can be performed in a few hours and in a single tube, and lends itself to
automation. Among the problems in the procedure are nonspecific hybridization, the number of steps involved
in the thermal cycling of the reaction, the number, and sometimes efficiency, of product molecules per cycle is
rather low thus necessitating an increased number of cycles to reach a sufficient amplification level. In addition,
RNA targets and DNA targets are indistinguishable in some mixed nucleic acid samples. However, new
developments in PCR techniques allow amplifications of over 100 billion-fold, meaning that a single molecule
can be amplified to a point of visibility on a simple agarose gel.
Several other target amplification procedures are also available. They are amplification by RNA
transcription (TAS, 3SR) and amplification by ligation (LAR/LAS). The comparative advantages and
disadvantages of these procedures and PCR are well described. It is likely that newer and even more useful
procedures will come in the years ahead.
Applications of Probe Technology
Nucleic acid probes are uniquely and specifically well suited to detect even minute amounts of their
homologous counterparts. Applications of this powerful new technology are rapidly increasing and virtually
limitless.
Detection of disease agent nucleic acids in diagnostic samples is an obvious application. Molecular
approaches to infectious disease diagnosis has been recently reviewed [6]. Systems must be specifically developed
for use on such varied diagnostic samples as tissues, cells, blood, urine, saliva, feces, mucosal swabs, skin
scrapings, semen or other specimen. All must be compared against and be more advantageous than current
conventional cultural, histopathologic, microscopic or immunologic diagnostic methods.
Nucleic acid probes can also aid in the differentiation between closely related microorganisms.
Detection of the presence of a specific serovar or strain can influence the therapeutic or preventive measures
that should be taken. Clinical samples are often complicated by the presence of nucleic acids of closely related
nonpathogens or pathogens of such low virulence as to be unimportant.
The typing of DNA using probes has many practical applications. They include, but are not limited to,
the detection of genetic defects, genome mapping, forensic uses, sex determination in sperm cells, paternity
determination and microbial classification.
198
Some applications of nucleic add probe methodology have been made to the detection and prognosis
of certain malignancies. Some conditions have been associated with viral infections, such as cervical cancer and
human papilloma. Others, such as certain breast and ovarian carcinomas, have been associated with oncogene
amplification. Certain leukemias and lymphomas have been connected to specific clonal gene arrangements and
chronic myelogenous leukemia has been shown by DNA probe use to be a specific gene translocation. Thus
nucleic acid probes offer considerable promise as malignancy diagnostic, prognostic and perhaps even therapeutic
applications.
An additional application of nucleic acid probe technology is in the area of quality control of biologics.
Probes represent a powerful new tool to screen for and detect adventitious microorganisms in biologicals.
Certain microorganisms are commonly found in cell cultures or production materials, like serum, and are quite
difficult to detect by current and more conventional methods.
At this point in the development of nucleic acid probe technology, the most common application is in
research. Probes are powerful research tools used for a wide variety of nondiagnostic purposes. They have
contributed significantly to the gathering of new information on pathogenesis, epidemiology, latency, microbial
genetics, taxonomy and microbial ecology. Probes are also used to develop new and improved immunogens,
diagnostic tests and/or therapeutic agents.
Probe Products in Human Diagnostics
The market was surveyed to determine the number and types of nucleic acid probes available for
diagnosis of human diseases. All companies in a recent product listing [7] were contacted, literature was
surveyed and numerous contacts were made with Food and Drug Administration officials.
Over 200 DNA or RNA probes are currently commercially available. These products are marketed by
23 companies and the American Type Culture Collection with most specified for research purposes only. They
cover such diverse areas as viruses (Human Immunodeficiency Virus, Human eapillomavirus, Herpes Simplex
Virus, Epstein-Barr Virus and Hepatitis A and B viruses), bacteria (Legionella, Mycobacteria, Campylobacter,
Mycoplasma and Haemophilus), mycotic agents (Histoplasma, Blastomyces and Coccidioides), drugs (digoxin
and phenobarbital), neoplasias (Chronic Myeloganous Leukemia, Acute Lymphocytic Leukemia and certain
Lymphomas), genetic diseases (Cystic Fibrosis and Duchene Muscular Dystrophy) as well as a host of oncogenes,
proto-oncogenes, all 24 human chromosomes and over 100 probes for specific human Restriction Fragment
Length Polymorphisms.
Nucleic acid diagnostic probes are considered to be devices by the FDA and as such are required to
receive premarket approval if marketed for diagnostic use. None are ficensed by FDA but 61 have received FDA
approval in the past 6 years. One company, Gen-Probe Inc., San Diego, has received approval for 30 probes.
Probes marketed for research purposes only receive limited FDA oversight.
It is difficult to compile information on probe product sales revenues. However, two companies active
in this area (Enzo Biochem, Inc. and Oncogane Science) reported gross sales from all products in 1990 of $19
million and $5 million respectively [8]. It is not known how much of these gross sales were derived from probe
sales. The market for probes in the second half of this decade has been estimated to be $500 million [9].
Probe development and use for human diagnostics has grown exponentially in the last few years. It is
likely that many new and perhaps more powerful probes will be developed in the very near future. Promising
new probes are being developed for Cryptosporidia [10], Bo_elia burgdorferi [11], neoplastic tissue [12], E. coil
in foods [13], Salmonella in foods [14], Listeria in foods [15], and Human Papillomavirus in Pap smears [16].
Ultimately, the value of these tests and any other probe tests that reach the market will be determined in the
same way as for all new diagnostic products - by independent scrutiny of their clinical performance, convenience
and relative cost [17].
199
Probe Products in Veterinary_ Dia2nostics
A similar market survey was conducted to determine the number and types of nucleic acid probes
available for diagnosis of animal diseases. Literature and scientific publications were surveyed and contacts were
made with U.S. Department of Agriculture officials in the Animal and Plant Health Inspection Service.
Over 100 probes developed against a wide range of animal viral and bacterial pathogens have been
reported [18]. Although premarket approval is not mandatory, one probe has received USDA approval to be
marketed for veterinary diagnostic purposes. It is a DNA probe developed by IDEXX Corporation of Portland,
ME for the detection of Mycobacterium paratuberculosis in fecal samples. A second probe, developed by
scientists at Montana State University is used by Agricultural Bioengineering, Inc. of Bozeman, MT to test
samples submitted to it for TritriehQmonas foetus, the flagellated protozoan that causes the reproductive disease
bovine trichomoniasis. Little information is available on these probe product sales since both were introduced
this year.
The future looks promising that additional probes will enter the veterinary diagnostics market. They
will cover an expanding number of pathogens and diseases. Many will have patent protection and exclusive
license provisions. Several of the more promising probes are specific for such diverse organisms as Trevonema
hyodvsenteriae [19], Campylobacter fetus and Campvlobacter hyointestinalis [20], Anaplasma maronale [21],
Bovine Virus Diarrhea Virus [22], Eperythrozoon suis [23] and Bovine Leukocyte Adhesion Deficiency [24], a
genetic disease of cattle.
SUMMARY
A powerful new technology utilizing labeled nucleic acid is being applied to human and veterinary
diagnostics. Over 300 nucleic acid probes have been developed in the past few years. This new technology will
be increasingly transferred into the U.S. and global marketplace with considerable promise of improvement in
human and animal health.
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Nucleic acid probes in diagnostic medicine

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