Identifying organisms by detecting intronic nucleic acids

Details Identifying organisms by detecting intronic nucleic acids Identifying organisms by detecting intronic nucleic acids

2000-08-23

2003-07-29

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S024330, C536S024300

Reexamination Certificate

active

06599701

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the field of nucleic acid and protein detection and, more specifically, to the rapid and accurate identification of organisms by detecting differences in nuclear and organellar introns.
BACKGROUND OF THE INVENTION
Microorganisms are the cause of damaging infections in both plants and animals. About 1.3% of patients admitted to hospitals in the U.S. have positive fungal cultures. In particular,
Candida albicans
is one of the most frequently observed pathogens in immunocompromised patients. Most individuals are colonized with
C. albicans
as a commensal organism, and when the individual becomes immunocompromised, the organism can establish an infection. Systemic Candida infections extend hospital stays and contribute to increased mortality.
There is a need for epidemiological and diagnostic tools to detect infectious microorganisms in situations where they are hard to distinguish or where the nature of the agent is still under investigation. This is particularly true in fungal diseases where considerable effort has gone into studying and combating such diseases in immunocompromised human patients and in diseases of crops.
Epidemiological and diagnostic tools for classifying plant infecting and mammalian infecting fungi have been used to identify the origin of fungal infections and to track the progression of disease after treatment with antifungal drugs. In the case of mammalian fungal pathogens, there are at least 19 species of Aspergillus and at least seven species of Candida that cause infection. Almost all the “species” in these genera are defined solely by morphological and nutritional characteristics. These tests are laborious and expensive and have not provided sufficient discrimination to date to classify all infectious organisms.
A variety of detection and identification methods have more recently been developed for detecting
Candida albicans
, including the germ tube test, carbohydrate assimilation test, antigen test, serology, fluorescein-conjugated lectin visualization, and nucleic acid detection by polymerase chain reaction (PCR). Despite these tests, current diagnosis of Candida continues to rely on differential culturing, because non-culture tests are costly, requiring multiple enzymatic or hybridization steps and, in the case of PCR, a series of different reaction cocktails and conditions. This additional work diminishes the throughput of a clinical laboratory and increases the chance of error.
There are no less than 30 genera of fungi involved in plant diseases and the relationships among these various species and genera of fungi is still not fully understood. Almost all the “species” in plant fungal genera are presently defined by morphological features or by host range. However, the lack of good morphological characters in fungi has led to often opposing classifications based on host plants, as for in “forma specialis,” or other characters for sub-species groupings. Furthermore, in some cases, fungal morphological features can only be discerned when infections are well established on the plant host and symptoms are visible, or when the fungi are present in large enough quantities to be cultured from the plant. Thus, diagnostics of plant infecting fungi is at a rudimentary stage and early detection in asymptomatic plants is not possible using these methods.
Molecular-based methods have been applied to a very limited number of plant pathogenic fungi (reviewed by Swaminathan et al., in
Diagnostic Molecular Microbiology, Principles and Applications
, D H Persing et al. eds., ASM Press, Washington, D.C., pp 26-50 (1993)). For example, immunoassays have been devised for earlier detection of Pythium (Miller et al.,
Phytopathol
. 78:1516 (1988)), Phytophthora and Rhizoctonia (MacDonald et al.,
Plant Disease
74:655-659 (1990)) and
Mycosphaerella fijiensis
(Novartis, AG Crop Protection Division, Basal Switzerland). Also, commercial kits are available and certified testing laboratories provide enzyme-linked immunoadsorbent assay (ELISA)-based assays for detection of some fungal species.
Furthermore, a variety of nucleic acid protocols have been used to detect plant pathogens, including plasmid content, pulsed field gel electrophoresis, nucleic acid hybridization, restriction digestion, and PCR (reviewed in Maclean et al.,
Adv. Plant Path
., 10:207-244 (1993); van Belkum et al.,
Clin. Infect. Dis
., 18:1017-1019 (1994); and Tang et al.,
Clin. Chem
., 43:2021-2038 (1997)). A few examples of the application of these approaches to fungal pathogens in plants include the arbitrarily primed PCR (“APPCR” or random amplified polymorphic DNA: “RAPD”)—based identification for epidemiology and population studies of intersterility groups in
Heterobasidion annosum
(Garbelotto et al.,
Can. J. Bot
., 71:565-569 (1993)) and RAPD-based identification of pathogenic versus non-pathogenic isolates of
Fusarium oxysporum
formal specialis (f. sp.) dianthi (Manulis et al.,
Phytopath
., 84:98-101 (1994)).
In addition, probes developed from tandem repeat loci within satellite DNA have been used to detect polymorphisms among
Heterobasidion annosum
isolates (DeScenzo et al.,
Phytopath
., 84:534-540 (1994)).
Although each of these methods are useful, there currently is no single effective approach for detection and classification. Moreover, many of the methods require some foreknowledge of the particular species of organism likely to be present. It is apparent that a need exists for improved molecular methods that avoid the increased costs and reduced speed associated with present diagnostic and epidemiological tests for determining infection of plants and animals.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide an approach to identify nucleic acid sequences and associated proteins that are useful for readily characterizing target organisms, such as differentiating between taxonomic groupings of target organisms, identifying the taxonomic group to which an organism belongs, etc. It also is an object of the present invention to use such nucleic acid sequences to rapidly and effectively identify organisms that are present in a sample. It is another object of the present invention to provide isolated nucleic acids comprising intronic regions useful in the methods of the invention. It is yet another object to provide kits suitable for practicing the methods of the invention.
To accomplish these and other objectives, there has been provided, according to one aspect of the present invention, a method for characterizing nuclear and organellar intronic regions that differ between or among various taxonomic groupings of organisms.
In one embodiment, an intronic region is selected from aligned nucleotide sequences of one or more gene homologs.
In another embodiment, a primer pair is generated for amplifying the intronic region and an amplified product is generated in a primer extension reaction. The amplified product from intronic regions of known organisms are analyzed to determine if the intronic region will be useful for characterizing unknown organisms. In one embodiment, the intronic region-specific primers flank more than one intron insertion site while in another embodiment, the intron region-specific primers flank a single intron insertion site.
In yet another embodiment, the intronic region is selected from gene sequences of organisms that reflect a broader taxonomic grouping than the taxonomic grouping of the target organisms sought to be characterized.
In still yet another embodiment, the target organisms sought to be characterized are from a single genus or very related genera and the organisms from which gene sequences are obtained are from different taxonomic classes or subclasses of organisms.
In further embodiments, the analysis of the amplified products from primer extension reactions include determining the presence or absence of the intronic region, the length of the intronic region, the nucleotide sequence of the intronic region, or restriction fragment length polymorphism. In s

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