Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-17
2001-10-09
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S810000, C536S023100, C536S024320, C536S024330
Reexamination Certificate
active
06300072
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention is concerned with speciation of organisms, for the purpose of improving differential diagnosis of disease. The assays currently available to distinguish between or among species have not always met the expectations of consumers because they are either too costly, cumbersome or unavailable.
Polymerase chain reaction (PCR) and serological assays are currently used to distinguish among species. Serological tests present problems with cross-reactivity and available PCR tests are complicated. Typically, PCR-based assays require three steps: 1) conducting PCR using a primer set which distinguishes among members of different genera, but not among members of the same genus; 2) digesting the PCR products with restriction enzymes and 3) distinguishing among species on the basis of restriction digest patterns. One assay uses several sets of species-specific primers instead of digestion with restriction enzymes, with identification of the PCR products made by size. Minnick and Barbian, 31
J Microb Meth
51 (1997).
One genus of microorganisms, Bartonella, causes a variety of species-dependent disease states in humans, and is therefore important to speciate prior to treatment. Humans infected with bacteria from the genus Bartonella display a variety of pathogies, and appropriate treatment has been surmised as dependant on the species causing the pathology. For instance, the species
B. henselae
(relatively common in flea-infested areas) presents as cat scratch disease or atypical cat scratch disease, and health care professionals continue to debate the appropriate antibiotic treatment. Bass et al., 16
Pediatr. Infect. Dis. J
., 163 (1997).
B. clarridgeiae
, another causative agent of cat scratch disease, can be treated with antibiotics, but it is not clear which are the most appropriate. ibid.
B. bacilliformis
is the causative agent for Carrion's disease (Oroya fever), and is typically treated with chloramphenicol, penicillins or tetracyclines. ibid. Another species,
B. elizabethae
has been associated with cardiac valve abnormalities, and is so rare that appropriate antibiotics have yet to be determined. ibid.
B. quintana
causes trench fever (rare except for unsanitary living conditions or in the immunocompromised), and has been successfully treated with penicillins, tetracyclines and cephalosporins. Kordick et al., 35(7)
J. Clin. Microb
. 1813 (1997).
B. vinsonii sub vinsonii
and
B. vinsonii sub berkhoffii
have only been found in dogs and voles.
Available Bartonella PCR diagnostics require several steps, and are therefore inconvenient for laboratory analysis of samples. For instance, PCR assays on the basis of differences in citrate synthase sequences have been performed using a first step of conducting PCR and a second step of digesting the PCR products with restriction enzymes, followed by gel electrophoresisis to distinguish among species. Joblet et al., 33(7)
J. Clin. Microb
. 1879 (1995); Norman et al., 33(7)
J. Clin. Microb
. 1797 (1995). PCR assays on the basis of differences in 16S rRNA sequences have also been conducted, using restriction enzymes to distinguish among species. Birtles, 129
FEMS Microbiol. Letters
261 (1995).
Likewise, primers have been used to amplify the region between the 16S and 23S genes (called “the intergenic region”) of Bartonella. In those assays, restriction enzymes were also used to cut and distinguish the PCR products. Matar et al., 31(7)
J. Clin. Microb
. 1730 (1993) and Roux and Raoult, 33(6)
J. Clin. Microb
. 1573 (1995). In Roux, a difference in size of PCR products (prior to digestion by restriction enzymes) was noted (page 1576); however, the differences are so small as to be indistinguishable on a gel. Moreover, no suggestion is made in Roux to use the pre-digestion PCR product size differences for the purpose of differentiation. In Matar, page 1732 that all three species had “an approximately 1,600-bp fragment” and bacilliformis had a 1,000 bp fragment prior to digestion.
In a different approach, Minnick and Barbian, 31 J Microb Meth 51 (1997) designed one set of primers from the 16S/23S intergenic region of Bartonella, and amplified fragments from
B. bacilliformis, B. elizabethae, B. henselae
and
B. vinsonii
. The fragments were of different, but indistinguishable sizes (FIG.
2
), and the researchers therefore conducted a second, species-specific amplification using different sets of primers for each species represented (FIG.
3
). Minnick, at 55 (1997).
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of these documents.
SUMMARY OF THE INVENTION
The present invention requires only a single step to generate amplicons which identify a specific species.
It is therefore an object to provide a simplified assay for distinguishing between or among species of a genus.
It is a specific object to provide a simplified assay for distinguishing between or among Bartonella species.
It is yet another object to provide materials (nucleic acids, vectors, cells, etc.) related to the methods disclosed, including primers and the full sequence of the 16S/23S region of two species of Bartonella.
In all of the above embodiments, it is an object to provide methods to diagnose disease using the materials and methods provided.
It is also an object to provide methods for identifying primers useful to conduct PCR assays which capitalize on the species-specific size differences in an intergenic region of a prokaryote.
It is also an object to provide methods for identifying primers useful to conduct PCR assays which capitalize on the species-specific size differences in the 16S/23S intergenic region of Bartonella.
Finally, it is an object of the invention to provide a kit for convenient use of the materials and methods herein provided.
Definitions: For the purposes of the present invention, the following terms shall have the following meanings:
“Amplicon(s)” shall mean a nucleic acid(s) produced through use of primers in PCR.
“Genus-specific primer(s)” shall mean primers capable of amplifying an amplicon from at least a portion of the 16S/23S intergenic region of at least two species of the same genus, and no other genera, and wherein the size of the amplicon is unique to the species.
“Bartonella genus-specific primer(s)” shall mean primers capable of amplifying an amplicon from at least a portion of the 16S/23S intergenic region of at least two Bartonella species, and no other genera, and wherein the size of the amplicon is unique to the species.
When the term “Genus-specific primer(s)” or “Bartonella genus-specific primer(s)” is used to describe primers used in PCR assays, it is assumed that said primers are also being in amounts sufficient to amplify at least one ascertainable fragment.
A “set” of primers means at least one forward and at least one reverse primer, that, when used in a PCR assay in appropriate amounts, is capable of amplifying an amplicon.
REFERENCES:
patent: 5869335 (1999-02-01), Munderloh et al.
patent: 5952194 (1999-09-01), Stiegler
patent: 5958414 (1999-09-01), Regnery, et al.
Dauga et al. Journal of Molecular Microbiology. 45: 192-199 (No date), 1996.*
Kordick, et al., 46-3Int J Syst Bacteriol704 (1996).
Minnick & Barbian., 31J Microb Meth51 (1997).
Bass, et al., 16Pediatr. Infect. Dis. J. 163 (1997).
Kordick, et al., 35(7)J. Clin. Microb. 1813 (1997).
Joblet, et al., 33(7)J. Clin. Microb. 1879 (1995).
Norman, et al., 33(7)J. Clin. Microb. 1797 (1995).
Birtles, 129 FEMSMicrobiol. Letters261 (1995).
Matar, et al., 31(7)J. Clin. Microb. 1730 (1993).
Roux & Raoult, 33(6)J. Clin. Microb. 1573 (1995).
Rikihisa, et al., 35(4)J. Clin. Microb. 823 (1997).
Messick, et al., 36(2)J. Clin. Microb. 462 (1998).
Dawson, et al., 156Arch Intern Med137 (1996).
Warner & Dawson,Ge
Heska Corporation
Heska Corporation
Myers Carla J.
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