Haemobartonella PCR methods and materials

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C435S091200, C536S023700, C536S024320, C536S024330

Reexamination Certificate

active

06518020

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 amplicon size. Minnick and Barbian, 31
J Microb Meth
51 (1997).
Haemobartonella felis,
which causes infectious feline anemia, has two known subspecies: the California subspecies and the Ohio/Florida (herein called “Ohio”) subspecies. Other organisms also cause anemia (eg. Bartonella and Ehrlichia), but treatment of the anemia is ideally directed to the causative organism. PCR technology has been used to detect
Haemobartonella felis,
although distinguishing between anemia-causing subspecies has not been accomplished.
In Rikihisa et al., 35 (4)
J. Clin. Microb.
823 (1997), there is disclosed the use of portions of conserved 16S sequences as primers in order to sequence the 16S genes of
H. felis
California and
H. felis
Ohio, and evolutionarily compare them to each other as well as to other organisms. It also discloses a method for distinguishing
H. felis
strains from one another, which method requires restriction enzyme cleavage and gel electrophoresis. In Messick et al., 36 (2)
J. Clin. Microb.
462 (1998), there are disclosed primers useful to identify (selectively)
H. felis
Ohio. It does not identify an assay for distinguishing
H. felis
Ohio and
H. felis
California or other organisms from
H. felis
Ohio.
In another organism, Bartonella, PCR assays have been discussed which use differences in citrate synthase sequences. These assays use a first step of conducting PCR and a second step of digesting the PCR products with restriction enzymes 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 in Bartonella have also been conducted, using restriction enzymes to distinguish among species. Birtles, 129
FEMS Microbiol. Letters
261 (1995).
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 Haemobartonella species.
It is yet another object to provide materials related to the methods disclosed, including primer sets.
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 the 16S region of Haemobartonella.
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 definitions:
“Amplicon(s)” shall mean a nucleic acid produced through use of primers in PCR.
“Genus-specific primer(s)” shall mean primers being capable of amplifying an amplicon from at least a portion of the 16S region of at least two Haemobartonella species, and no other genera, and wherein the size of the amplicon is unique to the species.
When the term “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 and in the presence of amplifiable nucleic acid, is capable of amplifying nucleic acid.
“Species” means any species or subspecies, or other subset of species or subspecies.
DETAILED DESCRIPTION OF THE INVENTION
In broad terms, the present invention includes materials and methods useful to distinguish between and among species of a genus. The methods simplify and are therefore more cost-effective than previous methods. In addition, because the present methods are simpler than previous methods, the risk of operator error, contamination, or any other technical problem is reduced, making the present invention inherently more reliable than previous methods.
The present invention also includes methods to detect Haemobartonella species in a test sample, comprising: a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of genus-specific primers; and b.) detecting Haemobartonella species in the test sample in the event a Haemobartonella-sized amplicon is present. A method as described, wherein step b.) comprises gel electrophoresis is preferred, although any method for detecting amplicon(s) (e.g. size-differentiating chromatography) is within the scope of the present invention.
For instance, the above method can be used to identify both the specific presence, or the specific absence of a certain species of Haemobartonella. As an example, the present method could be used to test a sample using a primer set (one forward sequence, one reverse sequence, in amounts necessary to conduct PCR) designed to amplify, both
H. felis
Ohio and
H. felis
California, although the size of the amplicons would differ. In that instance, it is possible that the primers would amplify an amplicon unique for
H. felis
Ohio, and not
H. felis
California. The result would indicate the presence of
H. felis
Ohio as well as the absence of
H. felis
California. In fact, methods as described, wherein the primers are capable of amplifying uniquely-sized amplicons from
H. felis
Ohio and
H. felis
California is a preferred embodiment of the present invention. However, methods wherein the primers are capable of amplifying uniquely-sized amplicons for every Haemobartonella species are also preferred.
Moreover, the present invention is not limited to the use of only one set of genus-specific primers. The methods herein also include those wherein a second set of primers is used, for example, for nested PCR. However, methods wherein PCR is conducted using one set of genus-specific primers is preferred.
Methods which utilize primers designed using conserved sequences in or flanking the Haemobartonella 16S region are within the scope of the present invention. A preferred region for designing forward primers for the present invention is the region spanning nucleotides 175-425. Not all bases are identical in these regions, but those in the art are aware of primer design strategy in light of non-identical sequences. A preferred region for designing reverse primers for the present invention is the region spanning nucleotides 455-700. Not all bases are identical in these regions, but those in the art are aware of primer design strategy in light of non-identical sequences.
Methods as above wherein the genus-specific forward primer comprises a sequenc

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