PCR materials and methods useful to detect canine and feline...

Details PCR materials and methods useful to detect canine and feline... PCR materials and methods useful to detect canine and feline...

2001-07-10

2003-07-22

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200

Reexamination Certificate

active

06596492

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the identification of clonal populations of canine T and B-cells that correlate with canine and/or feline lymphoma, and more particularly to the use of PCR to amplify certain clonal rearrangements that correlate with lymphoma.
BACKGROUND OF THE INVENTION
Polymerase chain reaction (PCR) has been used for amplification of DNA for over a decade. It is a well-characterized tool that has been shown to be useful in many assays, including clonal expansion-related assays. Various PCR assays have been developed by correlating unique primers with a disease state, or by identifying conditions that are uniquely capable of high levels of amplification.
In the past, canine and feline lymphomas have been identified by fairly inconclusive means, including physical examination, histology and cytology. Moreover, typical procedures currently used for obtaining sufficient material are invasive and expensive.
Thus, a need exists for less invasive and inexpensive diagnostic methods for identifying such lymphomas. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention generally provides methods for detecting canine or feline clonal rearrangement. The present invention further relates to correlating such clonal rearrangements to a diagnosis of T-cell lymphoid malignancies in canids and felids or B-cell lymphoid malignancies in canids.
Accordingly, one aspect of the present invention is directed to a method of detecting clonal rearrangement in a test sample, comprising:
(a) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of primers having at least one forward and at least one reverse primer, wherein the set of primers is selected from the group consisting of canine IgH CDR3-specific primers and canine T cell receptor gamma-specific primers; and
(b) detecting clonal rearrangement in the test sample in the event one or more dominant and discrete DNA polymerase chain reaction products are present.
According to another aspect of the invention, a method to diagnose lymphoid malignancy in a canid or felid, comprising:
(a) obtaining a test sample from the canid or felid;
(b) conducting polymerase chain reaction using starting materials which comprise the test sample and at least one set of primers having at least one forward and at least one reverse primer, wherein the set of primers is selected from the group consisting of canine IgH CDR3-specific primers and canine T cell receptor gamma-specific primers; and
(c) detecting clonal rearrangement in the test sample in the event one or more dominant and discrete DNA polymerase chain reaction products are present, wherein the presence of said DNA polymerase chain reaction products is diagnostic of lymphoid malignancy in the canid or felid.
Particularly useful primers for use in the above methods include, for example, those identified herein as SEQ.ID.NO:1, SEQ.ID.NO:2, SEQ.ID.NO:3, SEQ.ID.NO:4, SEQ.ID.NO:5, and SEQ.ID.NO:6. More specifically, useful forward primers include SEQ.ID.NO:1 and SEQ.ID.NO:6, while useful reverse primers include SEQ.ID.NO:2, SEQ.ID.NO:3, SEQ.ID.NO:4 and SEQ.ID.NO:5.
In addition, various techniques for detecting clonal rearrangment can be used, including, for example, gel electrophoresis, HPLC and other means known to those skilled in the art.
The above methods can be used to detect canine or feline T-cell clonal rearrangements and to diagnose canine or feline T-cell lymphoid malignancies by using canine T-cell receptor gamma-specific primers, including, for example, those identified as SEQ.ID.NO: 4, SEQ.ID.NO: 5 and SEQ.ID.NO:6.
Similarly, the above methods can be used to detect canine B-cell clonal rearrangements and to diagnose canine B-cell lymphoid malignancies by using canine IgH CDR3-specific primers, including, for example, those identified as SEQ.ID.NO:1, SEQ.ID.NO:2, and SEQ.ID.NO:3.
The present methods can also be used to distinguish between T-cell lymphomas and B-cell lymphomas in which canine IgH CDR-specific primers and canine T-cell receptor gamma-specific primers are used in the methods and detecting the presence of any PCR products obtained.
In a further aspect of the invention, kits containing desired starting materials and associated components for performing the methods described herein are also provided. Other objects and features of the present invention will be apparent from the following detailed description of the invention.


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