WO1999025874A1

WO1999025874A1 - Polymerase chain reaction test for detecting feline leukemia virus

Info

Publication number: WO1999025874A1
Application number: PCT/US1998/024433
Authority: WO
Inventors: Cheryl Lynn Swenson; Aivars Vilnis; Roger K. Maes; Patrick J. Venta
Original assignee: Board Of Trustees Operating Michigan State University
Priority date: 1997-11-18
Filing date: 1998-11-16
Publication date: 1999-05-27
Also published as: AU1412799A

Abstract

Improved methods for detecting feline leukemia virus are provided. The methods comprise using a high-temperature-activated Taq DNA polymerase or a hot-start procedure to perform PCR on a viral DNA segment unique to feline leukemia virus of exogenous origin, followed by detection of the PCR-amplified DNA.

Description

POLYMERASE CHAIN REACTION TEST FOR DETECTING FELINE LEUKEMIA VIRUS

FIELD OF THE INVENTION

This invention relates generally to methods for detecting feline leukemia virus (FeLV) and more specifically, to methods for detecting FeLV employing polymerase chain reaction.

BACKGROUND OF THE INVENTION The feline leukemia virus (FeLV) infection is the most common pathogen in cats and is the leading cause of infectious illness and death of domestic cats. FeLV is a horizontally transmitted retrovirus that can produce persistent viremia of up to 30% of exposed cats, 80% of which die within three years of exposure. D.W. Macy, "Testing Cats for the Feline Leukemia Virus," Veterinary Medicine, 278-288 (March, 1991 ). FeLV infection is a global veterinary problem and the feline care industry has called for the accurate assessment of the FeLV status of all cats. Editorial: "Recommendations for Feline Leukemia Virus Testing," Feline Practice 24(2): 14 (1996). However, current assays for the presence of acute or chronic FeLV infection are generally limited to those testing for proteins including: 1 ) serum antibodies produced against the FeLV itself; 2) serum antibodies produced against the feline oncornavirus-associated cell membrane antigen (FOCMA) which is produced in response to FeLV infection; and 3) the FeLV viral protein, p27, in the blood, bone marrow, saliva, plasma or serum (though research methods detected other FeLV components, such as the viral protein, gp70). The common tests for these proteins include live-cell and indirect immunofluorescence assay (IFA) tests, enzyme-linked immunosorbent assay (ELISA) tests, and concentrated immunoassay technology (CITE) tests, with ELISA being considered the test of choice for general use and IFA being considered the more sensitive assay. Ibid.; D.L. Macy, id.

However, the tests mentioned above bear a high risk of false positives and false negatives (id.) and they are insufficiently sensitive to detect either latent or atypical, low level FeLV infections. Id.; K.A. Hayes et al., "Atypical Localised Viral Expression in a Cat with Feline Leukemia," Veterinary Record 124:344-345 (1989). Thus, it has been recognized that polymerase chain reaction (PCR) techniques may provide a basis for designing tests that are more sensitive, since PCR might be used to amplify viral nucleic acids to more readily detectable levels, in particular nucleic acids unique to FeLV of exogenous origin. The U3 region of FeLV is a known indication of the presence of exogenous FeLV. This U3 region is a portion of the FeLV long terminal repeat (LTR) whose DNA sequence is reported by M.A. Stewart et al., in J. Virol. 58:825-834 (1986); and by B.T. Berry et al., in J. Virol. 62:3631-3641 (1988). Unfortunately, PCR has been seen as an invalid method of testing because of a lack of standardization of oligonucleotide primers and testing protocols and also because of inconsistent results. Editorial: "Recommendations for Feline Leukemia Virus Testing," Feline Practice 24(2): 14 (1996). Moreover, PCR has not been reported to detect FeLV infection in pre-tumorous cats testing negative for p27 or FOCMA by existing tests, specifically ELISA and IFA (even though there is one report of PCR detection of FeLV in immunochemistry tumor biopsies from FeLV-associated feline lymphosarcomas; see M.L. Jackson et al., "Feline Leukemia Virus Detection . . . from Cats with Lymphosarcoma," Can. J. Vet. Res. 57:269-276 (1993)). Rather, current PCR-based tests, including that used to detect FeLV in ELISA(-) lymphosarcoma tissue, have failed to detect FeLV viral DNA from ELISA(-) feline peripheral blood, bone marrow, and other tissues, and from ELISA(-) pre-tumorous cats in general. See, e.g., T. Miyazawa and O. Jarrett, in Arch. Virol. 142:323-332 (1997); M.L. Jackson et al., in J. Vet. Diagn. Invest. 8:25-30 (1996).

SUMMARY OF THE INVENTION Improved methods for detecting feline leukemia virus are provided. The methods comprise using a high-temperature-activated Taq DNA polymerase or a hot- start procedure to perform PCR on a viral DNA segment unique to feline leukemia virus of exogenous origin, followed by detection of the PCR-amplified DNA. The methods of the present invention have been found both to be superior to prior standard and PCR-type tests for FeLV and to be able to detect exogenous FeLV in ELISA(-) cats where prior PCR methods have failed to do so.

Additional objects, advantages, and features of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

The various advantages of the present invention will become apparent to one skilled in the art by reading the following specification and subjoined claims and by referencing the following drawings in which:

Figures 1 A-1 C present the nucleotide sequence of the envelope gene and long terminal repeat of FeLV Subtype A; the U3 region and the Specific Example 1 amplification target DNA are shown as dsDNA. The U3 DNA region is shown in bold. The nucleotide sequences of the Specific Example 1 primers are underlined and the nucleotide sequence of the Specific Example 1 probe is double underlined. The sequence framed by the primers (base pairs 3071-3280) is the 210bp sequence of the Specific Example 1 amplification target region.

Figures 2A-2C present the nucleotide sequence of the envelope gene and long terminal repeat of FeLV Subtype A. The U3 region and the Specific Example 1 amplification target DNA are shown as dsDNA. The U3 DNA region is shown in bold. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The methods of the present invention comprise the steps of amplifying feline genomic DNA by means of PCR using a high-temperature-activated, thermostable Taq DNA polymerase or a hot-start procedure, and analyzing the resulting PCR- amplified feline DNA. Where the feline DNA contains a region unique to exogenous FeLV - the U3 region of the Long Terminal Repeat (LTR) sequence of FeLV Subtype A - this region will be amplified and then qualitatively or quantitatively detected. The FeLV Subtype A U3 region was selected because its presence has a 100% correlation with the presence of exogenous FeLV, unlike DNA from FeLV Subtype B or C (whose presence has less than a 50% correlation with the presence of exogenous FeLV). See J.W. Casey et al., "The U3 Portion of Feline Leukemia Virus DNA Identifies Horizontally Acquired Proviruses in Leukemic Cats," Proc. Nat'l Acad. Sci. USA 78:7778-7782 (1981 ).

DNA is obtained from a sample of biological material e.g., from a subject cat. The DNA can be used as a PCR template in its unpurified state or after it has been purified from other components present in the sample. Any nucleic acid-containing tissue or biological material may provide the sample. In a preferred embodiment, feline circulating white blood cells or feline bone marrow cells are harvested by any method commonly known in the art and genomic DNA is extracted from these cells by any method commonly known in the art, e.g., by use of a phenol/chloroform method or by use of a spin column DNA isolation method (such as may be obtained from Qiagen GmbH of Hilden, Germany). In an alternate embodiment, the DNA may be obtained from the biological sample by use of a reverse transcriptase method in which a reverse transcriptase enzyme is first used to convert any viral RNA or mRNA present in the sample to DNA. Preferred reverse transcriptase enzymes (RTs) include Moloney murine leukemia virus RTs and their RT derivatives, avian myeloblastosis virus RT, Thermus thermophilus RT, and Thermus flavus RT. Any method known in the art as effective for allowing an RT to reverse transcribe RNA may be used; preferred methods include those described in C.W. Dieffenbach and G.S. Dveksler, PCR Primer 287-292 (1995) and F.M. Ausubel et al. (eds.), Short Protocols in Molecular Biology (1995) (unit 15.4). The DNA may then be used as a template for PCR, or the DNA may first be digested, by methods commonly known in the art, using one or more restriction enzymes. In a preferred embodiment, the restriction enzyme(s) used have recognition sites which are all located external to any FeLV U3 region present in the DNA; and more preferably located external to any FeLV sequences present in the DNA. A preferred restriction enzyme is EcoR I. After the enzyme has digested the DNA into fragments, these are then used as the template for PCR. In an alternate embodiment, the genomic DNA may be digested using a restriction enzyme that hydrolyses the PCR-targeted (U3-containing) DNA sequence at a single point internal to the target region and falling between the recognition sequences of the PCR primers; in this embodiment, an additional step is performed during the PCR reaction in which, after each round of PCR (or after all rounds of PCR are completed), a ligation is performed using a DNA ligase, e.g., T4 DNA ligase, to reconnect the amplified halves of the target DNA. Alternatively, a restriction enzyme may be used that cuts the U3-containing target region of the DNA in more than one place, provided that the target region remains intact.

In the PCR reaction, oligonucleotide primers are used which have been designed (and are effective) to allow amplification of all or part of the FeLV U3 region. Preferably these are designed to allow amplification of all or part of the FeLV U3 region shown in Figure 1. The primers may both be complementary to (different) nucleotide sequences within the U3 region or to non-U3 nucleotide sequences located in DNA segments adjacent to and associated with the U3 region; alternatively, the primers for one of the PCR-target DNA strands may be complementary to a sequence within the U3 region, while the primers for the other strand (of the PCR-target DNA) may be complementary to a non-U3 sequence located in a DNA segment adjacent to and associated with the U3 region. In a preferred embodiment, the downstream primer (toward the 5' end of the target sequence) is complementary to a U3 sequence and the upstream primer (toward the 3' end of the target sequence) is complementary to a non-U3 sequence. Thus, a preferred embodiment will employ: 1 ) a U3 primer recognizing a nucleotide sequence found within base pairs 2884-3224 of Figure 1 ; and 2) a non-U3 primer recognizing a nucleotide sequence found beyond base pair 3224 of Figure 1 , more preferably one found within base pairs 3225-3367. Oligonucleotide primers may be designed, based on the FeLV nucleic acid sequences, using any methods known in the art, whether designed, e.g., manually or by using a computer program effective for the purpose, e.g., an Oligonucleotide Selection Program such as is described by L. Hillier and P. Green, "A Computer Program for Choosing PCR and DNA Sequence Primers," PCRMeth. Appl. 1:124-128 (1991 ). A preferred pair of primers is one in which the first primer comprises the sequence:

5'-CAGCAGAAGTTTCAAGGC-3', and the second comprises the sequence: 5'-GAGGTTTATTCGTACACGG-3'. More preferred are primers consisting of these sequences, i.e. having no other nucleotides.

In a preferred embodiment, either the sense primers or the antisense primers are linked to a moiety that can be selectively reacted with another molecule to form a linkage. For example and without limitation, the moiety may be an immunoreactive molecule, such as an antigen, an antibody, or biotin, wherein the selective reaction partner of the moiety will then be, e.g. , an antibody, an antigen or an antibody-specific immunoglobulin, or avidin or streptavidin. The moiety will, upon reaction with its selective reaction partner, allow the later retrieval of any U3 DNA-containing sequences which have been successfully amplified by PCR. For example, an amplified DNA sequence linked, by its primer, to an antigen may later be retrieved by contacting it with an antigen-specific antibody which has been immobilized upon a solid support. A preferred moiety is biotin. Preferably, the moiety will be covalently attached to the 5' end of the primer.

In an alternate embodiment, the amplified DNA sequence may be retrieved by contacting it with a solid support-immobilized oligonucleotide whose sequence is complementary to a nucleotide sequence which is specific to the amplified DNA, and more preferably, specific to a terminal portion of the amplified DNA. In one version of this "hybridizing immobilization" embodiment, neither primer is labeled with a moiety. Detection of the immobilized, amplified DNA may then be accomplished, e.g., via use of an immunohistochemical method by contacting the double-stranded portion of the hybridizing-immobilized DNA with an antibody specific to double-stranded DNA (dsDNA), preferably a monoclonal antibody. In an alternate version of the "hybridizing immobilization" embodiment, at least one primer is biotin-labeled and the solid support-immobilized oligonucleotide is complementary to a terminal portion of the strand of amplified DNA that represents an elongation of the biotinylated primer; then, the biotin thereof is available to be utilized in detection, as by complexing it with reagents that cause a visible color change. In another alternate embodiment in which no biotinylated primers need be used, amplified DNA may be retrieved by contacting it with a biotinylated oligonucleotide whose sequence is complementary to a nucleotide sequence which is specific to the (preferably terminal end of the) amplified DNA; then the biotin may be used to immobilize the amplified DNA to a solid support. In this embodiment, the amplified target DNA may be detected by having previously hybridized it to a labeled oligonucleotide probe (at the same time as, or before, annealing the target DNA to the biotinylated oligonucleotide), where the probe recognizes a sequence spaced apart from that recognized by the oligonucleotide probe.

Where an oligonucleotide-to-moiety linkage is utilized, any linking chemistry such as is known in the art as capable of covalently linking such a moiety to an oligonucleotide may be used, provided the oligonucleotide remains intact and the moiety remains functional. A preferred linking chemistry is phosphoramidite chemistry. More preferred is the use of N-aliphatic or N,N-dialiphatic (e.g., N,N- diisopropyl) phosphoramidite linkers and their derivatives. Even more preferred are /?-cyanoalkyl phosphoramidites; especially preferred are ?-cyanoethyl phosphoramidites, for example, O=P[O(CH₂)₂CN][N(CH₃CHCH₃)J. The phosphoramidite linker may first be linked to the moiety or to the 3' hydroxy group or 5' phosphate substituent on a terminal deoxyribose of the oligonucleotide. When β- cyanoethyl phosphoramidite is used, preferably it is first linked to the deoxyribose and then to the moiety.

Using the primers and the genomic DNA or DNA fragments, a PCR reaction is then carried out using DNA polymerase. A preferred DNA polymerase is Taq DNA polymerase. More preferred is a high-temperature-activated (i.e. non-polymerizing at temperatures significantly below about 70°C), thermostable (i.e. retaining activity at least up to a point within the range of about 95°C to about 100°C) Taq DNA polymerase; an especially preferred DNA polymerase of this type is Taq Gold DNA polymerase (available from Perkin-Elmer of Branchburg, NJ). Alternatively, the DNA polymerase may be "high-temperature-activated" because it has been complexed to an inhibitor that dissociates from the enzyme at or near the temperature used to melt or that used to amplify the template DNA. For example, a DNA polymerase may be complexed to an anti-DNA polymerase antibody (preferably a monoclonal antibody) which becomes denatured and/or dissociates from the enzyme at such a temperature. A preferred anti-DNA polymerase antibody is the TaqStart antibody (available from CloneTech). The PCR reaction may be a standard reaction such as is known in the art or a "hot start" PCR reaction. In a hot start PCR reaction, a high-temperature-activated Taq DNA polymerase may, but need not, be used. In the hot start procedure, all reactants except one, preferably the template DNA, are mixed together and overlain with a layer of wax that melts at or just below the optimal temperature for DNA polymerase activity and the wax is allowed to harden. Any wax which has such melting behavior may be used; a preferred wax is paraffin wax. After the wax has hardened, the template DNA is then placed upon the wax and the reaction vial is raised to a temperature to begin the PCR reaction. As the reaction mixture approaches or attains the optimal reaction temperature, the wax melts, allowing the template DNA to mix with the rest of the reactants.

In another embodiment of the hot start reaction, the template DNA may be mixed with the wax as it cools, or may be placed between two layers of wax, so that it becomes trapped inside the wax layer; alternatively, the template DNA may be premixed with wax in the form of small, wax beads which are added to the reaction vial, in which case no solid wax layer initially need cover the reactant mixture. Other hot start PCR methods may also be employed, e.g., wherein the DNA polymerase is added to the reaction vial only after the vial has been raised to the reaction temperature; or wherein an antibody-complexed DNA polymerase is utilized. The phrase "hot start" as used herein, thus encompasses both the wax-based hot start reactions as well as the antibody-based and other variations of PCR such as are known in the art. Additional PCR methods known to those skilled in the art, which may also be employed, include, without limitation, "touchdown" PCR, "long distance" PCR, and the various PCR methods described in C.W. Dieffenbach and G.S. Dveksler, PCR Primer (1995) and in F.M. Ausubel et al. (eds.), Short Protocols in Molecular Biology (1995).

Preferably the PCR reaction is performed in a buffered, aqueous mixture of sufficient pH containing sufficient concentrations of dATP, dCTP, dGTP, and dTTP to allow the DNA polymerase to synthesize DNA efficiently. Any buffer effective to allow the PCR reaction to proceed may be used; a preferred buffer for use in the reaction mixture is PCR Buffer II (Perkin-Elmer). Preferably, about 0.1 to about 1.0 mM of each dNTP, and more preferably about 0.2mM of each dNTP, will be used. The reactant mixture will preferably also contain an amount of Mg²⁺ effective to support the activity of DNA polymerase. Preferably the Mg²⁺ will be provided by MgCI₂. Preferably, about 1-5mM MgCI₂ (more preferably, about 3mM) will be used. Also, a sufficient quantity of the primers to produce a final concentration effective for the desired number of cycles of amplification will be added to the reactant mixture. At least one copy of each primer must be added, preferably about 10-100nM of each, and more preferably about 40-50 nM of each. Preferably a concentration of DNA polymerase effective to perform PCR for the desired number of amplification cycles is used. Preferably about 1-5 units of DNA polymerase, and more preferably about 2.5 units of DNA polymerase is added. In any non-hot start PCR method used, the reaction will begin with the addition of the DNA polymerase. At the start of the reaction, at least about 50bp of a single copy of template DNA must be present (whether in the reactant mixture or in or on a wax layer or in, e.g., wax beads); preferably at least about 50ng of the template DNA (preferably template DNA fragments) will be present, more preferably about 75ng to about ^μg, and even more preferably, about 100ng.

The reaction vial containing the reactant mixture (with or without a solid wax template DNA separator), will be temperature cycled according to any temperature cycling method known in the art to be effective for DNA synthesis and primer-assisted amplification. Preferably at least one, and more preferably at least two, amplification cycles will be performed; even more preferably about 30-50 cycles, and still more preferably about 35-40 cycles will be performed. A preferred amplification cycle will comprise: about 15 seconds at about 94°C for denaturation, about 30 seconds at about 50°C for annealing, and about 30 seconds at about 72°C for elongation. After the amplification cycles are completed, the amplification phase of the PCR reaction is followed by one round of completion/quenching in which the reaction is brought to completion at about 72°C and then quenched at about 4°C.

After the PCR reaction is complete, the amplified DNA may be used as template DNA in a subsequent round of PCR amplification. In such a "nested PCR" procedure, the subsequent round of PCR uses PCR oligonucleotide primers which recognize sequences of the (amplified) template DNA which are between (and either inclusive or exclusive of) the recognition sequences of the original primers.

After completion of the PCR reaction - in any embodiment where the reactant mixture contained no doubly labeled (i.e. reporter label/quenching label linked) probe - - after the PCR reaction has been completed, any successfully amplified FeLV US- containing DNA sequences must be detected either qualitatively or quantitatively. Any such method as is known in the art may be used. For example, the DNA from each sample vial and each calibration standard vial may be electrophoresed on an agarose gel, along with molecular weight standards, and the bands may be stained with ethidium bromide. A visual comparison of bands corresponding to the molecular weight of the amplified target DNA region may then be made between sample and calibration standards to give a semi-quantitative result. Alternatively, the bands may be photographed and the photographic bands may then be quantified by use of a densitometer. In a qualitative version of this detection method, the presence, in a sample, of bands of the appropriate molecular weight for amplification target DNA may be taken as an indicator of the presence of exogenous FeLV. Alternately, e.g., a Southern transfer of the amplified DNA bands may be performed and the DNA bands may then be hybridized to a target DNA-specific, detectable probe for quantitative or qualitative analysis.

In a preferred embodiment, the amplified FeLV U3-containing DNA sequences can be directly quantitated by measuring the increase in fluorescence caused by the binding of a fluorescent reporter to double-stranded DNA. An example of a reporter is SYBR Green (Perkin-Elmer). The fluorescence reporter is added at the start of PCR DNA amplification reaction. As amplification progresses and the amount of double-stranded DNA increases, there is a concomitant increase in fluorescence as the reporter binds to the double-stranded DNA. This increase can be monitored spectrofluorometrically using instrumentation such as, but not limited to, the AB1 Prism 7700 Sequence Detector. The double-stranded DNA can be quantitated by using an internal standard or a standard curve. Detection using a fluorescent probe for double-stranded DNA can be used with, for example, but not limited to, conventional PCR, hot start PCR and RT-PCR.

In a preferred embodiment, detection will involve annealing a labeled oligonucleotide probe to a PCR-amplified target DNA sequence and detecting the probe's label. The probe is designed to be complementary, in whole or in part, to a portion of the amplified target DNA falling between (and exclusive of) the nucleotide sequences upon which the primers bind. Preferably, at least part of the probe is complementary to U3-specific DNA. Such probes may be designed and synthesized based on the nucleotide sequence of the U3 region (Figure 1 ) and synthesized in a DNA synthesizer by methods known in the art. A preferred probe comprises the sequence:

5'-CAGCAGTCTCCAGGCTCCCCAGTTGAC-3'. Even more preferred is a probe consisting of this sequence. Where the sense or antisense primers are biotinylated, preferably, the probe will be complementary to whichever DNA strand (sense or antisense) represents an elongation of the primer that was biotinylated. In an alternate embodiment, a target DNA-complementary probe is designed so that, once annealed to its complementary target DNA sequence, its Tm is at least 5 °C higher than the Tm of whichever annealed, primer-and-target DNA strand pair has the higher melting temperature. The probe is preferably covalently linked to a detectable label. This label may be or may comprise at least one fluorescent, luminescent (including, e.g., chemiluminescent, electroluminescent, orelectrochemiluminescent), phosphorescent, colored, colorable (e.g., pH or redox indicator), radioactive (e.g., ³H, ¹⁴C, or ³²P- containing), immunoreactive, or NMR-detectable component. Methods appropriate for quantitatively, semi-quantitatively, or qualitatively detecting the label are widely available and selection of an appropriate method is well within the knowledge of one of ordinary skill in the art. For example, the amplified target DNA may be isolated (e.g., by means of a primer-plus-biotin-to-avidin linkage) and hybridized to the labeled probe, and excess labeled probe then washed away or otherwise separated from the hybrids (or quenched relative to the hybridized labeled probe). Then the labeled probe-target DNA hybrids can be qualitatively or quantitatively detected, as by using, for example and without limitation: a radiographic, direct isotopic decay, or scintillation counting detection method for a radioactive-label; and, a visual or colorimetric detection method for a colored or colorizable label; and, a visual, fluorometric, or photometric detection method for an (appropriately stimulated) fluorescent or luminescent probe. A preferred label is a luminescent label. A preferred luminescent label will comprise a chelant-isotope complex. Especially preferred is a tris(hydroxymethyl) aminomethyl-2',2'-bicyclopyridine ruthenium (II) complex ("TBR"). Preferably, phosphoramidite linking chemistry is used to attach the label to the probe. When TBR is utilized as the label, a preferred linking chemistry is β- cyanoethyl-phosphoramidite chemistry. This chemistry may be used to attach the label to either the 3' or 5' end of the probe. Preferably, the probe is end-labeled at its 5' end as follows: ?-cyanoethyl-phosphoramidite is first reacted with the 5' phosphate substituent of the deoxyribose located at the 5' end of the probe; then the TBR is covalently attached to the phosphoramidite-activated probe. When a TBR complex is used, the preferred method of quantitation will be by measurement of DNA-derived electroluminescence. Such quantitation may be, and preferably is, performed in a Perkin-Elmer QPCR 5000 detection system, according to manufacturer's instructions.

Once constructed, the labeled probes are hybridized with the amplified DNA sequences under any conditions known in the art as effective for melting and then annealing DNA. Preferred conditions include, e.g., denaturing the DNA at about 95°C for about 5 minutes in a buffered aqueous solution and then annealing the DNA and probes at about 55°C for about 15 minutes. If any U3 DNA has been amplified, this procedure will result in the formation of labelled probe-DNA hybrids. Any concentration of probes effective for hybridizing to DNA may be used; preferably a molar concentration in excess of that of the amplified DNA will be used. In a preferred embodiment, at least about 0.01 μM of the labeled probe is added to the amplified DNA, more preferably about 0.1 to about 1 μM, even more preferably about 0.2 to about 0.5 μM and still more preferably, about 0.4 μM.

In a preferred embodiment, the labelled probe-DNA hybrids are then retrieved by reacting the moiety of the, e.g., biotinylated, PCR primer with its reactive pair, e.g., streptavidin. Preferably, the reactive pair (e.g., streptavidin) has been covalently immobilized upon a solid support. Solid support chemistries and immobilization methods are well known in the art and any such support chemistries and immobilization methods may be used. Especially preferred for use with biotinylated primers are commercially available magnetic beads upon which are immobilized streptavidin molecules. Preferably streptavid in-coated Dynal m-450 magnetic beads (available from Dynal, Inc. of Great Neck, NY) are utilized. These beads are combined with the labelled probe-DNA hybrids and the reactive pair is allowed to complex under any conditions known in the art to be effective for reactive pair complexing. For the biotin-avidin or biotin-streptavidin system, preferred conditions include bringing the bead/probe-DNA system to about 55°C for about 30 minutes. In another embodiment, the probes may be contacted with and hybridized to the amplified DNA sequences after linkage of the DNA to a solid support, except where a hybridization immobilization method has been used to immobilize the amplified DNA. Afterwards, the bead/DNA-probe-label system is quantitated, e.g., by washing the beads to remove unannealed labeled probes and then quantitatively detecting the remaining labels, e.g., by fluorometry. In a preferred embodiment, this quantitative measurement will take place by loading the samples into the Perkin-Elmer QPCR 5000 according to the manufacturer's instructions and allowing the machine to wash the beads of any unannealed labeled probes and measuring the electroluminescence of the remaining labeled probes. In an alternate embodiment, the probe is labeled preferably by linking (most preferably, end-linking) it to two labels: a reporter label at one end of the probe, e.g., a fluorescent label (whose fluorescence may be induced, e.g., by laser stimulation), and a quenching label at the other end of the probe, i.e. which quenches detection of, e.g., any detectable energetic emissions (such as fluorescence) from the reporter label. A preferred reported label is a fluorescent label and a preferred quenching label is a rhodamine-type dye. Preferably, the quenching label is attached to the 3' end of the probe. Effective labels and reagents for labeling the probe, as well as instructions for performing the labeling reaction(s), may be obtained in a TaqMan PCR Reagent Kit (Perkin-Elmer). In this embodiment, the labeled probe is added to the PCR reaction in which the FeLV DNA is amplified, before addition of the DNA polymerase (in this method, neither PCR primer need be labelled, e.g., biotinylated). When a fluorescent reported label is used, the PCR reaction is preferably performed in the presence of a fluorescence detector (e.g., a spectrographic fluorescence detector) while being intermittently or continuously bathed in light of a sufficient wavelength to stimulate fluorescence from the reporter label. As the nuclease activity of the DNA polymerase hydrolyses the probe, the fluorescence probe is released so as to allow a fluorescent signal to be detected from the reporter dye. Enough labeled probe is introduced before the start of the PCR reaction to allow labeled probes to anneal to the DNA amplified during each round of PCR. In this way, an increasing fluorescence signal will be detected only when the target DNA recognized by the probe is present and being PCR-amplified. Such a system is the ABI PRISM 7700 Sequence Detection System (available from Perkin-Elmer). In a preferred embodiment of this detection method, the annealed probe will have a Tm of at least about 5 °C higher than that of the annealed primers; effective probes may be obtained from the TaqMan Probe Synthesis Service (Perkin-Elmer). The detection of, e.g., fluorescence, may be taken as a qualitative indication of the presence of viral DNA or the degree of fluorescence may be measured and correlated with the number of copies of viral target DNA present initially in each sample, as a quantitative measurement, e.g., by comparison to a standard curve generated from data obtained from viral DNA calibration standards. TagMan Universal PCR Master Mix. PE Applied Biosystems (1998).

In another embodiment, continuous flow PCR on a microchip utilizing the DNA of the present invention can be used to detect the presence of FeLV. The microchip is designed such that the sample is constantly moving through individual temperature zones allowing melting, annealing and extension of the template DNA and primers. Kopp, M.U. et al., Science 280:1046-1048 (1998). The primers and DNA of the present invention can be used with this method. The amplified product can then be analyzed.

In another embodiment, FeLV U3-containing RNA sequences can be detected by RT-PCR. Although RNA, not DNA is the template, essentially the same DNA primers are used. Either a two-step or one-step PCR protocol can be utilized. In the former, the first step comprises amplification by reverse transcriptase and the second step, for the DNA amplification with taq polymerase. In a one-step RT-PCR, reverse transcriptase and taq polymerase are both present. The amplified DNA can be qualitatively or quantitatively detected by the same methods described for PCR.

It will be appreciated by those skilled in the art that any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products. An assay kit may comprise the primers necessary to amplify the U3-coding region of FeLV. The primers may also be labeled primers. The kit may further comprise other necessary components to allow detection of the amplified DNA product. Alternatively, the kit may comprise labeled probes for detection and quantitation of the DNA product. The kit may further comprise necessary reagents such as, but not limited to, buffers, dNTP's and DNA polymerase. Such kits may also include instructions for detecting FeLV in a DNA sample. Any of the preferred embodiments may be performed as a quantitative test.

One preferred method of doing so is by running calibration standards, containing known numbers of copies of FeLV DNA, concurrently with the samples as described above. The detection data for the standards may then be plotted against known copies of virus to generate a standard curve against which the concentration of virus in the samples may be interpolated.

In will be appreciated that the methods of the present invention may also be used to detect viruses other than FeLV, such as, but not limited to, HIV. The appropriate primers may be designed and after amplification by PCR, the viral genes can be quantitated by the methods of the present invention. It will also be appreciated that the methods of the present invention may be used outside the veterinarian arena, e.g., for testing human DNA samples.

The foregoing and other aspects of the invention may be better understood in connection with the following examples, which are presented for purposes of illustration and not by way of limitation. SPECIFIC EXAMPLE 1

From 98 random-source cats, peripheral blood samples (anti-coagulated) were collected and used in performing ELISA antigen tests and FOCMA antibody tests. Three of the cats tested positive by ELISA (for antigen) and positive by FOCMA (for antibody), four more tested negative by ELISA and positive by FOCMA, and two more were negative by ELISA and FOCMA (for a total of 9 cats). The other 89 cats 41 more were ELISA(-) and FOCMA(+) and 48 were negative both by ELISA and FOCMA.

In order to perform a quantitative version of a preferred method of the present invention, jugular vein blood samples (EDTA-anticoagulated) and femoral bone marrow samples were obtained from all 98 cats. From these samples, white blood cells and bone marrow cell total genomic DNA was extracted using a phenol/chloroform method and the isolated DNA was digested with EcoR I, which does not cut FeLV U3.

PCR reactions were prepared as follows. A set of PCR primers comprising oligonucleotides having the sequence 5'-CAGCAGAAGTTTCAAGGC-3' and oligonucleotides having the sequence 5'-GAGGTTTATTCGTACACGG-3', were synthesized. The 5'-GAGGTTTATTCGTACACGG-3' primers were covalently linked to biotin at their 5' ends, using /?-cyanoethyl phosphoramidite (available from Baron Analytical Services of Milford, CT and Applied Biosystems, Inc. of Foster City, CA). A maximum of three reaction vials were prepared for each sample, depending on the amount of DNA available from the subject. Each reaction vial was loaded with 90 μL of an aqueous mixture that contained: 10 //L of 10x PCR Buffer II (from Perkin-Elmer of Branchburg, NJ); 0.5 μL (i.e. 2.5 units) of Taq Gold DNA Polymerase (also from Perkin-Elmer); 0.2 mM concentrations of each of dATP, dCTP, dGTP, and dTTP (from Pharmacia Biotech, Piscataway, NJ); 3mM MgCI₂; 55 μL distilled deionized H₂O; and 0.25 μM of each of the two primers. To start the reaction, each vial received 100ng of the EcoR l-digested DNA fragment preparation in 10 μL aqueous solution.

Five calibration standards were also prepared as above, but these received 10 μL of either water or a Bfa l-digested DNA fragment preparation made from pCRII plasmids carrying the 210bp FeLV amplification target region whose sequence is illustrated in Figure 1. These vectors had been constructed as follows. A PCR reaction was performed using a feline genomic DNA template and the two PCR primers described above. The resulting amplified 210bp fragments were isolated on a 3% agarose gel and the band containing the fragments was cut from the gel; the fragments were then purified using a QiaQuick Gel Extraction kit (available from Qiagen GmbH of Hilden, Germany). An Original TA Cloning Kit (obtained from the Invitrogen Corp. of San Diego, CA) was used to ligate the fragments into pCRII vectors, transform E. coli bacterial host cells, and culture the transformed cells on LB agar-ampicillin plates, according to the manufacturer's instructions. Plasmids were harvested from a bacterial cell colony which was confirmed to contain the FeLV insert. DNA was extracted from the cellular material using a Plasmid Maxi kit (from Qiagen GmbH) and quantified using a fluorometer (available from Hoefer Pharmacia Biotech, Inc. of San Francisco, CA). The isolated plasmid DNA was then digested with the restriction endonuclease, Bfa I. Each calibration set contained either: water (i.e. no copies of digested vector DNA) or (on average) 22.5 copies, 225 copies, 2,253 copies, or 22,533 copies of digested vector DNA.

After initiation of the reaction, all reaction vials were thermocycled according to the following protocol:

Step #Cvcles Temperature (°C) Time

1. Initial Denaturation 1 94 10 min.

2. Amplification 38

Denaturation 94 15 sec.

Annealing 50 30 sec.

Elongation 72 30 sec.

3. Completion 1 72 5 min.

4. Quenching 1 4

A labeled probe was prepared by synthesizing an oligonucleotide complementary to a U3 region present among the PCR products. This oligonucleotide had the sequence: 5'-CAGCAGTCTCCAGGCTCCCCAGTTGAC-3'.

The oligonucleotide was covalently labeled (using 8-cyanoethyl phosphoramidite), at its 5' end, with a tris(hydroxymethyl) aminomethyl-(2',2'-bipyridine) ruthenium (II) chelate (available from Baron Analytic Services of Milford, CT and Applied Biosystems, Inc. of Foster City, CA). ln order to complex the labeled probe to a complementary region of the PCR product DNA, reaction vials were prepared with 28 μL of 1x PCR Buffer II (from Perkin-Elmer), 0.4 μM of the labeled oligonucleotide probe, and 2 μL of the PCR product DNA, in a total volume of 50 μL. The DNA was denatured at 95°C for 5 minutes and then annealed at 55°C for 15 minutes to allow the probe to hybridize to the DNA. Next, 30 μL of streptavidin-coated Dynal m-450 magnetic beads (from Dynal, Inc. of Great Neck, NY) were added to each reaction vial and the biotin-tagged strands were bound to the immobilized streptavidin at 55°C for 30 minutes. The contents of each vial was then analyzed in a Perkin-Elmer QPCR 5000, DNA-derived electroluminescence detection system, according to manufacturer's instructions, and the average of triplicate luminosity measurements was determined.

The log of measured luminosity for each calibration standard was plotted against the log of FeLV copy number in each to create a standard curve. The copy number for samples was then interpolated from this standard curve, based on each sample's luminosity data. Of the 98 cats studied, eleven tested positive by this quantitative version of the present test: nine by peripheral blood and two by bone marrow alone. These results show that the PCR assay of the present invention is superior to standard FOCMA and ELISA tests for the presence of exogenous FeLV. Moreover, this data shows that the methods of the present invention are able to detect exogenous FeLV infection from peripheral blood and in pre-tumorous cats testing negative for antigen by ELISA as well as in cats testing negative for antibody by FOCMA. Prior art PCR tests have not been able to do this. Thus, the methods of the present invention offer the feature of early detection of FeLV infection from readily obtained tissues such as blood. The methods of the present invention thus represent a significant advancement.

SPECIFIC EXAMPLE 2 In this embodiment, the PCR reaction exploits the 5' nuclease activity of Taq DNA Polymerase to cleave a TaqMan probe (Perkin-Elmer) during PCR. The TaqMan probe contains a reporter dye at the 5' end of the probe and a quencher dye at the 3' end of the probe. During the reaction, cleavage of the probe separates the reporter dye and the quencher dye, which results in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites.

The samples were Bfa /digested DNA fragment preparations made from pCRII plasmids carrying the 210 bp FeLV amplification target region and prepared as described in Specific Example 1. Samples were either digested with E. coli prior to the assay or left undigested. A set of PCR primers comprising oligonucleotides having the sequence 5'-TTCTGCTATAAAACGAGCCATCAG-3' and 5'- GGCGGTCAAGTCTCAGCAAA-3' were synthesized. A probe comprising the oligonucleotides 5'-CCCAACGGGCGCGCAAGT-3' and labeled on the 5' end with a fluorescent reporter and the 3' end with a quencher was also synthesized. Each tube of a Micro-Amp Optical 96-Tube Reaction Format contained a final reaction mixture of 1x PE buffer (from Perkin-Elmer), 3.5 mM MgCI₂, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.4 mM dUTP, 450 nM each primer, 700 nM probe, 0.5 units AmpErase UNG, 1.25 units PE Gold Taq (from Perkin-Elmer), DNA sample templates and dd H₂O to a final volume of 50 μ\. After initiation of the reaction by addition of the DNA sample template, all reaction vials were thermocycled according to the following protocol:

Step # Cycles Temperature (°C) Time

1. Hold 1 50 2 min

2. Hold 1 95 10 min

3. Denaturation 40 95 15 sec

4. Annealing — 60 1 min

The increase in fluorescence due to elongation of the primers and subsequent hydrolysis of the labelled nucleotide, was monitored spectrophotometrically on an ABI Prism 7700 Sequence Detector. The resulting amplified DNA contained 65 bp. A linear response at all DNA sample template concentrations up to 225,330 copies was obtained. The E. coli digested or undigested samples were run in triplicate and results were reproducible, with the minimum number of copies detected at about 22.53 copies. These results show that the PCR assay of the present invention is superior to standard FOCMA and ELISA tests for the presence of exogenous FeLV.

The foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the accompanying drawings and claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims. All references cited herein including literature references are incorporated by reference as if fully set forth.

Claims

What is claimed is:

1. A method for detecting a feline leukemia virus gene in a DNA sample comprising the steps of:

(a) amplifying the DNA sample by polymerase chain reaction to produce polymerase chain reaction products, wherein the primers used in the polymerase chain reaction amplify the U3-coding region; and

(b) detecting the polymerase chain reaction products, thereby detecting a feline leukemia virus gene.

2. The method of Claim 1 , wherein the genomic DNA sample is initially treated with a restriction endonuclease.

3. The method of Claim 2, wherein the restriction endonuclease is EcoRI.

4. The method of Claim 1 , wherein the primers are on opposite ends of the U3-coding region.

5. The method of Claim 1 , wherein the primers comprise a nucleotide sequence of 5'-CAGCAGAAGTTTCAAGGC-3'and 5'-GAGGTTTATTCGTACACGG-3'.

6. The method of Claim 1 , wherein at least one of the primers has a covalently attached label, said label being capable of binding to a second moiety.

7. The method of Claim 6, wherein the label is biotin.

8. The method of Claim 7, wherein detection of the amplified product further comprises:

(a) forming a complex between the amplified products and streptavidin;

(b) isolating the complex; and

(c) quantitating the amount of product from the complex.

9. The method of Claim 8, wherein the streptavidin is immobilized.

10. The method of Claim 8, wherein the streptavidin is covalently labeled with a detectable label.

1 1. The method of Claim 1 , wherein the polymerase chain reaction is hot start PCR.

12. The method of Claim 1 , wherein the detection of the amplified product is quantitative.

13. The method of Claim 1 , wherein the amplified DNA product is detected by the hybridization of a oligonucleotide probe to the amplified product.

14. The method of Claim 13, wherein the probe is a U3-specific probe.

15. The method of Claim 14, wherein the probe comprises a nucleotide sequence of 5'-CAGCAGTCTCCAGGCTCCCCAGTTGAC-3'.

16. The method of Claim 13, wherein the probe is covalently linked to at least one detectable label.

17. The method of Claim 16, wherein the detectable label is selected from the group consisting of fluorescent, luminescent, phosphorescent, colored, colorable, radioactive, immunoreactive, and NMR-detectable labels.

18. The method of Claim 17, wherein the detectable label is a luminescent label.

19. The method of Claim 18, wherein the luminescent label is tris(hydroxymethyl)aminomethyl(2',2'-bipyridine) ruthenium (II).

20. The method of Claim 1 , wherein the amplified product is detected by the binding of a fluorescent probe specific for double stranded DNA.

21. The method of Claim 20, wherein the fluorescent probe is SYBR Green.

22. A method for detecting a feline leukemia virus gene in a DNA sample comprising the steps of:

(a) amplifying the DNA sample by polymerase chain reaction to produce polymerase chain reaction products, wherein the primers used in the polymerase chain reaction amplify the U3-coding region, and a DNA-specific oligonucleotide probe is used in the polymerase chain reaction wherein the probe comprises contiguous nucleotides of a nucleic acid sequence which is between and is exclusive of the primer recognition sequences; and

23. The method of Claim 22, wherein the primers comprise a nucleotide sequence of 5'-TTCTGCTATAAAACGAGCCATCAG-3' and 5'-GGCGGTCAAGTCTCAGCAAA-3'.

24. The method of Claim 22, wherein the probe is a U3-specific probe.

25. The method of Claim 24, wherein the probe comprises a nucleotide sequence of 5'-CCCAACGGGCGCGCAAGT-3'.

26. The method of Claim 24, wherein the probe further comprises:

(a) a fluorescent reporter label covalently linked to the 5' terminus of the probe; and (b) a quenching label covalently linked to the 3' terminus of the probe.

27. A method for detecting a feline leukemia virus gene in an RNA sample comprising the steps of:

(a) translating the RNA to DNA by reverse transcriptase polymerase chain reaction; (b) amplifying the DNA by polymerase chain reaction; and

(c) detecting the amplified DNA, thereby detecting a feline leukemia virus gene.

28. The method of Claim 27, wherein the steps of translation and amplification are performed in one reaction mixture.

29. An assay kit for screening for a feline leukemia virus gene in a DNA sample comprising polymerase chain reaction primers that amplify the DNA of the US- coding region by polymerase chain reaction.

30. The kit of Claim 29, wherein the primers comprise a nucleotide sequence of 5'-CAGCAGAAGTTTCAAGGC-3' and 5'-GAGGTTTATTCGTACACGG-3'.

31. The kit of Claim 29, wherein at least one of the primers has a covalently attached label, said label being capable of binding a second moiety.

32. The kit of Claim 31 , further comprising an immobilized moiety capable of complexing with the primer label.

33. The kit of Claim 29, further comprising a probe for detecting the amplified DNA.

34. The kit of Claim 33, wherein the probe is a U3-specific probe.

35. The kit of Claim 33, wherein the probe is covalently linked to at least one detectable label.

36. The kit of Claim 35, wherein the detectable label is selected from the group consisting of fluorescent, luminescent, phosphorescent, colored, colorable, radioactive, immunoreactive, and NMR-detectable labels.

37. The kit of Claim 29, further comprising a fluorescent label specific for double-stranded DNA for quantitating amplified DNA.

38. The kit of Claim 29, further comprising a DNA-specific oligonucleotide probe, wherein the probe comprises contiguous nucleotides of a nucleic acid sequence which is between and is exclusive of the primer recognition sequences.

39. The kit of Claim 38, wherein the probe is a U3-specific probe.

40. The kit of Claim 39, wherein the probe further comprises:

(a) a fluorescent reporter label covalently linked to the 5' terminus of the probe; and

(b) a quenching label covalently linked to the 3' terminus of the probe.