Methods of utilizing the TT virus

Abstract

The present invention relates to nucleic oligomer primers or probes useful for detection of TTV in test samples. Also provided are assays which utilize these primers and probes, as well as test kits which contain these oligomer primers and/or. In addition, the present invention encompasses the use of TTV nucleotide sequences as nucleic acid vectors and as markers for determining transmission between individuals as well as the route thereof. Additionally, the present invention invention encompasses a method of detecting TTV infection prior to xenotransplantation of a tissue or organ.

We claim:  
     
         1 . A probe or primer specific for TT virus selected from the group consisting of SEQ ID NO:29, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70 and SEQ ID NO:71.  
     
     
         2 . The probe or primer of    claim 1   , wherein said probe or primer is selected from the group consisting of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO: 63.  
     
     
         3 . The probe or primer of    claim 1   , wherein said probe or primer is selected from the group consisting of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67.  
     
     
         4 . The probe or primer of    claim 1   , wherein said probe or primer is selected from the group consisting of SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71.  
     
     
         5 . A method for detecting the presence of TTV target nucleotides which may be present in a test sample comprising the steps of: 
 (a) contacting a test sample suspected of containing a target TTV nucleotide sequence with a TTV primer pair consisting of: 1) SEQ ID NO:60 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71, to form a reaction mixture which generates a product;    (b) contacting said reaction mixture with a TTV primer pair consisting of: 1) SEQ ID NO:62 and 2) a primer selected from the group consisting of SEQ ID SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71, wherein the nucleotide sequence of said selected primer hybridizes with said product of said reaction mixture of (a); and    (c) detecting the presence of the TTV target nucleotide in said test sample.    
     
     
         6 . The method of    claim 5    wherein said primer pair of step (a) consists of SEQ ID NO:60 and SEQ ID NO:61, and said primer pair of step (b) consists of SEQ ID NO:62 and SEQ ID NO:63.  
     
     
         7 . A method for detecting the presence of TTV target nucleotides which may be present in a test sample comprising the steps of: 
 (a) contacting a test sample suspected of containing a target TTV nucleotide sequence with a TTV primer pair consisting of: 1) SEQ ID NO:64 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71, to form a reaction mixture which generates a product;    (b) contacting said reaction mixture with a TTV primer pair consisting of: 1) SEQ ID NO:66 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71, wherein the nucleotide sequence of said selected primer hybridizes with said product of said reaction mixture;    (c) detecting the presence of the TTV target nucleotide in said test sample.    
     
     
         8 . The method of    claim 7    wherein said primer pair of step (a) consists of SEQ ID NO:64 and SEQ ID NO:65, and said primer pair of step (b) consists of SEQ ID NO:66 and SEQ ID NO:67.  
     
     
         9 . A method for detecting the presence of TTV target nucleotides which may be present in a test sample comprising the steps of: 
 (a) contacting a test sample suspected of containing a target TTV nucleotide sequence with a TTV primer pair consisting of: 1) SEQ ID NO:68 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO;67, SEQ ID NO:69 and SEQ ID NO:71, to form a reaction mixture which generates a product;    (b) contacting said reaction mixture with a TTV primer pair consisting of: 1) SEQ ID NO:70 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71, wherein the nucleotide sequence of said selected primer hybridizes with said product of said reaction mixture; and    (c) detecting the presence of said target TTV nucleotide in said test sample.    
     
     
         10 . The method of    claim 9    wherein said primer pair of step (a) consists of SEQ ID NO:68 and SEQ ID NO:69, and said primer pair of step (b) consists of SEQ ID NO:70 and SEQ ID NO:71.  
     
     
         11 . The method of claims  5 , 7 or 9 wherein said test sample is isolated from a human or an animal.  
     
     
         12 . A test kit for detecting target TTV nucleotides in a test sample, comprising: 
 (a) a container containing a primer pair specific for a TTV target nucleotide, wherein said primer pair consists of 1) SEQ ID NO:60 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71;    (b) a container containing a primer pair specific for TTV, wherein said primer pair consists of 1) SEQ ID NO:62 and 2) a primer selected from the group consisting of is SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO;69 and SEQ ID NO:71.    
     
     
         13 . The test kit of    claim 12    wherein said primer pair of (a) consists of SEQ ID NO:60 and SEQ ID NO:61, and said primer pair of (b) consists of SEQ ID NO:62 and SEQ ID NO:63.  
     
     
         14 . A test kit for detecting target TTV nucleotides in a test sample, comprising: 
 (a) a container containing a primer pair specific for a TTV target nucleotide, wherein said primer pair consists of 1)SEQ ID NO:64 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65. SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71;    (b) a container containing a primer pair specific for TTV, wherein said primer pair consists of 1) SEQ ID NO:66 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71.    
     
     
         15 . The test kit of    claim 14    wherein said primer pair of (a) consists of SEQ ID NO:64 and SEQ ID NO:65, and said primer pair of (b) consists of SEQ ID NO:66 and SEQ ID NO:67.  
     
     
         16 . A test kit for detecting target TTV nucleotides in a test sample, comprising: 
 (a) a container containing a primer pair specific for a TTV target nucleotide, wherein said primer pair consists of: 1) SEQ ID NO:68 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71;    (b) a container containing a primer pair specific for TTV, wherein said primer pair consists of: 1) SEQ ID NO:70 and 2) a primer selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71.    
     
     
         17 . The test kit of    claim 16    wherein said primer pair of (a) consists of SEQ ID NO:68 and SEQ ID NO:69, and said primer pair of (b) consists of SEQ ID NO:70 and SEQ ID NO:71.  
     
     
         18 . The method of claims  12 ,  14 , or  16  wherein said test sample is isolated from a human or an animal.  
     
     
         19 . A TTV-based vector comprising: 1) a promoter; 2) a heterologous DNA sequence; and 3) a nucleotide sequence encoding TTV, a fragment of said nucleotide sequence or a complement of said nucleotide sequence or said fragment, wherein said heterologous DNA sequence is operably linked to said promoter.  
     
     
         20 . The vector of    claim 19    wherein said promoter is derived from TTV or from a heterologous source.  
     
     
         21 . The vector of    claim 20    wherein said heterologous DNA sequence comprises a polynucleotide sequence that is complementary to a targeted RNA sequence.  
     
     
         22 . The vector of    claim 20    wherein said heterologous DNA sequence encodes a protein.  
     
     
         23 . The vector of    claim 20    wherein said vector is capable of being packaged into TTV particles for stable maintenance or expression of said heterologous DNA sequence.  
     
     
         24 . A host cell comprising said vector of    claim 19   .  
     
     
         25 . The host cell of    claim 24    wherein said cell is eukaryotic.  
     
     
         26 . A method of expressing said heterologous DNA sequence of    claim 19    or a product encoded by said heterologous DNA sequence, in a host, comprising introducing said vector of    claim 19    into said host for a time and under conditions sufficient for expression of said heterologous DNA sequence or product encoded thereby.  
     
     
         27 . A method of detecting transmission of TTV from one individual to another comprising the steps of: 
 (a) obtaining a biological sample from an individual having TTV;    (b) isolating a TTV DNA sequence from said biological sample;    (c) obtaining a biological sample from a second individual having TTV;    (d) isolating a TTV DNA sequence from said biological sample of said second individual;    (e) comparing said TTV DNA sequence of said first individual with said TTV DNA sequence of said second individual, identity between said DNA sequence of said first individual and said DNA sequence of said second individual indicating transmission of TTV from one individual to the other.    
     
     
         28 . A method of determining TTV-infection in a tissue or organ prior to transplantation or xenotransplantation of said tissue or organ comprising the steps of: 
 (a) contacting a biological sample suspecting of containing a TTV target nucleotide sequence, from a potential donor human or animal, with a TTV primer pair represented by SEQ ID NO:60 and SEQ ID NO:61 to form a first reaction mixture;    (b) contacting said reaction mixture with a TTV primer pair represented by SEQ ID NO:62 and SEQ ID NO:63 in order to form a second reaction mixture; and    (c) detecting the presence of the TTV target nucleotide in said test sample, presence of said nucleotide indicating TTV-infection in said biological sample and in said tissue or organ.    
     
     
         29 . A method of determining TTV-infection in a tissue or organ prior to transplantation or xenotransplantation of said tissue or organ comprising the steps of: 
 (a) contacting a biological sample suspecting of containing a TTV target nucleotide sequence, from a potential donor human or animal, with a TTV primer pair represented by SEQ ID NO:64 and SEQ ID NO:65, to form a first reaction mixture;    (b) contacting said reaction mixture with a TTV primer pair represented by SEQ ID NO:66 and SEQ ID NO:67; and    (c) detecting the presence of the TTV target nucleotide in said test sample, presence of said nucleotide indicating TTV-infection in said biological sample and in said tissue or organ.    
     
     
         30 . A method of determining TTV-infection in a tissue or organ prior to transplantation or xenotransplantation of said tissue or organ comprising the steps of: 
 (a) contacting a biological sample suspecting of containing a TTV target nucleotide sequence, from a potential donor human or animal, with a TTV primer pair represented by SEQ ID NO:68 and SEQ ID NO:69, to form a first reaction mixture;    (b) contacting said reaction mixture with a TTV primer pair represented by SEQ ID NO:70 and SEQ ID NO:71; and    (c) detecting the presence of the TTV target nucleotide in said test sample, presence of said nucleotide indicating TTV-infection in said biological sample and in said tissue or organ.    
     
     
         31 . The method of claims  28 ,  29 , or  30  wherein said biological sample is selected from the group consisting of blood, tissue and an organ.  
     
     
         32 . A method of detecting the presence of target TTV nucleotides in a test sample, comprising the steps of: 
 (a) contacting a test sample suspecting of containing a target TTV nucleotide with a primer pair represented by SEQ ID NO:60 and SEQ ID NO:61, to form a reaction mixture;    (b) contacting said reaction mixture with at least one TTV probe selected from the group consisting of SEQ ID NO:62 and SEQ ID NO:63; and    (c) detecting the presence of said target TTV nucleotide in said test sample.    
     
     
         33 . The method of    claim 32   , wherein said at least one probe is conjugated to a detectable signal-generating compound.  
     
     
         34 . The method of    claim 33    wherein said detectable signal generating compound is selected from the group consisting of a chemiluminescent compound, fluorescein and an enzyme.  
     
     
         35 . The method of    claim 32    wherein said at least one probe is conjugated to an antibody.  
     
     
         36 . A method of detecting the presence of target TTV nucleotides in a test sample, comprising the steps of: 
 (a) contacting a test sample suspecting of containing a target TTV nucleotide with a primer pair represented by SEQ ID NO:64 and SEQ ID NO:65 to form a reaction mixture;    (b) contacting said reaction mixture with at least one TTV probe selected from the group consisting of SEQ ID NO:66 and SEQ ID NO:67; and    (c) detecting the presence of said target TTV nucleotide in said test sample.    
     
     
         37 . The method of    claim 36   , wherein said at least one probe is conjugated to a detectable signal-generating compound.  
     
     
         38 . The method of    claim 37    wherein said detectable signal generating compound is selected from the group consisting of a chemiluminescent compound, fluorescein and an enzyme.  
     
     
         39 . The method of    claim 36    wherein said at least one probe is conjugated to an antibody.  
     
     
         40 . A method of detecting the presence of target TTV nucleotides in a test sample, comprising the steps of: 
 (a) contacting a test sample suspecting of containing a target TTV nucleotide with a primer pair represented by SEQ ID NO:68 and SEQ ID NO:69 to form a reaction mixture;    (b) contacting said reaction mixture with at least one TTV probe selected from the group consisting of SEQ ID NO:70 and SEQ ID NO:71; and    (c) detecting the presence of said target TTV nucleotide in said test sample.    
     
     
         41 . The method of    claim 40   , wherein said at least one probe is conjugated to a detectable signal-generating compound.  
     
     
         42 . The method of    claim 41   , wherein said at least one probe is conjugated to a detectable signal-generating compound.  
     
     
         43 . The method of    claim 40   , wherein said at least one probe is conjugated to a detectable signal-generating compound.  
     
     
         44 . A method of detecting TTV target nucleotides which may be present in a test sample comprising: 
 (a) contacting said test sample suspected of containing a target TTV nucleotide sequence with a TTV primer pair consisting of 1) a primer selected from the group consisting of: SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, and SEQ ID NO:70, and 2) a primer selected from the group consisting: SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, and SEQ ID NO:71, to form a reaction mixture which generates a product;    (b) contacting said reaction mixture with a TTV primer pair consisting of: 1) a primer selected from the group consisting of: SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, and SEQ ID NO:70, wherein the nucleotide sequence of said selected primer hybridizes with said product of said reaction mixture and 2) a primer selected from the group consisting: SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, and SEQ ID NO:71, wherein the nucleotide sequence of said selected primer hybridizes with said product of said reaction mixture, and (c) detecting the presence of the TTV target nucleotide in said test sample.