Diagnostic assays for parvovirus B19

Abstract

Human parvovirus B19 primers and probes derived from conserved regions of the parvovirus B19 genome are disclosed. Also disclosed are nucleic acid-based assays using the primers and probes.

1 . A method for detecting human parvovirus B19 in a biological sample, the method comprising: 
 isolating nucleic acids from a biological sample suspected of containing human parvovirus;    amplifying the nucleic acids using a sense and an antisense primer wherein each of the primers is not more than 60 nucleotides in length and is sufficiently complementary to a portion of the sense and antisense strands, respectively, of the isolated nucleic acid to hybridize therewith; and    (a) the sense primer comprises SEQ ID NO:60 or a nucleotide sequence having at least 90% sequence identity thereto;    (b) the antisense primer comprises SEQ ID NO:59 or a nucleotide sequence having at least 90% sequence identity thereto; and    detecting the presence of the amplified nucleic acids as an indication of the presence of human parvovirus B19 in the sample,    wherein amplifying uses a fluorogenic 5′ nuclease assay using the sense primer and the antisense primer and detecting is done using at least one probe comprising a detectable label.    
     
     
         2 . The method of  claim 1 , wherein the nucleic acids are isolated from the biological sample by a method comprising: 
 (a) contacting a solid support comprising capture nucleic acids associated therewith with the biological sample under hybridizing conditions wherein target nucleic acid strands, if present in the biological sample, hybridize with the capture nucleic acids; and    (b) separating the solid support from the sample.    
     
     
         3 . The method of  claim 2 , wherein the solid support comprises beads.  
     
     
         4 . The method of  claim 3 , wherein the beads are magnetic beads.  
     
     
         5 . The method of  claim 2 , wherein the capture nucleic acids further comprise a homopolymer chain to link the capture nucleic acids to the solid support.  
     
     
         6 . The method of  claim 5 , wherein the homopolymer chain is a polyA chain.  
     
     
         7 . The method of  claim 6 , wherein the capture nucleic acids comprise one or more oligonucleotides selected from the group consisting of SEQ ID NOS:49-55 and a nucleotide sequence having at least 90% sequence identity to SEQ ID NOS:49-55.  
     
     
         8 . The method of  claim 1 , wherein the detectable label is a fluorescent label selected from the group consisting of 6-carboxyfluorescein (6-FAM), tetramethyl rhodamine (TAMRA), and 2′, 4′, 5′, 7′,-tetrachloro-4-7-dichlorofluorescein (TET).  
     
     
         9 . The method of  claim 8 , wherein the probe further comprises detectable labels at the 5′-end and at the 3′-end.  
     
     
         10 . The method of  claim 9 , wherein the at least one probe is not more than 50 nucleotides in length and comprises at least 10 nucleotides from SEQ ID NO:61.  
     
     
         11 . The method of  claim 1 , wherein an internal control sequence is present.  
     
     
         12 . The method of  claim 11 , wherein the internal control sequence is derived from the nucleotide sequence of SEQ ID NO:92.  
     
     
         13 . The method of  claim 12 , further comprising a detectably labeled probe sequence for the internal control sequence.  
     
     
         14 . A method for detecting human parvovirus B19 in a biological sample using a fluorogenic 5′ nuclease assay, the method comprising: 
 (a) isolating nucleic acids from a biological sample suspected of containing human parvovirus B19 by contacting magnetic beads comprising capture nucleic acids associated therewith with the biological sample under hybridizing conditions wherein target nucleic acid strands, if present in the biological sample, hybridize with the capture nucleic acids, wherein the capture nucleic acids comprise one or more oligonucleotides selected from the group consisting of SEQ ID NOS:49-55, and separating the magnetic beads from the sample;    (b) amplifying the nucleic acids using a sense and an antisense primer wherein each of the primers is not more than 60 nucleotides in length and is sufficiently complementary to a portion of the sense and antisense strands, respectively, of the isolated nucleic acid to hybridize therewith, wherein the sense primer comprises SEQ ID NO:60 and the antisense primer comprises SEQ ID NO:59; and    (c) detecting the presence of the amplified nucleic acids using a probe not more than 50 nucleotides in length and that comprises the nucleotide sequence of SEQ ID NO:61, as an indication of the presence of human parvovirus B19 in the sample.    
     
     
         15 . The method of  claim 14 , wherein an internal control sequence is present.  
     
     
         16 . The method of  claim 15 , wherein the internal control sequence is derived from the nucleotide sequence of SEQ ID NO:92.  
     
     
         17 . The method of  claim 16 , further comprising a detectably labeled probe sequence for the internal control sequence.  
     
     
         18 . The method of  claim 7 , wherein the capture nucleic acids comprise the oligonucleotide of SEQ ID NO:55.  
     
     
         19 . The method of  claim 18 , wherein the capture nucleic acids comprise the oligonucleotide of SEQ ID NO:55 and one or more of the oligonucleotides of SEQ ID NOS:49-54.  
     
     
         20 . The method of  claim 19 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:49 and 55.  
     
     
         21 . The method of  claim 19 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:50 and 55.  
     
     
         22 . The method of  claim 19 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:51 and 55.  
     
     
         23 . The method of  claim 19 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:52 and 55.  
     
     
         24 . The method of  claim 19 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:53 and 55.  
     
     
         25 . The method of  claim 19 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:54 and 55.  
     
     
         26 . The method of  claim 19 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:49, 52, 53 and 55.  
     
     
         26 . The method of  claim 14 , wherein the capture nucleic acids comprise the oligonucleotide of SEQ ID NO:55 and one or more of the oligonucleotides of SEQ ID NOS:49-54.  
     
     
         27 . The method of  claim 26 , wherein the capture nucleic acids comprise the oligonucleotides of SEQ ID NOS:49, 52, 53 and 55.  
     
     
         28 . A kit for detecting human parvovirus B19 in a biological sample, the kit comprising: 
 capture nucleic acids comprising one or more oligonucleotides selected from the group consisting of SEQ ID NOS:49-55;    a pair of primer oligonucleotides wherein each of the primer oligonucleotides is not more than 60 nucleotides in length and one of the primer oligonucleotides comprises SEQ ID NO:60 and the other primer oligonucleotide comprises SEQ ID NO:59; and    written instructions for identifying the presence of human parvovirus B19.    
     
     
         29 . The kit of  claim 28 , further comprising a polymerase and buffers.  
     
     
         30 . The kit of  claim 28 , further comprising at least one probe oligonucleotide comprising a detectable label.  
     
     
         31 . The kit  claim 30 , wherein the detectable label is a fluorescent label selected from the group consisting of 6-carboxyfluorescein (6-FAM), tetramethyl rhodamine (TAMRA), and 2′, 4′, 5′, 7′,-tetrachloro-4-7-dichlorofluorescein (TET).  
     
     
         32 . The kit of  claim 31 , wherein the probe further comprises detectable labels at the 5′-end and at the 3′-end.  
     
     
         33 . The kit of  claim 32 , wherein the at least one probe is not more than 50 nucleotides in length and comprises the nucleotide sequence of SEQ ID NO:61.  
     
     
         34 . The kit of  claim 28 , further comprising an internal control derived from the nucleotide sequence of SEQ ID NO:92.