Kits for diagnosis and monitoring of viral infection by analysis of viral transrenal nucleic acids in urine
The present invention relates to methods for diagnosis or monitoring of viral infection by detecting the presence of transrenal viral nucleic acids or nucleic acids of viral origin in urine sample, with or without isolation of nucleic acids from a urine sample. The analysis of the nucleic acids is performed through hybridization of the nucleic acids with specific probes, or through a chain amplification reaction with specific primers. The methods are applicable to all viral pathogenic agents, including RNA, DNA, episomal, or integrated viruses.
1. A kit, comprising:
a) two or more reagents or materials for isolation and purification of cell-free nucleic acids of a virus from a urine sample of a subject, wherein said reagents or materials comprise a matrix including a silica-based resin, wherein said silica-based resin isolates nucleic acids that are less than 300 nucleotides and wherein said reagents or materials comprise an agent that inhibits the degradation of said cell-free nucleic acids; and
b) two or more primers for detecting said cell-free nucleic acids of a virus in said urine sample, wherein the two or more primers comprise a forward primer consisting of SEQ ID NO: 5 and a reverse primer that is selected from the group consisting of SEQ ID NOs: 6-8.
2. The kit according to claim 1 , further comprising reagents for performance of a technique selected from the group consisting of hybridization of nucleic acids, cycling probe reaction, polymerase chain reaction, nested polymerase chain reaction, ligase chain reaction, and strand displacement amplification,
3. The kit according to claim 2 , further comprising primers that are specific for nucleic acids that encode a viral agent.
4. The kit according to claim 1 , further comprising a chaotropic agent.
5. The kit according to claim 1 , further comprising agents selected from the group consisting of ion-chelating agents, denaturing agents, and ionic detergents.
6. The kit according to claim 5 , wherein said ion-chelating agent is EDTA; said denaturing agent is selected from the group consisting of guanidine HC 1 and guanidine isothiocyanate; or said ionic detergent is selected from the group consisting of N-lauryl sarcosine and sodium dodecyl sulfate.
7. A kit comprising:
a) two or more reagents or materials for the detection and quantitation of cell-free nucleic acids of a virus from a urine sample of a subject, wherein said reagents or materials comprise a matrix including a silica-based resin, wherein said silica-based resin isolates nucleic acids that are less than 300 nucleotides and wherein said reagents or materials comprise an agent that inhibits the degradation of said cell-free nucleic acids in urine; and
b) two or more primers for detecting and quantitating said cell-free nucleic acids of a virus in said urine sample, wherein the two or more primers comprise a forward primer consisting of SEQ ID NO: 5 and a reverse primer that is selected from the group consisting of SEQ ID NOs: 6-8.
8. The kit according to claim 7 further comprising reagents for performance of a technique selected from the group consisting of hybridization of the nucleic acids, cycling probe reaction, polymerase chain reaction, nested polymerase chain reaction, ligase chain reaction, and strand displacement amplification.
9. The kit according to claim 7 , further comprising a chaotropic agent.
10. The kit according to claim 7 , wherein said agents are selected from the group consisting of ion-chelating agents, denaturing agents, and ionic detergents.
11. The kit according to claim 10 , wherein said ion-chelating agent is EDTA; said denaturing agent is selected from the group consisting of guanidine HCl and guanidine isothiocyanate; or said ionic detergent is selected from the group consisting of N-lauryl sarcosine and sodium dodecyl sulfate.