ASSAY FOR DETECTION OF JC VIRUS DNA
In one aspect, the disclosure provides methods for isolating nucleic acid from a Cerebrospinal Fluid (CSF) sample. In one aspect, the disclosure provides methods for determining the amount of JC virus DNA in a sample.
1 . A method for isolating nucleic acid from a Cerebrospinal Fluid (CSF) sample, the method comprising:
adding carrier nucleic acid and protease to a CSF sample,
incubating the sample comprising the carrier nucleic acid and the protease,
applying the incubated sample to a nucleic acid binding column,
washing the column to which the sample was applied, and
applying eluent to the column resulting in the isolation of the nucleic acid.
2 . The method of claim 1 , wherein the volume of the CSF sample is at least 1 ml.
3 . The method of claim 1 , wherein the carrier nucleic acid is carrier RNA.
4 . The method of claim 3 , wherein the resulting concentration of the carrier RNA in the CSF sample is 2.8 microgram/ml or less.
5 . The method of claim 1 , wherein incubating the sample comprises a first step of incubating the sample at room temperature (RT) and a second step of incubating the sample at a temperature that is above RT.
6 . The method of claim 5 , wherein the incubating steps are 15 minutes long.
7 . The method of claim 5 , wherein the temperature above RT is 56° C.
8 . The method of claim 1 , wherein washing the column comprises adding a washing buffer to the column and spinning the column at a centrifugal force of 4000 g.
9 . The method of claim 1 , wherein applying eluent comprises applying the eluent to the column for at least two times.
10 . The method of claim 1 , wherein the eluent is incubated on the column for 5 minutes.
11 . The method of claim 1 , wherein 30 microliter of eluent is applied.
12 . The method of claim 1 , wherein the nucleic acid in the CSF sample is DNA.
13 . The method of claim 12 , wherein the DNA is viral DNA.
14 . The method of claim 13 , wherein the viral DNA is JC virus DNA.
15 . The method of claim 14 , further comprising performing a real-time polymerase chain reaction (Real-time PCR) to determine the amount of JC virus DNA.
16 . The method of claim 15 , wherein the Real-time PCR primers and probe are directed to the JC virus T antigen.
17 . The method of claim 16 , wherein the sequences of the Real-time PCR primers and probe are SEQ ID NOs:1-2 and SEQ ID NO:3, respectively.
18 . A method for determining the amount of JC virus DNA in a sample, the method comprising:
performing Real-time PCR on the sample, wherein the Real-time PCR primers and probe are directed to the JC virus T antigen.
19 . The method of claim 18 , wherein the sequences of the Real-time PCR primers and probe are SEQ ID NOs:1-2 and SEQ ID NO:3, respectively.
20 . A kit for isolating nucleic acid from a Cerebrospinal Fluid (CSF) sample, the kit comprising a protease, carrier nucleic acid, a nucleic acid binding column and instructions for use.
21 - 25 . (canceled)