Assay for detection of jc virus dna

A JC virus, virus technology, applied in the field of nucleic acid detection

Inactive Publication Date: 2014-05-28
BIOGEN MA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Detection of JCV in cerebrospinal fluid (CSF) is confirmatory for PML but is technically challenging

Method used

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  • Assay for detection of jc virus dna
  • Assay for detection of jc virus dna
  • Assay for detection of jc virus dna

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: DNA extraction from cerebrospinal fluid (CSF)

[0063] Materials and Method

[0064] -Modified the QIAamp MinElute Virus Spin Kit (Cat#57704, QIAGEN) protocol for processing human CSF samples. The following buffers are used for DNA extraction

[0065] -Buffer AW2 was prepared by adding 30 mL of ethanol (96%-100%) to a bottle containing 13 mL of buffer AW2 concentrate and mixing well. Store the buffer at ambient temperature.

[0066] -Prepare QIAGEN protease by adding 1.4 mL of buffer AVE to a bottle of lyophilized QIAGEN protease and mixing gently. Store the protease at 2°C-8°C.

[0067] -Carrier RNA solution (1 μg / μL): Prepared by adding 310 μL of buffer AVE to a tube of freeze-dried carrier RNA to prepare a 1 μg / μL solution and mixing by pulse vortexing. The carrier RNA was stored at -20+10°C and not subjected to more than three freeze-thaw cycles. The final concentration of carrier RNA in buffer AL was 5.6 μg / mL. For example, for n samples, [(1.1)×(5.6)×(n)] ...

Embodiment 2

[0076] Example 2: Real-time PCR assay for JCVDNA quantification

[0077] Materials and Method

[0078] Primers and probes are designed for conserved regions of the T antigen gene of the JC virus genome, and BLAST searches are performed to ensure cross-reactivity. The sequences of primers and probes are as follows:

[0079]

[0080] ABI7900HT sequence detection system (Applied Biosystems) was used to perform Taqman real-time quantitative PCR. Use Taqman Universal PCR Master Mix (Applied Biosystems) to run real-time PCR, and prepare each reaction according to the following table:

[0081] Table 1

[0082]

[0083] For each response, in Add 40 μL of the above master mix to 10 μL of DNA eluate on the Optical 96-well reaction plate (Cat#N8010560, Applied Biosystems), and subject it to PCR analysis according to the following steps:

[0084] 1.50℃ for 2 minutes-1 cycle

[0085] 2.95℃ for 10 minutes-1 cycle

[0086] 3.95°C for 15 seconds; 60°C for 1 minute to 50 cycles

[0087] Use JC virus (Ca...

Embodiment 3

[0097] Example 3: Comparison

[0098] The results of the method described in Example 1 were compared with the method described in the "standard" protocol provided by QIAamp MinElute Virus Spin Kit (Cat#57704, Qiagen). See, for example, pages 59-60 of the DNA Mini Kit manual and pages 19-21 of the QIAamp MinElute Virus Spin Kit manual. Different amounts of JC virus DNA copies were added to the CSF sample, and the "standard" protocol and the protocol described in Example 1 were used to isolate the DNA. The RT-PCR protocol described in Example 2 was used to determine the copy number of JC virus DNA in a sample containing the isolated DNA.

[0099] The "standard" extraction method produces an assay sensitivity of 500 copies / mL. The method described in Example 1 produces a detection of 10 copies / mL. (See table below)

[0100] Table 4: Comparison between the method of Example 1 and the standard scheme

[0101]

[0102] Equivalent

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Abstract

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.

Description

[0001] Related application [0002] This application claims the rights and interests of U.S. Provisional Application No. 61 / 513,483 filed on July 29, 2011 in accordance with Section 119(e) of Title 35 of the United States Code. The content of the provisional application is hereby incorporated by reference in its entirety. Invention field [0003] The invention belongs to the field of detection of nucleic acids in biological samples. Background of the invention [0004] JC virus (JCV) is a human polyoma virus known to cause a rare disorder of the central nervous system (CNS) called progressive multifocal leukoencephalopathy (PML). The detection of JCV in cerebrospinal fluid (CSF) is a confirmation of PML, but it is technically challenging. Therefore, there is a need for improved assays for the detection and quantification of JCV in CSF. Summary of the invention [0005] Different aspects of the invention (especially) provide methods and kits for isolating nucleic acids, such as JC v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12P19/34C07H21/00
CPCC12N15/1003C12Q1/701C07H21/00C07H1/06C12N2710/22011C12N15/101C12P19/00
Inventor 索马·雷
Owner BIOGEN MA INC
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