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Detection of polyomavirus

A polyoma virus, a technology for detecting samples, applied in the field of detection, can solve problems such as hindering the development of reliable methods for detecting infection

Inactive Publication Date: 2011-05-18
岩城公辅
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, a major obstacle in the development of efficient assays for polyomaviruses is the large number of intraspecific polymorphisms in the BKV and JCV nucleotide sequences
To date, insertions and deletions of nucleotide polymorphisms (such as SNPs) have hampered the development of reliable methods for detecting infection

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] The detection of embodiment 1-polyomavirus

[0086] The real-time amplification assay was performed using primers SEQ ID NO:2 and SEQ ID NO:3 and probes SEQ ID NO:4 and SEQ ID NO:5. The assay includes DNA amplification by polymerase chain reaction (PCR) and real-time detection using a fluorescently labeled donor probe of SEQ ID NO: 4 and an LC610-labeled acceptor probe of SEQ ID NO: 5, which The needles are designed to specifically hybridize to BKV DNA under stringent conditions. Different concentrations of BKV DNA and JCV DNA were detected together with a negative control containing no DNA samples.

[0087] In LightCycler Real-time PCR amplification was performed on a 480 PCR machine (Roche, Basel, Switzerland), and data analysis was performed using LCS480 version 1.2.9.11 software provided by the manufacturer. Reagents from Roche (Basel, Switzerland) were used for all reactions. Each 20-μl PCR reaction contained 1× Fast-Start Hyb Probe mixed master solution (R...

Embodiment 2

[0093] Example 2 - Evaluation of samples containing BKV and JCV

[0094] Samples containing BKV and JCV were evaluated using the general conditions described in Example 1. Well D1 is a negative control that does not contain viral DNA. Microwells D2 to D6 contain 8×10 5 Copy, 8×10 4 Copy, 8×10 3 Copy, 8×10 2 Copy and 8×10 1 Copies of BKV DNA. Microwells D7 to D12 are duplicates of microwells D1 to D6, respectively. Well E1 is a negative control that does not contain viral DNA. Microwells E2 to E6 respectively contained BKV DNA and JCV DNA at a ratio of 1:1 at the following concentrations, E2:10 5 BKV DNA copies and 10 5 JCV DNA copy; E3:10 4 BKV DNA copies and 10 4 JCV DNA copy; E4:10 3 BKV DNA copies and 10 3 JCV DNA copy; E5:10 2 BKV DNA copies and 10 2 JCV DNA copy; E6:10 1 BKV DNA copies and 10 1 A copy of the JCV DNA. Microwells E7 to E12 are duplicate wells of microwells E1 to E6, respectively.

[0095] Figure 4 , Figure 5 as well as Figure 6 Am...

Embodiment 3

[0099] Example 3 - Clinical Trial

[0100] The following primer / probe combinations were used for routine clinical sample testing: combination 1 consisting of primers SEQ ID NO: 6 and SEQ ID NO: 4 and probe sequence SEQ ID NO: 14; combination 1 consisting of primers SEQ ID NO: 6 and SEQ ID NO: 6 and SEQ ID NO: Combination 2 consisting of ID NO: 2 and probe sequence SEQ ID NO: 14; combination 3 consisting of primer BKV_5.2 and SEQ ID NO: 4 and probe sequence SEQ ID NO: 14; and combination 3 consisting of primer BKV_5.2 and Combination 4 consisting of SEQ ID NO: 2 and the probe sequence SEQ ID NO: 14.

[0101] The PCR reaction had a final reaction volume of 40 μl: 10 μl of sample and 30 μl of mixed master mix. For a total volume of 40 μl per sample microwell, the mixed mother liquor composition (30 μl) contained a concentration of 3.125 μM of forward primer, a concentration of 3.125 μM of reverse primer, a concentration of 2.0 to 2.5 μM of the MGB Taqman probe, 20 μl LightCyc...

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Abstract

Methods and kits are provided for testing for the presence or absence of a polyomavirus, such as BKV, in a sample. The methods and kits are useful for quantifying BKV and differentiating BKV from JCV.

Description

[0001] Cross references to related patent applications [0002] This application claims the benefit of the priority date of U.S. Provisional Patent Application Serial No. 61 / 064,166, filed February 20, 2008, which is incorporated herein by reference in its entirety. Background technique [0003] Human polyomaviruses JC and BK are widespread in the human population. Primary infection with these viruses is usually asymptomatic and can lead to transient viruria. Both JC virus (JCV) and BK virus (BKV) establish a latent period in kidney tissue and B lymphocytes following primary infection (G. Lecatsas, B.D. Schoub, A.R. Rabson, and M. Jfe, Letter, Lancet 2:907 -908, 1976). Polyomavirus-associated disease is primarily associated with immune impairment, and rapid detection and differentiation of pathogens in immunocompromised patients is important to aid clinical management. JCV is the causative agent of the neurological disease progressive multifocal leukoencephalopathy, which ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70
CPCC12Q1/701C12Q1/6888C12R2001/91
Inventor 岩城公輔
Owner 岩城公辅
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