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Highly sensitive method for detection of viral HIV DNA remaining after antiretroviral therapy of aids patients

a hiv dna and antiretroviral therapy technology, applied in the field of ##ds for detecting polynucleotides, can solve the problem that the classical inhibitors used in art cannot achieve the eradication of viral infection, and achieve the effect of improving the sensitivity of pcr

Inactive Publication Date: 2013-06-06
MONTAGNIER LUC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]A method of improving the sensitivity of PCR by 10 to 100 times by processing samples with serial dilutions (1 / 10 at each step) and vigorous vortexing between each dilution step. Additionally, RNase treatment of the filtered original sample can be combined with the serial dilution process.

Problems solved by technology

This would explain why the classical inhibitors used in ART cannot achieve eradication of the viral infection.

Method used

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  • Highly sensitive method for detection of viral HIV DNA remaining after antiretroviral therapy of aids patients
  • Highly sensitive method for detection of viral HIV DNA remaining after antiretroviral therapy of aids patients
  • Highly sensitive method for detection of viral HIV DNA remaining after antiretroviral therapy of aids patients

Examples

Experimental program
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Effect test

example 1

Measurement of Electromagnetic Signals

[0045]The plasma or DNA solution [1-4 ng / ml] is dissolved in Phosphate Buffered Saline (PBS) at the concentration of 10−2, then filtered on Millipore 0.45 micrometer filter and the filtrate is refiltered on Anotop Whatman filter of 20 nanometer porosity. The filtrate is then diluted in distilled water in 1.5 ml Eppendorf conical plastic tubes in serial 1 part sample:9 parts diluent [decimal] dilutions ranging from 10−2 to 10−15 and strongly agitated on a vortex for at least 15 seconds.

[0046]Plasma is prepared by centrifugation of heparinized blood of patients presenting with conditions of: 1) Asymptomatic, untreated; 2) Symptomatic, not yet treated, with high virus load; or 3) Symptomatic, treated by antiretroviral therapy with no detectable virus load by commercial kits (<200 RNA copies / ml).

[0047]EMS was only detected in the plasma of the third category (30 out of 30), in plasma dilutions ranging from 10−5 to 10−8. Results with the two first ca...

example 2

The Decay with Time of EMS Production in Plasma Stored at +4° C.

[0050]The capacity to emit EMS in plasma can last for several days, sometimes for several weeks of storage, indicating a relative stability of the nanostructures that emit EMS in the plasma proteinic environment. In vitro studies indicated that filtration of the plasma (usually at the 1 / 100 dilution in PBS or saline) through 20 nM filters was a prerequisite for detecting the signals in further dilutions of water. In some rare cases, weaker signals can be detected at lower dilutions after filtration through 100 nM porosity filters. Positive signals were usually found in the range of the 10−3 to 10−9 dilutions.

example 3

Evidence that Positive Signals Come from DNA

[0051]Experiments were conducted to determine if nucleic acids carrying the genetic information for HIV, either residual viral RNA or proviral DNA, could be the sources of signals in the plasma of infected patients. Three groups of patients: infected and not treated in the asymptomatic stage; infected and not treated in the symptomatic stages; and infected and treated with ART with no detectable viral load.

[0052]Plasma was diluted 1 / 100 in PBS and the nucleic acids were extracted by the phenol-chloroform method. The solution was precipitated with ethanol and the precipitates were solubilized in water. The solution was filtered through a 20 nM filter at a concentration ranging from 1 ng / ml to 4 ng / ml.

[0053]EMS emissions were detected only in the group of patients treated by antiretroviral therapy and having an undetectable virus load. The signals were produced in the same range of aqueous dilutions than fresh plasma. Filtration of the origi...

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Abstract

Methods for detecting polynucleotides, especially the DNA replicated from samples obtained from subjects infected with pathogenic viruses such as human immunodefiency virus, by detecting electromagnetic signals (“EMS”) emitted by such polynucleotides, and methods for improving the sensitivity of the polymerase chain reaction (“PCR”).

Description

CROSS-REFERENCE TO RELATED APPLICATION(S)[0001]This application claims priority under 35 U.S.C. §120 to U.S. Ser. No. 12 / 797,826, filed Jun. 10, 2010 and under 35 U.S.C. §119(e) to U.S. Provisional 61 / 186,610, filed Jun. 12, 2009, each of which is incorporated by reference in its entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]Methods for detecting polynucleotides, especially the DNA replicated from samples obtained from subjects infected with pathogenic viruses such as human immunodeficiency virus, by detecting electromagnetic signals (“EMS”) emitted by such polynucleotides, and methods for improving the sensitivity of the polymerase chain reaction (“PCR”).[0004]Electromagnetic signals of low frequency have been shown to be produced in aqueous dilutions by Human Immunodeficiency Virus DNA. In vivo, HIV DNA signals are detected only in patients previously treated by antiretroviral therapy and having no detectable viral RNA copies in their blood. It is sugges...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N27/327C12P19/34
CPCC12Q1/6806C12Q1/6816C12Q1/703G01N37/005G01N27/3275C12P19/34C12Q2523/303C12Q2527/146
Inventor MONTAGNIER, LUC
Owner MONTAGNIER LUC
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