Highly sensitive method for detection of viral hiv dna remaining after antiretroviral therapy of aids patients

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”).

1 . A method for detecting a polynucleotide comprising:
 isolating nucleic acid from a sample;   diluting the isolated nucleic acid in an aqueous solvent to produce a sample in a form suitable for measurement of low frequency electromagnetic emissions from nucleic acid or associated nanostructures in the sample over time;   measuring or detecting said low frequency electromagnetic emissions over time; and   determining the presence of said nucleic acid in the sample by detecting an electromagnetic emission signal (EMS) associated with said polynucleotide;   wherein said signal is not produced by a sample isolated from an otherwise identical source that does not contain the nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein said nucleic acid is DNA produced from an isolated viral polynucleotide or from a biological sample obtained from a subject infected with a virus encoding said viral polynucleotide using PCR or another nucleic acid amplification technique. 
     
     
         3 . The method of  claim 1 , wherein said nucleic acid is DNA produced from a viral polynucleotide isolated from a biological sample selected from the group consisting of blood, plasma, serum, seminal fluid, vaginal fluid, saliva, sweat, urine, and feces of said subject; or wherein said polynucleotide is obtained from a sample of potable water. 
     
     
         4 . The method of  claim 1 , wherein said nucleic acid is viral DNA encoded by human immunodeficiency virus. 
     
     
         5 . The method of  claim 1 , wherein said nucleic acid is viral DNA encoded by a human immunodeficiency virus isolated from a subject who is undergoing ART (anti-retroviral treatment), undergoing treatment with one or more inhibitors of reverse transcriptase, or is viral DNA encoded by a proviral form of a human immunodeficiency virus. 
     
     
         6 . The method of  claim 1 , wherein said nucleic acid is viral DNA encoded by infectious material isolated from a subject infected with human immunodeficiency virus that passes through a 20 nM filter. 
     
     
         7 . The method of  claim 1 , wherein said diluting step dilutes the nucleic acid by about 10 −7  to 10 −13  compared to its original concentration. 
     
     
         8 . The method of  claim 1 , wherein said measuring comprises placing the diluted nucleic acid near an antenna adapted to receive electromagnetic signals having a frequency approaching about 0 Hz to about 20 kHz and receiving the electromagnetic signals from the antenna. 
     
     
         9 . The method of  claim 1 , further comprising performing a time domain to frequency domain transformation on the measured or detected signal, optionally on the signal components of the measured or detected signal having frequencies between about 1 and 20,000 Hz. 
     
     
         10 . The method of  claim 1 , wherein measuring or detecting said low frequency electromagnetic signal emissions over time further comprises transmitting, outputting, displaying, printing, or producing a data structure representing the electromagnetic signal emissions or analyzed electromagnetic signal emissions; or further comprises outputting, displaying, or printing a three dimensional histogram of the electromagnetic signal emissions after Fourier transformation. 
     
     
         11 . The method of  claim 1  for detecting an animal having a pathogenic infection, which comprises:
 a) obtaining a body fluid from an animal suspected of having a pathogenic infection, 
 b) filtering the body fluid to obtain a filtered body fluid, 
 c) serial diluting of the filtered body fluid until obtaining a dilution to test for EMS;
 wherein, the serial diluting comprises multiple cycles of:
 vortexing the filtered body fluid and 
 diluting the filtered body fluid at a dilution of 1:9; 
 
 
 d) measuring an EMS from the diluted body fluid in step d), 
 e) analyzing the EMS, 
 f) determining if the EMS corresponds to an EMS produced by a pathogen. 
 
     
     
         12 . The method of  claim 1  for detecting a reservoir of human immunodeficiency virus in a subject comprising:
 (a) obtaining a sample of body fluid from an animal, 
 filtering the sample, 
 vortexing the sample, 
 diluting the sample at a dilution of 1:9, 
 measuring an EMS from the diluted sample, 
 analyzing the EMS, and 
 determining if the EMS corresponds to HIV virus; 
 (b) obtaining a sample of body fluid from an animal, 
 filtering the sample, 
 serial diluting of the sample until obtaining a dilution to test for EMS; 
 wherein, the serial diluting comprises multiple cycles of:
 vortexing the filtered body fluid and 
 diluting the filtered body fluid at a dilution of 1:9; 
 
 measuring an EMS from the diluted sample, 
 analyzing the EMS, and 
 determining if the EMS corresponds to HIV virus; 
 (c) obtaining a sample of body fluid from an animal, 
 filtering the sample, 
 treating filtered sample with an RNase, 
 vortexing the sample, 
 diluting the sample at a dilution of 1:9, and 
 analyzing diluted sample with RT-PCR; or 
 (d) obtaining a sample of body fluid from an animal, 
 filtering the sample, 
 treating filtered sample with an RNase, 
 serial diluting of the filtered sample with the RNase body until obtaining a dilution to test for EMS; 
 wherein, the serial diluting comprises multiple cycles of:
 vortexing the filtered body fluid and 
 diluting the filtered body fluid at a dilution of 1:9; 
 
 analyzing diluted sample with RT-PCR; 
 (e) obtaining a sample of body fluid from an animal, 
 filtering the sample, 
 treating filtered sample vortexing the sample, 
 diluting the sample at a dilution of 1:9, and 
 analyzing diluted sample using HIV primers with nested PCR; or 
 (f) obtaining a sample of body fluid from an animal, 
 filtering the sample, 
 treating filtered sample, 
 serial diluting of the filtered sample until obtaining a dilution to test for EMS; 
 wherein, the serial diluting comprises multiple cycles of:
 vortexing the filtered body fluid and 
 diluting the filtered body fluid at a dilution of 1:9; 
 
 analyzing diluted sample using HIV primers with nested PCR. 
 
     
     
         13 . The method of  claim 1  for determining the efficacy of a treatment for a pathogenic disease, to determine whether a subject has been cured of human immunodeficiency virus, detecting viral DNA in a subject with undetectable viral RNA, to assess eradication of a viral infection, or to confirm EMS generation by human immunodeficiency virus, respectively, comprising:
 (a) determining efficacy of treatment of a pathogenic infection comprising: 
 measuring an EMS in a person corresponding to an EMS from a pathogenic particle; 
 treating the person with a treatment for which an efficiency is being determined; 
 measuring an EMS in the person treated with the treatment; and 
 determining the relationship between the EMS before treatment and the EMS after treatment; 
 (b) determining whether a subject has been cured of a human immunodeficiency virus infection comprising: 
 measuring an EMS in a person corresponding to an EMS from a HIV virus; 
 treating the person with a treatment for which a cure is expected; and 
 not detecting an EMS in the person corresponding to the EMS from the HIV virus; 
 (c) detecting viral DNA in a patient with undetectable viral RNA comprising: 
 obtaining a sample of body fluid from a patient, 
 filtering the sample, 
 treating filtered sample with an RNase, 
 serial diluting of the filtered sample with the RNase until obtaining a dilution to test for EMS;
 wherein, the serial diluting comprises multiple cycles of:
 vortexing the filtered body fluid and 
 diluting the filtered body fluid at a dilution of 1:9; and 
 
 
 analyzing diluted sample with RT-PCR; 
 (d) assessing eradication of a viral infection based on reduction of viral DNA comprising: 
 measuring an EMS in a person corresponding to an EMS from a viral DNA; 
 treating the person with a treatment for which an efficiency is being determined; 
 measuring an EMS in the person treated with the treatment; and 
 determining the relationship between the EMS before treatment and the EMS after treatment; or 
 (e) confirming EMS generation by human immunodeficiency virus comprising: 
 obtaining a sample from a patient, 
 filtering the sample, 
 treating filtered sample with an RNase, 
 vortexing the sample, 
 diluting the sample at a dilution of 1:9, and 
 amplifying the diluted sample with RT-PCR using a PCR primer for a HIV gene sequence 
 
     
     
         14 . A method for amplifying a DNA sample comprising:
 filtering a sample containing DNA,   optionally, treating said sample with RNase,   diluting or serially diluting the filtered sample, wherein said diluting or serially diluting comprises vigorous agitation or vortexing of the filtered sample, and   amplifying DNA from said diluted sample by a method selected from the group consisting of polymerase chain reaction (PCR), nested-PCR, RT-PCR, nested RT-PCR, and other conventional DNA amplification methods.   
     
     
         15 . The method of  claim 14 , wherein said sample is treated with RNase after filtration. 
     
     
         16 . The method of  claim 14 , wherein said sample is serially diluted by a factor of 1/10 (one part sample to nine parts diluent) to a dilution of 10 −7  to 10 −13  based on the concentration of DNA in the original sample. 
     
     
         17 . The method of  claim 14 , wherein the sample is obtained from a subject infected with a human immunodeficiency virus. 
     
     
         18 . The method of  claim 14 , wherein said sample is material that passes through a 20 nM filter that is isolated from a subject infected with human immunodeficiency virus. 
     
     
         19 . A composition comprising a filtered, vortexed, diluted sample of DNA, prepared according to the method of  claim 1 , wherein the filtered, vortexed, diluted sample of DNA has a detectable electromagnetic signal. 
     
     
         20 . An apparatus to analyze a sample obtained from a subject having a pathogenic infection comprising:
 a sample loading device;   a sample filtering device; a sample diluting device;   a sample vortexing device;   a sample measuring device for EMS;   an EMS analyzer; and   a data display device.