Abstract:
A method for detecting electromagnetic waves derived from bacterial DNA, comprising extracting and purifying nucleic acids from a sample; diluting the extracted purified nucleic acids in an aqueous solvent; measuring a low frequency electromagnetic emission over time from the diluted extracted purified nucleic acids in an aqueous solvent; performing a signal analysis of the low frequency electromagnetic emission over time; and producing an output, based on the signal analysis, in dependence on the DNA in the sample. The DNA may be extracted from at least one of blood, feces, urine, saliva, tears, seminal fluid, sweat, seminal and vaginal fluids of a patient, or water to determine, e.g., potability. The samples may be frozen. The extracting and purifying may comprise diluting the sample with an aqueous buffer and mixing; degrading proteins in the diluted sample; precipitating DNA from the buffer solution; and resuspending the precipitated DNA in an aqueous solution.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     The present application is a Continuation of Ser. No. 12/560,772, filed Sep. 16, 2009, issued Mar. 26, 2013 as U.S. Pat. No. 8,405,379, which claims benefit of priority from U.S. Provisional Patent Application No. 61/098,405, filed Sep. 9, 2008, and U.S. Provisional Patent Application No. 61/098,227, filed Sep. 18, 2008, each of which is expressly incorporated herein by reference in its entirety. 
    
    
     BACKGROUND OF THE INVENTION 
     In previous patent applications, U.S. patent application Ser. No. 12/097,204, and PCT/FR2007/001042, filed Jun. 22, 2007, each of which expressly incorporated herein by reference in their entirety, the present applicant has described a new method for detecting some low electromagnetic frequency electromagnetic signals in diluted filtrates of the culture medium of certain bacteria and viruses, as well as in diluted plasma of patients infected by the same agents. The electromagnetic signals (EMS) were believed to be produced by certain defined nanostructures induced by the microorganisms, in high dilutions of in water, the nanostructures being previously removed by filtration. 
     See (each of which is expressly incorporated herein by reference):
     U.S. Pat. No. 6,541,978, WO 00/17638 A (Digibio; Benveniste, Jacques; Guillonnet, Didier) 30 Mar. 2000 (2000 Mar. 30).   U.S. Ser. No. 09/787,781, WO 00/17637 A (Digibio; Benveniste, Jacques; Guillonnet, Didier) 30 Mar. 2000 (2000 Mar. 30).   U.S. Ser. No. 09/720,634, WO 00/01412 A (Digibio; Benveniste, Jacques; Guillonnet, Didier) 13 Jan. 2000 (2000 Jan. 13).   FR 2,811,591 A (Digibio) 18 Jan. 2002 (2002 Jan. 18).   FR 2,700,628 A (Benvenistre Jacques) 22 Jul. 1994 (1994 Jul. 22).   Benveniste J. et al: “Remote Detection Of Bacteria Using An Electromagnetic/Digital Procedure”, Faseb Journal, Fed. Of American Soc. For Experimental Biology, Bethesda, Md., US, No. 5, Part 2, 15 Mar. 1999 (1999 Mar. 15), page A852, XP008059562 ISSN: 0892-6638.   Thomas et al: “Activation Of Human Neutrophils By Electronically Transmitted Phorbol-Myristate Acetate” Medical Hypotheses, Eden Press, Penrith, US, vol. 54, no. 1, January 2000 (2000-01), pages 33-39, XP008002247, ISSN: 0306-9877.   Benveniste J. et al.: “Qed And Digital Biology” Rivista Di Biologia, Universita Degli Studi, Perugia, IT, vol. 97, no. 1, January 2004 (2004-01), pages 169-172, XP008059428 ISSN: 0035-6050.   Benveniste J. et al.: “A Simple And Fast Method For In Vivo Demonstration Of Electromagnetic Molecular Signaling (Ems) Via High Dilution Or Computer Recording” FASEB Journal, Fed. Of American Soc. For Experimental Biology, Bethesda, Md., US, vol. 13, no. 4, Part 1, 12 Mar. 1999 (1999 Mar. 12), page A163, Abstr. No. 016209, XP008037356 ISSN: 0892-6638.   Benveniste J: “Biological effects of high dilutions and electromagnetic transmission of molecular signal” [Progress In Neonatology; 25th National Conference Of Neonatology] S. Karger Ag, P.O. Box, Allschwilerstrasse 10, CH-4009 Basel, Switzerland; S. Karger Ag, New York, N.Y., USA Series: Progres En Neonatologie (ISSN 0251-5601), 1995, pages 4-12, XP009070841; and 25ES Journees Nationales De Neonatologie; Paris, France; May 26-27, 1995 ISSN: 3-8055-6208-X.   Benveniste et al.: “Abstract 2392” FASEB Journal, Fed. Of American Soc. For Experimental Biology, Bethesda, Md., US, 22 Apr. 1998 (1998 Apr. 22), page A412, XP009070843 ISSN: 0892-6638.   Benveniste et al.: “Abstract 2304” FASEB Journal, Fed. Of American Soc. For Experimental Biology, Bethesda, Md., US, 28 Apr. 1994 (1994 Apr. 28), page A398, XP009070844 ISSN: 0892-6638.   See also, U.S. Pat. Nos. 7,412,340, 7,081,747, 6,995,558, and 6,952,652, each of which is expressly incorporated herein by reference.   

     SUMMARY OF THE INVENTION 
     The present invention concerns the identification of the molecules present in, for example, bacteria which are the sources of the signals. In particular, some particular forms and sequences of DNA from potentially pathogenic bacteria may give rise to these signals. The extraction of DNA has the advantage of facilitating the detection of electromagnetic signals from complex biological fluids, even when they have been kept frozen at low temperature. The method is particularly appropriate for the detection, identification and treatment of chronic bacterial infections in diseases associated with autoimmune components, such as rheumatoid arthritis and multiple sclerosis, or in neuropathies of unknown origin such as Alzheimer&#39;s and Parkinson&#39;s diseases. 
     In preliminary experiments, the inventor had observed that a pretreatment of a suspension of  Escherichia coli  ( E. Coli ) by 1% formaldehyde did not alter its capacity to induce the electromagnetic signals, while killing the bacteria. This treatment alters the surface proteins of the bacterial cells without attacking their genetic material, i.e., double-helical DNA. This suggested that the source of the signals may be the DNA itself. Indeed, DNA extracted from the bacterial suspension by the classical phenol:chloroform extraction technique was able, upon filtration and appropriate dilutions in water, to emit electromagnetic signals similar to those produced by intact bacteria under the same conditions. DNAse treatment of the extracted DNA solution abolishes the capacity to emit signals, provided one eliminates the source of signal coming from the nanostructures previously induced by the DNA. 
     It is therefore an object of the invention to provide a method for detecting electromagnetic waves derived from bacterial DNA, comprising extracting and purifying nucleic acids from a sample; diluting the extracted purified nucleic acids in an aqueous solvent; measuring a low frequency electromagnetic emission over time from the diluted extracted purified nucleic acids in an aqueous solvent; performing a signal analysis of the low frequency electromagnetic emission over time; and producing an output, based on the signal analysis, in dependence on the DNA in the sample. The output may vary in dependence on DNA in the sample derived from pathogenic bacteria in plasma of patients suffering from chronic diseases. The sample may contain DNA extracted from at least one of blood or blood plasma, feces, urine, saliva, tears, seminal fluid, sweat, seminal and vaginal fluids of a patient. The sample may also contain DNA extracted from potable water. For example, the sample may contain DNA which is extracted from samples previously frozen and stored at temperatures between about −20° C. and −70° C. The diluting step may, for example, dilute the DNA by about 10 −7  to 10 −13 . 
     The extracting and purifying process may comprise: diluting the sample with an aqueous buffer and mixing; degrading proteins in the diluted sample; precipitating DNA from the buffer solution; and resuspending the precipitated DNA in an aqueous solution. Further, the resuspended DNA may be filtered through at least one submicron filter, wherein the sample measured comprises the filtrate. The filtrate may be diluted in an aqueous solution prior to measuring. The resuspended DNA may be diluted by 10 2  to 10 20  prior to measurement. 
     The measuring may comprise placing the diluted extracted purified nucleic acids near an antenna adapted to receive electromagnetic signals having a frequency approaching about 0 Hz and up to 20 kHz, and receiving the electromagnetic signals from the antenna. 
     The signal analysis may comprise performing a time domain to frequency domain transformation on the signal. Transformed signals from two different samples may be compared. For example, the transform may be a frequency domain (such as an FFT or DFT, generally using an appropriate window function), wavelet domain, or other reversible or irreversible transform. The signal analysis may comprise applying a threshold function to the frequency domain transformed signal. The threshold function may be a static or dynamic or adaptive threshold, and may be uniform or changing over a spectrum of characteristics. The signal analysis may comprise a three-dimensional histogram. The signal analysis may comprise reducing a background noise component of the signal. The signal analysis may comprise selective analysis of signal components having frequencies between about 500-3000 Hz. 
     The signal analysis may be performed on a general purpose computer, for example having a processor, memory for storing program instructions and data, an operating system, application software, and inputs and outputs, and the output may be presented through a graphic user interface. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a schematic diagram of the apparatus for recording electromagnetic signals. 
         FIGS. 2A-2O  show, respectively time domain electromagnetic signals (EMS) emitted over 6 seconds by serial decimal dilutions from 10 −2  to 10 −15  of a solution (filtered through a 100 nm pore size filter, except  FIG. 2O , which was unfiltered).  FIGS. 2A ,  2 B,  2 C,  2 D,  2 E,  2 F,  2 L,  2 M,  2 N, and  2 O are control samples, showing a noise pattern, and  FIGS. 2G ,  2 H,  2 I,  2 J,  2 K, are dilutions of a filtered solution of DNA extracted from  E. Coli  K1. 
         FIGS. 3A-3O  show, respectively, show a zoom analysis (millisecond scale) of one part of the recording shown of a respective dilution EMS signal shown in  FIGS. 2A-20 . 
         FIGS. 4A-4O  show, respectively, a tridimensional Fourier transform as output from Matlab software of the recording, in which the Abscissa shows a range of frequencies from 20 to 20,000 Hz, the ordinate represents the intensity of the waves, and the third dimension represents recordings at different times. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A typical experiment is described as follows: First, extreme precautions have to be taken at all steps of the process in order to avoid adventitious contamination from external sources: all manipulations have to be done in a class 100 or better laminar flow hood, and centrifugations are operated in stoppered sterile tubes. The water used for dilutions of reagents and samples has been obtained by distillation at 500° C. (pyrolysis) to destroy any macromolecular organic compound. 
     1) DNA Extraction: 
     The sample is first equilibrated at room temperature (15-20° C.), then it is diluted 1:100 in a volume of 400 μl final in sterile PBS 1× in a 2 ml microcentrifuge tube, and mixed by pulse-vortexing for 10 sec. 
     40 μl of a 10 mg/ml solution of Proteinase K are added (1 mg/ml final concentration) and then 20 μl of an aqueous solution of SDS 10% (Sodium Dodecyl Sulfate) are added (0.5% final concentration). The mixture is mixed by pulse-vortexing for 10 sec, and incubated 15 min at 56° C. 
     500 μl of Phenol:Chloroform:lsoAmyl Alcohol (25:24:1), are then added and mixed by pulse-vortexing for 20 sec. The mixture is centrifuged for 10 min at room temperature at 6000×9 (8000 rpm). The upper aqueous phase is collected (approximately 5000) and placed in a new 2 ml microcentrifuge tube. Cold ethanol, 2.5 volumes (1,125 μl) and 2.5M Sodium Acetate (pH 5.2), 1:10 of final volume (160 μl) are added to precipitate the DNA, mixed by inverting the tube carefully (5-10 times) and left for 15 min at 20° C. The sample is centrifuged for 30 min at 14,000 rpm at 4° C. and the supernatant is discarded. The pellet is washed twice by 500 μl of cold ethanol 70% and the suspension is centrifuged for 10 min at 14,000 rpm at 4° C. The supernatant is discarded and the pellet is dried at room temperature for 15 min. The pellet is then re-suspended in 60 μl Tris 10 mM (pH 7.6), mixed by pipetting and stored at 4° C. for immediate use in the SEM protocol or frozen at −20° C. or preferentially at −70° C. for further analysis. 
     2) EMS Measurement: 
     An aliquot of the DNA solution is diluted 1:100 vol/vol in water and the dilution is filtered first through a Millipore filter of pore size 0.45 μm (Millex). The filtrate is filtered again through a 0.1 μm Millipore filter. This filtration step is important, and it has been observed that no signals are detected in its absence at any dilution. In contrast to the microorganism suspensions where the filtration was supposed to retain the bacteria or viruses, this filtration let the DNA pass through, and the latter is therefore still present in the filtrate. 
     By definition, the “filtrate” is the liquid which has passed through the pores of the filter and is not retained by the filter. 
     The role of filtration for the DNA solution is probably to dissociate the network of nanostructures which are trapped in a gel at high concentration, thus allowing them to vibrate in resonance with the excitation produced by the electromagnetic background. 
     The filtration step must be immediately followed by the dilution steps 10 by 10 in water as previously described, for the filtrates of plasma or of microorganism suspensions. 
     For each dilution, 0.1 ml of the previous dilution is added to 0.9 ml of water in an Eppendorf conical tube, and strongly agitated for 15 seconds in a vortex mixer. Again, 0.1 ml of this solution is diluted in 0.9 ml of water, etc. 
     Usually, the range of dilutions is made between 10 −2  and 10 −15 , eventually 10 −20 . Capture and analysis of the EMS is proceeded as previously described in U.S. patent application Ser. No. 12/097,204, and as shown in  FIG. 1 . Briefly, this method is as follows: 
     The detection of signals is performed with equipment shown in a schematic view in  FIG. 1 . The equipment includes a reading solenoid coil ( 1 ) with high sensitivity between about 0 and 20,000 Hertz, positioned on a table made of an isolating material, e.g., shield ( 5 ). The solutions to be read are distributed in plastic Eppendorf® conical tubes ( 2 ), 1.5 milliliter in capacity. The liquid volume is, for example, 1 milliliter, though lesser amounts may be employed. Each sample is read for 6 seconds, twice in a row, and each reading is entered separately. The electric signals delivered by the solenoid (coil  1  of wire surrounding the sample tube  2 ) are amplified using a sound card ( 4 ) input to a personal computer ( 3 ) the appropriate software of which may provide a visual representation of the recorded elements. 
     An amplitude raw global representation is presented. Some background noise is generally present, which can then be filtered. A positive signal is detected when the amplitude exceeds at least 1.5 times the background noise. In general, the detected amplitude is twice and sometimes three times, the background noise. This detected signal is called an SEM electromagnetic signal. 
     A 3D histogram analysis may be performed, respectively of the background noise and the signal in presence of the sample. The recorded signal may be broken down into individual frequencies through a Fourier transform of the background noise and the signal respectively in the presence of the sample. Of course, other types of signal analysis may be employed, for example wavelet analysis, principal component analysis, or other linear, non-linear, statistical, discrete, etc. analysis techniques which analyze a signal amplitude with respect to time signals, particularly those which extract or emphasize information represented therein. 
     Each dilution tube is placed on the top of a solenoid transforming changes in the magnetic field into an electric current. The current is amplified 500 times by a SoundBlaster® card and analyzed on a computer employing three software applications: one for direct recording of the waves, and two for performing Fourier analysis of the harmonics, as shown in  FIGS. 4A-4O . 
     A positive signal is generally defined by an increase of higher frequencies (500-3,000 Hz) over the respective background sample, though the criteria may be different for different types of analysis; that is, using a Fourier analysis, the positive experimental show a significant difference from control with respect to an increase in signal energy in the 500-3,000 Hz band. Using other analysis techniques, the definition of a positive result will correspondingly be different. 
     Usually positive signals are found in the range of the 10 −7  to 10 −13  dilutions. At high dilutions (from 10 −12 ), calculations indicate that there is no DNA left and that the EMS should come for self-maintained nanostructures induced in water by the DNA from which they originated. 
     3) Effect of DNAse: 
     Destruction of DNA sequences by DNAse abolishes their capacity to induce EMS in water. Since the nanostructures formed in water are fully resistant to DNAse, but are sensitive to heat, the DNAse effect is shown according to the following protocol, designed to suppress this secondary source of EMS. The DNA preparation is heated at 100° C. for 30 min to destroy the nanostructures. After cooling at 37° C., DNAse I at a final concentration of 10 U/μg of DNA is added to the DNA solution and incubated in the presence of 5 mM MgCl2 for 16 Hours at 37° C. An aliquot of untreated DNA solution is kept as a positive control. The DNAse treated preparation is completely devoid of EMS emission at any dilutions. Therefore the main source of EMS is DNA. 
     4) Nature of the DNA Sequences at the Origin of EMS: 
     A survey of the main bacteria species involved in human infections indicated that the following species are producing signals, either as pure culture suspension, or as DNA: 
       Escherichia coli  (strain K1) 
       Streptococcus  B 
     
       Staphylococcus aureus  
     
     
       Proteus mirabilis  
     
     
       Pseudomonas aeruginosa  
     
     
       Bacillus aeruginosa  
     
     
       Mycoplasma pirum  
     
     Positive dilutions were all in the range of 10 −7  to 10 −13  and the signal frequency profiles were similar, although small differences may exist between species and may be revealed by a more refined signal analysis. By contrast, no signals were detected from certain strains of  E. Coli  used as plasmid vector for molecular cloning such as  E. coli  XL1 B (Stratagene) and  E. coli  TOP10 (Invitrogen) and also in bacteria known to be apathogenic, such as  Lactobacillus  and  Bacillus  Calmette-Guerin. This indicated that only some specific sequences of bacterial DNA were at the origin of EMS. As shown in  FIGS. 2A-2O , the EMS are recorded over 6 seconds. The data is then subject to a frequency domain transform such as a Fast Fourier Transform (FFT) as shown in  FIGS. 4A-4O . The samples represent serial decimal dilution concentrations from 10 −2  to 10 −15  of a solution (filtered through a 100 nm pore size filter, except  FIG. 2O , which was unfiltered).  FIGS. 2A ,  2 B,  2 C,  2 D,  2 E,  2 F,  2 L,  2 M,  2 N, and  2 O are control samples (containing no DNA), showing a noise pattern, and  FIGS. 2G ,  2 H,  2 I,  2 J,  2 K, are dilutions of a filtered solution of DNA extracted from  E. Coli  K1. As can be seen, as compared to the control samples, the spectral energy of the EMS from the experimental samples of  FIGS. 4G-4K , at frequencies greater than about 75 Hz up to about 2,000 Hz is clearly higher than the control samples. This distinction is even clearer at frequencies above 500 Hz. In the time domain plots of  FIGS. 2A-2O  and the zoom detail of  FIGS. 3A-3O , it is visually apparent that  FIGS. 2G ,  2 H,  2 I,  2 J and  2 K and  FIGS. 3G ,  3 H,  3 I,  3 J and  3 K have higher amplitudes of high frequency signal components than the control samples. 
     This data suggests that a simplified analysis of spectral energy at about 1,500 Hz or more generally in the 1,400-2,000 Hz band may be sufficient to determine the presence of the effect, and therefore than a relatively simple instrument, without requiring complex analytical software, may be used for screening samples. 
     In an attempt to analyze the nature of the sequences involved,  Mycoplasma pirum  species was used, in which a gene indispensable for its eventual pathogenic role was cloned, the gene coding for the adhesin protein, a protein necessary for the attachement of the bacterium to eukaryotic cells. 
     This gene was cloned in a plasmid carried by an  E. Coli  strain in two fragments corresponding respectively to the N-terminus and the C-terminus of the protein. 
     The vector  E. Coli  strain (XL1 B) lacking the plasmid did not produce EMS as well as its extracted DNA. 
     By contrast, when the bacterial strain was transformed by the plasmids carrying the adhesin gene fragments, its extracted DNA produced EMS as well as the purified plasmid DNA. 
     The two gene fragments were excised by appropriate restriction enzymes from the plasmid and purified by electrophoresis in agarose gel. The electrophoretic bands corresponding to their molecular weight (respectively 1.5 Kb for the N-terminus and 3.5 Kb for C-terminus) were cut from the gel and the DNA fragments were eluted. 
     Again, upon the procedure previously described (filtration with 0.45 μm and 0.1 μm filters) the two pure DNA fragments corresponding to the adhesin gene each yielded typical EMS. 
     This result indicates that the present method is able to distinguish in a given bacterium between the specific DNA sequences which are responsible for emitting the electromagnetic signals. 
     The adhesin gene of  M. pirum  is, in its native state, part of the chromosomal DNA, indicating that whatever be its location, plasmidic or integrated, its sequences by themselves are able to induce EMS. 
     This procedure opens the way to a more refined intragenic analyzis of the minimal sequences of a gene which are responsible for the EMS. 
     It is noted that, while the signals themselves may be similar for various DNA samples which are associated with the EMS, the DNA sequences themselves are different. Therefore, the DNA sequence giving rise to a signal may be inferred based on similarities of the EMS to the corresponding EMS of authentic samples (either gathered contemporaneously or retrieved from a database), and/or by studies which target particular DNA sequences, and thereby modify signals associated with those sequences. For example, the EMS associated with DNA may be modified by small molecule agents, binding proteins and factors, DNA (e.g., primer-type sequences) or RNA (e.g., siRNA) or other nucleic acid targeting molecules, restriction endonucleases, antibodies and immunoglobulin-type agents, etc. Therefore, even if an EMS is not itself diagnostic for a pathological condition, the selective extinction or enhancement of the EMS based on selective modification of the sample may indeed play a role in the diagnosis. 
     However it is already applicable for the detection of infections by pathogenic bacteria in human and animal diseases, especially when there are no available molecular or serological methods of detection. 
     Patients suffering from chronic diseases such as rheumatoid arthritis, multiple sclerosis, Alzheimer&#39;s and Parkinson&#39;s diseases are generally positive for EMS in their plasma. However, freezing of the plasma will abolish the emission of EMS. 
     Therefore, the plasma has to be analyzed soon after separation from the drawn blood. 
     The present method described here is a significant improvement in that it allows detection of EMS from DNA extracted from plasma stored frozen.