Abstract:
A method for identifying an RNA form of a bacteria, comprising reverse transcribing RNA material; conducting PCR using primers for a first highly conserved genetic sequence generic of the bacteria; conducting nested PCR using primers for a second highly conserved genetic sequence within the first genetic sequence of the bacteria; and identifying the bacteria based on unconserved amplified sequences linked to the conserved sequences.

Description:
This application claims benefit under 35 U.S.C. 119(e) to Provisional Application 60/603,120, filed on Aug. 20, 2004. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the field of PCR methods, and specific primers therefore, as well as their use in the identification of any type of bacteria, and in particular RNA forms of bacteria. 
     BACKGROUND OF THE INVENTION 
     The use of biological fluids for therapeutic human application such as plasmas, albumin, live vaccines, stem cells requires that they are absolutely devoid of bacterial contamination. It has been found that filtering, and possibly other traditional methods, may fail to eliminate all forms of organisms, leading to possible contamination or experimental artifacts. 
     It is believed that certain pathologies are associated with bacteria or bacterial forms which are difficult to detect, and which may pass through nano-porous barriers. This leads to possible errors in diagnosis or causation, and which may lead to erroneous treatment and impede prevention. 
     See, Hopert, Anne, Uphoff, Cord C., Wirth, Manfred, Hauser, Hansjorg, and Drexler, Hans G., “Specificity and sensitivity of Polymerase Chain Reaction (PCR) in comparison with other methods for the detection of  mycoplasma  contamination in cell lines”, J. Immunological Methods, 164(1993):91-100, expressly incorporated herein by reference. 
     Kajander, E. O., et al., “Comparison of  Staphylococci  and novel Bacteria-Like Particles from blood”, Zbl. Bakt. Suppl. 26, 1994, expressly incorporated herein by reference. 
     Akerman, Kari K., “Scanning Electron Microscopy of Nanobacteria—Novel Biofilm Producing Organisms in Blood”, Scanning Vol. 15, Suppl. III (1993), expressly incorporated herein by reference. www.newcastle.edu.au/discipline/biology/projects/hons_cpru.html, expressly incorporated herein by reference. 
     Cifticioglu, Neva, et al., “Apoptotic effect of nanobacteria on cultured mammalian cells”, Mol. Biol. Cell. Suppl., Vol. 7 (1996):517a 
     Cifticioglu, Neva, et al., “A new potential threat in antigen and antibody products: Nanobacteria”, Vaccines 97, Brown et al. Ed., Cold Spring Harbor Laboratory Press, New York, 1997, expressly incorporated herein by reference. 
     Baseman, Joel B., et al., “ Mycoplasmas : Sophisticated, Reemerging, and Burdened by their Notoriety”, EID Vol. 3, N o  1 www.cdc.gov/ncidod/EID/vol3no1/baseman.htm, expressly incorporated herein by reference. 
     Relman, David A., “Detection and Identification of Previously Unrecognized Microbial Pathogens”, EID Vol. 4, N o  3 www.cdc.gov/ncidod/EID/vol4no3/relman.htm, expressly incorporated herein by reference. 
     Mattman, Lida H., Cell Wall Deficient Forms-Stealth Pathogens, 2nd Ed., CRC Press (1993), expressly incorporated herein by reference. 
     U.S. Pat. No. 5,688,646, expressly incorporated herein by reference, describes novel  mycoplasmas  which are prominent in patients who are thought to be suffering from AIDS. Devices are also provided for the in vitro detection of  mycoplasmas  in biological fluid by means of a reagent which is specific for the  mycoplasma  group without being specific for particular species within said group. Devices for testing  mycoplasma  sensitivity to antibiotics are also described. 
     See the following, each of which and cited references is expressly incorporated herein by reference in their entirety: 
     
       
         
               
               
             
               
               
             
           
               
                   
               
               
                 Pat. No 
                 Title 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 6,562,611 
                 FEN-1 endonucleases, mixtures and cleavage methods 
               
               
                 6,558,902 
                 Infrared matrix-assisted laser desorption/ionization mass spectrometric analysis of 
               
               
                   
                 macromolecules 
               
               
                 6,555,357 
                 FEN-1 endonuclease, mixtures and cleavage methods 
               
               
                 6,555,338 
                 NrdF from  Staphylococcus aureus   
               
               
                 6,537,774 
                 UPS (undecaprenyl diphosphate synthase 
               
               
                 6,492,113 
                 Detection of  Mycoplasma  genus and species in patients with chronic fatigue syndrome 
               
               
                   
                 and fibromyalgia 
               
               
                 6,489,139 
                 FabZ from  Staphylococcus aureus   
               
               
                 6,489,110 
                 EF-Tu mRNA as a marker for viability of bacteria 
               
               
                 6,458,572 
                 Phosphatidylglycerophosphate synthase from  Staphylococcus aureus   
               
               
                 6,458,535 
                 Detection of nucleic acids by multiple sequential invasive cleavages 02 
               
               
                 6,448,037 
                 PgsA 
               
               
                 6,432,703 
                 RATC from  Streptococcus pneumoniae   
               
               
                 6,410,286 
                 Asparaginyl tRNA synthetase from  Staphylococcus Aureus   
               
               
                 6,399,343 
                 inFB 
               
               
                 6,372,424 
                 Rapid detection and identification of pathogens 
               
               
                 6,361,965 
                 YfiI pseudouridine synthase 
               
               
                 6,353,093 
                 gidB 
               
               
                 6,350,600 
                 trmD 
               
               
                 6,348,582 
                 Prokaryotic polynucleotides polypeptides and their uses 
               
               
                 6,348,328 
                 Compounds 
               
               
                 6,348,314 
                 Invasive cleavage of nucleic acids 
               
               
                 6,346,397 
                 GyrA 
               
               
                 6,340,564 
                 yhxB 
               
               
                 6,331,411 
                 TopA 
               
               
                 6,326,172 
                 ytgP 
               
               
                 6,312,932 
                 Yfil pseudouridine synthase 
               
               
                 6,309,866 
                 6-phosphogluconate dehydrogenase 
               
               
                 6,303,771 
                 Pth 
               
               
                 6,294,652 
                 Response regulator 
               
               
                 6,294,357 
                 FabF from  Staphylococcus aureus   
               
               
                 6,287,807 
                 MurF 
               
               
                 6,287,804 
                 nrdG 
               
               
                 6,277,595 
                 FabZ 
               
               
                 6,274,719 
                 Gcp 
               
               
                 6,274,361 
                 pth 
               
               
                 6,270,762 
                 tdk 
               
               
                 6,268,177 
                 Isolated nucleic acid encoding nucleotide pyrophosphorylase 
               
               
                 6,261,802 
                 Ups (ugc) 
               
               
                 6,261,769 
                 Intergenic spacer target sequence for detecting and distinguishing  Chlamydial  species 
               
               
                   
                 or strains 
               
               
                 6,255,075 
                 Bira 
               
               
                 6,251,631 
                 nadE from  Streptococcus pneumoniae   
               
               
                 6,251,629 
                 ABC transporter 
               
               
                 6,248,721 
                 Method of using mouse model for evaluation of HIV vaccines 
               
               
                 6,245,891 
                 nusB polypeptides and polynucleotides and methods thereof 
               
               
                 6,245,542 
                 tRNA methyltransferase from  Streptococcus pneumoniae   
               
               
                 6,238,900 
                 Polynucleotides encoding glutamyltrna synthetase from  staphylococcus aureus   
               
               
                 6,238,887 
                 Ribosome recycling factor (FRR) of  Staphylococcus aureus   
               
               
                 6,238,882 
                 GidA1 
               
               
                 6,228,625 
                 metK from  Streptococcus pneumoniae   
               
               
                 6,228,584 
                 DexB 
               
               
                 6,210,880 
                 Polymorphism analysis by nucleic acid structure probing with structure-bridging 
               
               
                   
                 oligonucleotides 
               
               
                 6,204,014 
                 DnaB 
               
               
                 6,197,549 
                 Ama 
               
               
                 6,197,300 
                 ftsZ 
               
               
                 6,194,170 
                 MurF of  Streptococcus pneumoniae   
               
               
                 6,190,881 
                 Ribonucleotide diphosphate reductase, nrdF, of  streptococcus pneumoniae   
               
               
                 6,168,797 
                 FabF 
               
               
                 6,165,992 
                 Histidine kinase 
               
               
                 6,165,991 
                 Sensor histidine kinase of  Streptococcus pneumoniae   
               
               
                 6,165,764 
                 Polynucleotides encoding tRNA methyl transferases from  Streptococcus pneumoniae   
               
               
                 6,162,619 
                 Sensor histidine kinase of  streptococcus pneumoniae   
               
               
                 6,162,618 
                 6-phosphogluconate dehydrogenase of  Streptococcus pneumoniae   
               
               
                 6,156,537 
                 Phospho-N-acetylmuramoyl-pentapeptide transferase of  Streptococcus pneumoniae   
               
               
                 6,146,863 
                   Staphylococcus aureus  3-hydroxyacyl-CoA dehydrogenase 
               
               
                 6,146,846 
                 Primosome protein a of  streptococcus pneumoniae   
               
               
                 6,140,079 
                 GidB 
               
               
                 6,140,061 
                 Response regulator 
               
               
                 6,111,074 
                 PyrH of  Streptococcus pneumoniae   
               
               
                 6,110,723 
                 Yfii pseudouridine synthase 
               
               
                 6,110,685 
                 infB 
               
               
                 6,090,543 
                 Cleavage of nucleic acids 
               
               
                 6,060,294 
                 Alanyl tRNA synthetase from  Staphylococcus aureus   
               
               
                 6,001,567 
                 Detection of nucleic acid sequences by invader-directed cleavage 
               
               
                 5,994,111 
                 Leucyl tRNA synthetase from  staphylococcus aureus   
               
               
                 5,985,557 
                 Invasive cleavage of nucleic acids 
               
               
                 5,882,643 
                 Lep 
               
               
                 5,851,764 
                 Human prostate tumor inducing gene-1 and uses thereof 
               
               
                 5,843,669 
                 Cleavage of nucleic acid acid using thermostable  methoanococcus jannaschii  FEN-1 
               
               
                   
                 endonucleases 
               
               
                 5,843,654 
                 Rapid detection of mutations in the p53 gene 
               
               
                 5,795,976 
                 Detection of nucleic acid heteroduplex molecules by denaturing high-performance 
               
               
                   
                 liquid chromatography and methods for comparative sequencing 
               
               
                 5,789,217 
                 DNA encoding asparaginyl tRNA synthetase from  staphylococcus aureus   
               
               
                 5,786,197 
                 Lep 
               
               
                 5,776,750 
                 Alanyl tRNA synthetase polynucleoyides of  staphylococcus   
               
               
                 5,763,246 
                 DNA encoding arginyl tRNA synthetase from  staphylococcus aureus   
               
               
                 5,750,387 
                 DNA encoing leucyl TRNA synthetase from  staphylococcus aureus   
               
               
                 5,688,646 
                   Mycoplasmas -agents for detecting and characterizing  mycoplasmas  in vitro (See 
               
               
                   
                 above) 
               
               
                 6,562,957 
                 Genomic sequence encoding endoglin and fragments thereof 
               
               
                 6,545,140 
                 DNA encoding an avian beta-defensin and uses thereof 
               
               
                 6,531,148 
                 Therapeutic agents 
               
               
                 6,495,661 
                 DNA encoding the outer membrane protein of  Pasteurella multocida   
               
               
                 6,475,990 
                 Drugs, foods or drinks with the use of algae-derived physiologically active substances 
               
               
                 6,451,601 
                 Transiently immortalized cells for use in gene therapy 
               
               
                 6,403,564 
                 Ribavirin-interferon alfa combination therapy for eradicating detectable HCV-RNA in 
               
               
                   
                 patients having chronic hepatitis C infection 
               
               
                 6,399,373 
                 Nucleic acid encoding a retinoblastoma binding protein (RBP-7) and polymorphic 
               
               
                   
                 markers associated with said nucleic acid 
               
               
                 6,368,600 
                 Parasitic helminth cuticlin nucleic acid molecules and uses thereof 
               
               
                 6,339,151 
                 Enzyme catalyzed therapeutic agents 
               
               
                 6,277,830 
                 5′-amino acid esters of ribavirin and the use of same to treat hepatitis C with interferon 
               
               
                 6,258,778 
                 Methods for accelerating bone and cartilage growth and repair 
               
               
                 6,248,329 
                 Parasitic helminth cuticlin nucleic acid molecules and uses thereof 
               
               
                 6,245,750 
                 Enzyme catalyzed therapeutic agents 
               
               
                 6,022,687 
                 Diagnosis of and therapy for hereditary haemorrhagic telangiectasia 
               
               
                 6,593,086 
                 Nucleic acid amplification methods 
               
               
                 6,583,266 
                 Nucleic acid and amino acid sequences relating to  mycobacterium tuberculosis  and 
               
               
                   
                 leprae for diagnostics and therapeutics 
               
               
                 6,573,068 
                 Claudin-50 protein 
               
               
                 6,569,647 
                 Nucleic acid amplification method: ramification-extension amplification method 
               
               
                   
                 (RAM) 
               
               
                 6,562,611 
                 FEN-1 endonucleases, mixtures and cleavage methods 
               
               
                 6,558,953 
                 Grapevine leafroll virus proteins and their uses 
               
               
                 6,558,909 
                 Haemobartonella PCR methods and materials 
               
               
                 6,558,905 
                 Diagnostics and therapeutics for osteoporosis 
               
               
                 6,558,902 
                 Infrared matrix-assisted laser desorption/ionization mass spectrometric analysis of 
               
               
                   
                 macromolecules 
               
               
                 6,555,357 
                 FEN-1 endonuclease, mixtures and cleavage methods 
               
               
                 6,548,633 
                 Complementary DNA&#39;s encoding proteins with signal peptides 
               
               
                 6,531,648 
                 Grain processing method and transgenic plants useful therein 
               
               
                 6,524,795 
                 Diagnostics for cardiovascular disorders 
               
               
                 6,521,426 
                 Preparation of recombinant adenovirus carrying a rep gene of adeno-associated virus 
               
               
                 6,518,020 
                 Haemobartonella PCR methods and materials 
               
               
                 6,503,747 
                 Serotype-specific probes for Listeria monocytogenes 
               
               
                 6,495,325 
                 Detection and quantification of micro-organisms using amplification and restriction 
               
               
                   
                 enzyme analysis 
               
               
                 6,458,584 
                 Customized oligonucleotide microchips that convert multiple genetic information to 
               
               
                   
                 simple patterns, are portable and reusable 
               
               
                 6,458,535 
                 Detection of nucleic acids by multiple sequential invasive cleavages 02 
               
               
                 6,444,876 
                 Acyl CoA: cholesterol acyltransferase related nucleic acid sequences 
               
               
                 6,436,399 
                 Nucleic acid encoding the major outer membrane protein of the causative agent of 
               
               
                   
                 human granulocytic ehrlichiosis and peptides encoded thereby 
               
               
                 6,432,649 
                 Methods for detecting  Ehrlichia canis  and  Ehrlichia chaffeensis  in vertebrate and 
               
               
                   
                 invertebrate hosts 
               
               
                 6,423,499 
                 PCR primers for detection and identification of plant pathogenic species, subspecies, 
               
               
                   
                 and strains of acidovorax 
               
               
                 6,403,093 
                 Methods to detect granulocytic ehrlichiosis 
               
               
                 6,399,307 
                 Methods of quantifying viral load in an animal with a ribonuclease resistant RNA 
               
               
                   
                 preparation 
               
               
                 6,387,617 
                 Catalytic nucleic acid and methods of use 
               
               
                 6,372,424 
                 Rapid detection and identification of pathogens 
               
               
                 6,348,314 
                 Invasive cleavage of nucleic acids 
               
               
                 6,329,138 
                 Method for detection of the antibiotic resistance spectrum of mycobacterium species 
               
               
                 6,312,922 
                 Complementary DNAs 
               
               
                 6,312,903 
                 Simulataneous detection, identification and differentiation of eubacterial taxa using a 
               
               
                   
                 hybridization assay 
               
               
                 6,306,653 
                 Detection and treatment of breast disease 
               
               
                 6,300,091 
                 Herbicide target genes and methods 
               
               
                 6,300,072 
                 PCR methods and materials for detecting  bartonella  species 
               
               
                 6,287,779 
                 Detection of fermentation-related microorganisms 
               
               
                 6,268,142 
                 Diagnostics and therapeutics for diseases associated with an IL-1 inflammatory 
               
               
                   
                 haplotype 
               
               
                 6,261,773 
                 Reagent for nucleic acid amplification and process for nucleic acid amplification 
               
               
                 6,252,130 
                 Production of somatic mosaicism in mammals using a recombinatorial substrate 
               
               
                 6,251,607 
                 PCR primers for the rapid and specific detection of  Salmonella typhimurium   
               
               
                 6,248,519 
                 Detection of fermentation-related microorganisms 
               
               
                 6,221,582 
                 Polynucleic acid sequences for use in the detection and differentiation of prokaryotic 
               
               
                   
                 organisms 
               
               
                 6,214,982 
                 Ribonuclease resistant RNA preparation and utilization 
               
               
                 6,214,548 
                 Diagnostic methods for Cyclospora 
               
               
                 6,194,145 
                 Genus and species-specific identification of  Legionella   
               
               
                 6,180,339 
                 Nucleic acid probes for the detection and identification of fungi 
               
               
                 6,103,468 
                 Rapid two-stage polymerase chain reaction method for detection of lactic acid bacteria 
               
               
                   
                 in beer 
               
               
                 6,090,543 
                 Cleavage of nucleic acids 
               
               
                 6,033,858 
                 Detection of transmissible spongiform encephalopathies 
               
               
                 6,025,132 
                 Probes targeted to rRNA spacer regions, methods and kits for using said probes, for 
               
               
                   
                 the detection of respiratory tract pathogens 
               
               
                 6,001,567 
                 Detection of nucleic acid sequences by invader-directed cleavage 
               
               
                 6,001,564 
                 Species specific and universal DNA probes and amplification primers to rapidly detect 
               
               
                   
                 and identify common bacterial pathogens and associated antibiotic resistance genes 
               
               
                   
                 from clinical specimens for routine diagnosis in microbiology laboratories 
               
               
                 5,994,066 
                 Species-specific and universal DNA probes and amplification primers to rapidly detect 
               
               
                   
                 and identify common bacterial pathogens and associated antibiotic resistance genes 
               
               
                   
                 from clinical specimens for routine diagnosis in microbiology laboratories 
               
               
                 5,985,557 
                 Invasive cleavage of nucleic acids 
               
               
                 5,976,805 
                 Neisseria gonorrhoeae specific DNA fragment-GC3 
               
               
                 5,958,693 
                 Extraction of DNA by boiling cells in an alkaline phenol/guanidine thiocyanate 
               
               
                   
                 solution 
               
               
                 5,948,888 
                 Neural thread protein gene expression and detection of Alzheimer&#39;s disease 
               
               
                 5,948,634 
                 Neural thread protein gene expression and detection of Alzheimer&#39;s disease 
               
               
                 5,942,391 
                 Nucleic acid amplification method: ramification-extension amplification method 
               
               
                   
                 (RAM) 
               
               
                 5,939,262 
                 Ribonuclease resistant RNA preparation and utilization 
               
               
                 5,922,538 
                 Genetic markers and methods for the detection of  Listeria monocytogenes  and  Listeria   
               
               
                   
                 spp 
               
               
                 5,919,625 
                 Ribonuclease resistant viral RNA standards 
               
               
                 5,912,117 
                 Method for diagnosis of lyme disease 
               
               
                 5,907,085 
                 Grapevine leafroll virus proteins and their uses 
               
               
                 5,876,924 
                 Nucleic acid amplification method hybridization signal amplification method (HSAM) 
               
               
                 5,843,669 
                 Cleavage of nucleic acid acid using thermostable  methoanococcus jannaschii  FEN-1 
               
               
                   
                 endonucleases 
               
               
                 5,830,670 
                 Neural thread protein gene expression and detection of Alzheimer&#39;s disease 
               
               
                 5,795,976 
                 Detection of nucleic acid heteroduplex molecules by denaturing high-performance 
               
               
                   
                 liquid chromatography and methods for comparative sequencing 
               
               
                 5,763,169 
                 Nucleic acid probes for the detection and identification of fungi 
               
               
                 5,747,257 
                 Genetic markers and methods for the detection of  escherichia coli  serotype-0157:H7 
               
               
                 5,744,306 
                 Methods for nucleic acid detection, sequencing, and cloning using exonuclease 
               
               
                 5,734,086 
                 Cytochrome P450.sub.lpr gene and its uses 
               
               
                 5,707,802 
                 Nucleic acid probes for the detection and identification of fungi 
               
               
                 5,693,467 
                 Mycoplasma polymerase chain reaction testing system using a set of mixed and single 
               
               
                   
                 sequence primers 
               
               
                 5,688,669 
                 Methods for nucleic acid detection, sequencing, and cloning using exonuclease 
               
               
                 5,677,124 
                 Ribonuclease resistant viral RNA standards 
               
               
                 5,660,981 
                 Selection of diagnostic genetic markers in microorganisms and use of a specific marker 
               
               
                   
                 for detection of  salmonella   
               
               
                 5,658,749 
                 Method for processing  mycobacteria   
               
               
                 5,656,740 
                 Nucleic acid fragments useful in the detection of  Salmonella   
               
               
                 5,643,723 
                 Detection of a genetic locus encoding resistance to rifampin in  mycobacterial  cultures 
               
               
                   
                 and in clinical specimens 
               
               
                 5,593,836 
                 Primers and probes for detecting  Pneumocystis carinii   
               
               
                 5,589,570 
                 Integrin alpha subunit cytoplasmic domain polypeptides and methods 
               
               
                 5,518,901 
                 Methods for adapting nucleic acid for detection, sequencing, and cloning using 
               
               
                   
                 exonuclease 
               
               
                 5,310,874 
                 Integrin .alpha. subunit cytoplasmic domain polypeptides and antibodies 
               
               
                 20030124545 
                 Quantitative assay for the simultaneous detection and speciation of bacterial infections 
               
               
                 20030118992 
                 ABC transporter 
               
               
                 20030108873 
                 Systems for the detection of target sequences 
               
               
                 20030082614 
                 Map 
               
               
                 20030065156 
                 Novel human genes and gene expression products I 
               
               
                 20030059771 
                 Compositions and methods related to the Rig tumor suppressor gene and protein 
               
               
                 20030054338 
                 Detection of target sequences by cleavage of non-target nucleic acids 
               
               
                 20030044864 
                 Cellular engineering, protein expression profiling, differential labeling of peptides, and 
               
               
                   
                 novel reagents therefor 
               
               
                 20030044796 
                 Reactions on dendrimers 
               
               
                 20030027286 
                 Bacterial promoters and methods of use 
               
               
                 20030017532 
                 ndp 
               
               
                 20020162123 
                 Combination immunogene therapy 
               
               
                 20020160447 
                 Ups (ugc) 
               
               
                 20020150963 
                 Map 
               
               
                 20020146790 
                 MurF 
               
               
                 20020146751 
                 fabF 
               
               
                 20020128437 
                 GidA1 
               
               
                 20020127596 
                 murF2 
               
               
                 20020123047 
                 dexB 
               
               
                 20020120116 
                   Enterococcus faecalis  polynucleotides and polypeptides 
               
               
                 20020119520 
                 FabZ 
               
               
                 20020119510 
                 gcp 
               
               
                 20020119454 
                 Polymorphism analysis by nucleic acid structure probing with structure-bridging 
               
               
                   
                 oligonucleotides 
               
               
                 20020115075 
                 nadE 
               
               
                 20020106776 
                 pth 
               
               
                 20020102700 
                 metK 
               
               
                 20020098544 
                 nrdG 
               
               
                 20020091237 
                 nusB 
               
               
                 20020082234 
                 Novel prokaryotic polynucleotides, polypeptides and their uses 
               
               
                 20020058799 
                 Novel pth 
               
               
                 20020052472 
                 ama 
               
               
                 20020048789 
                 birA 
               
               
                 20020025516 
                 NRDE 
               
               
                 20020004581 
                 gcp 
               
               
                 20020004580 
                 ftsZ 
               
               
                 20010027183 
                 tdk 
               
               
                 20010023064 
                 yfjO 
               
               
                 20010020010 
                 Histidine kinase 
               
               
                 20010016334 
                 MurF 
               
               
                 20010014670 
                 Treatment and diagnosis of Alzheimer&#39;s disease 
               
               
                 20010010912 
                 nrdF 
               
               
                 20030145347 
                 Grain processing method and transgenic plants useful therein 
               
               
                 20030144490 
                 Extended cDNAs for secreted proteins 
               
               
                 20030144234 
                 Methods for the treatment of chronic pain and compositions therefor 
               
               
                 20030143684 
                 Method of identifying inhibitors of C—C chemokine receptor 3 
               
               
                 20030143671 
                 Novel beta-defensins 
               
               
                 20030143618 
                 Method for easy cloning and selection of chimeric DNA molecules 
               
               
                 20030143600 
                 Detection of extracellular tumor-associated nucleic acid in blood plasma or serum 
               
               
                   
                 using nucleic acid amplification assays 
               
               
                 20030143593 
                 Quantative method for measuring gene expression 
               
               
                 20030143577 
                 High fidelity DNA polymerase compositions and uses therefor 
               
               
                 20030143553 
                 Nucleic acid amplification with direct sequencing 
               
               
                 20030143537 
                 Genotyping assay to predict CYP3A5 phenotype 
               
               
                 20030143534 
                 Probes for myctophid fish and a method for developing the same 
               
               
                 20030143531 
                 Detection and quantification of microorganisms using amplification and restriction 
               
               
                   
                 enzyme analysis 
               
               
                 20030143241 
                 Hepatitis E virus vaccine and method 
               
               
                 20030143219 
                 Nucleic acid molecules encoding a transmembrane serine protease 25, the encoded 
               
               
                   
                 polypeptides and methods based thereon 
               
               
                 20030143202 
                 Anemia 
               
               
                 20030139590 
                 DNA encoding SNORF25 receptor 
               
               
                 20030138925 
                 Novel human gene relating to respiratory diseases, obesity, and inflammatory bowel 
               
               
                   
                 disease 
               
               
                 20030138854 
                 Method for determining BAGE expression 
               
               
                 20030138816 
                 Methods and kits for diagnosing the occurrence or the phase of minimal change 
               
               
                   
                 nephrotic syndrome (MCNS) in a human 
               
               
                 20030138803 
                 Identification and use of molecules implicated in pain 
               
               
                 20030138798 
                 Macular degeneration diagnostics and therapeutics 
               
               
                 20030138783 
                 Aberrantly methylated genes as markers of breast malignancy 
               
               
                 20030138772 
                 Method of detecting and/or identifying adeno-associated virus (AAV) sequences and 
               
               
                   
                 isolating novel sequences identified thereby 
               
               
                 20030135879 
                 Methods and materials for making and using transgenic dicamba-degrading organisms 
               
               
                 20030134343 
                 Specific mucin expression as a marker for pancreatic cancer 
               
               
                 20030134310 
                 Cellular kinase targets and inhibitors, and methods for their use 
               
               
                 20030134301 
                 Identification and use of molecules implicated in pain 
               
               
                 20030134295 
                 Method for detection of pathogenic organisms 
               
               
                 20030134293 
                 Method for rapid and accurate identification of microorganisms 
               
               
                 20030134275 
                 Telomerase reverse transcriptase (TERT) genes 
               
               
                 20030131377 
                 DNA molecules from maize and methods of use thereof 
               
               
                 20030131375 
                 Plant regulatory sequences for selective control of gene expression 
               
               
                 20030129746 
                 Epitopes in viral envelope proteins and specific antibodies directed against these 
               
               
                   
                 epitopes: use for detection of HCV viral antigen in host tissue 
               
               
                 20030129724 
                 Class II human histone deacetylases, and uses related thereto 
               
               
                 20030129702 
                 DNA encoding galanin GALR2 receptors and uses thereof 
               
               
                 20030129687 
                 Keratinocyte growth factor-2 
               
               
                 20030129667 
                 Method of diagnosing transmissible spongiform encephalopathies 
               
               
                 20030129631 
                 Gene family with transformation modulating activity 
               
               
                 20030129599 
                 Human hematopoietic growth regulatory gene and uses 
               
               
                 20030129589 
                 DNA diagnostics based on mass spectrometry 
               
               
                 20030129577 
                 Kunitz domain polypeptide Zkun8 
               
               
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     SUMMARY OF THE INVENTION 
     The present invention allows improved detection of such bacterial contaminants, including unconventional forms such as filtering forms (nanoforms), nanobacteria, and L-forms. 
     Its principal applications are the:
         Detection of very low levels of mycoplasma contamination of cell lines and biological fluids   Identification of latent bacterial infections in various pathologies   Detection of live forms passing through filters having a pore size of between 100 and 20 nm   Direct detection of RNA-containing subunits of bacteria.       

     Different primers have been designed:
         MOLL primers have been designed initially to detect mollicutes ( mycoplasma ) species based on the conserved regions of the 16 s ribosomal RNA gene. In fact they can also detect gram positive bacteria       

     
       
         
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
           
               
                 SEQ ID NO:1 
               
             
          
           
               
                 1) Moll Outer Primer (sense) 
                 (AAYGGGTGAGTAACA 
               
               
                   
                 CGT), 
               
               
                   
               
             
          
           
               
                 SEQ ID NO:2 
               
             
          
           
               
                 2) Moll Outer Primer (antisense) 
                 (CCCGAGAACGTATTC 
               
               
                   
                 ACCG) 
               
               
                   
               
             
          
           
               
                 SEQ ID NO:3 
               
             
          
           
               
                 3) Moll Inner Primer (sense) 
                 (CTACGGGAGGCAGC 
               
               
                   
                 AGTA) 
               
               
                   
               
             
          
           
               
                 SEQ ID NO:4 
               
             
          
           
               
                 4) Moll Inner Primer (antisense) 
                 (GTATCTAATCCTRTTT 
               
               
                   
                 GCTCCCCA) 
               
             
          
         
       
         
         
           
             BACT primers will detect gram positive and gram negative bacteria. The sequence of MOLL primers is included in the degenerated sequence of the BACT primers. 
           
         
       
    
     
       
         
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
           
               
                 SEQ ID NO:5 
               
             
          
           
               
                 1) Bact Outer Primer (antisense) 
                 (CCCGRGAACGTATTCA 
               
               
                   
                 CSG), 
               
               
                   
               
             
          
           
               
                 SEQ ID NO:6 
               
             
          
           
               
                 2) Bact Inner Primer (sense) 
                 (CTACGGGAGGCWGCAG 
               
               
                   
                 TRRGGAAT), 
               
               
                   
               
             
          
           
               
                 SEQ ID NO:7 
               
             
          
           
               
                 3) Bact Inner Primer (antisense) 
                 (WGGGTATCTAATCCTR 
               
               
                   
                 TTTGMTCCCCW) 
               
             
          
         
       
     
     The GNEG set of primers is specific of gram negative bacteria. It differs from the Moll 16 out S by a single nucleotide
     1) Gneg Outer Primer (sense) SEQ ID NO: 8 (RAYGGGTGAGTAAYGYMT),   

     The present invention therefore provides a method for identifying an RNA form of a bacteria, comprising reverse transcribing RNA material; conducting PCR using primers for a first highly conserved genetic sequence generic of the bacteria; conducting nested PCR using primers for a second highly conserved genetic sequence within the first amplified genetic sequence of the bacteria; and identifying the bacteria based on unconserved amplified sequences linked to the conserved sequences. 
     It is believed that the Nanoforms are a stable, low metabolic rate form of bacteria, which may be related to pathology, which have characteristic DNA which is generally undetectable by PCR or nested PCR. However, these organisms do have characteristic RNA, and therefore these can be detected by nested RT-PCR. Likewise, because these are now detectable according to the present invention, it is therefore possible to monitor and optimize treatments directed toward clearing these from infected subjects. 
     It is believed that Nanoforms are involved in human pathology, and further that these low metabolic organisms are involved in a constellation of chronic human diseases. Further, it is believed that some of these Nanoforms may be subcellular, that is, incomplete, and therefore may require association with other Nanoform, or other organisms or cells, for replication or reconstitution as a complete DNA bacterial form. Preliminary evidence suggests that the genetic material within a single Nanoforms is insufficient to reconstitute the entirety of a related bacterial (DNA) form, and therefore that multiple Nanoforms may be required in order to be self-replicating for the complete organism. 
     For example, multiple Nanoforms may infect a single cell, together constituting a complete genome for the associated DNA bacteria. Reverse transcriptase activity, for example, due to retroviruses, endogenous retroviral sequences, DNA pol I activity, etc., may be sufficiently active to generate the bacterial genome. 
     These Nanoforms may be biologically associated with retroviruses, such as HIV, which would therefore increase their likelihood of replication, since they would then carry their own reverse transcriptase, and potentially account for replication of sub-cellular fragments. The retroviruses may be passengers within the Nanoforms, and the Nanoforms represent an infectious particle for the virus. 
     The present invention reveals that the Nanoforms retain conserved sequences of 16S rRNA, and therefore may be targeted on this basis, for example by tetracycline analog antibiotics, especially administered over extended durations. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Material and Methods 
     Oligonucleotide Primers. 
     The oligonucleotide primers used were: 
     
       
         
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
           
               
                 SEQ ID NO:1 
               
             
          
           
               
                 1) Moll Outer Primer (sense) 
                 (AAYGGGTGAGTAACAC 
               
               
                   
                 GT), 
               
               
                   
               
             
          
           
               
                 SEQ ID NO:8 
               
             
          
           
               
                 2) Gneg Outer Primer (sense) 
                 (RAYGGGTGAGTAAYGY 
               
               
                   
                 MT), 
               
               
                   
               
             
          
           
               
                 SEQ ID NO:5 
               
             
          
           
               
                 3) Bact Outer Primer (antisense) 
                 (CCCGRGAACGTATTCA 
               
               
                   
                 CSG), 
               
               
                   
               
             
          
           
               
                 SEQ ID NO:6 
               
             
          
           
               
                 4) Bact Inner Primer (sense) 
                 (CTACGGGAGGCWGCAG 
               
               
                   
                 TRRGGAAT), and 
               
               
                   
               
             
          
           
               
                 SEQ ID NO:7 
               
             
          
           
               
                 5) Bact Inner Primer (antisense) 
                 (WGGGTATCTAATCCTR 
               
               
                   
                 TTTGMTCCCCW) 
               
               
                 where R = G or A, S = G or C, W = A or T, M = A or C, and Y = T or C. 
               
             
          
         
       
     
     Expected lengths of amplicons are ˜200 bp and ˜450 bp with the outer and inner primers, respectively. The primers employed were formulated as equal amounts of each primer within the class identified by the sequence. 
     Nucleic Acid Preparation and PCR/RT-PCR. 
     Cell line supernatants (400 μl), human plasma (200-400μl) and human peripheral blood mononuclear cells (PBMC, 3-10 millions cells) were lyzed with 10 mMTris, pH7.4, 10 mM EDTA, 150 mM NaCl, 0.4% SDS, and 10 μg Proteinase K at 60° C. for 1 h. Nucleic samples were extracted three times with one volume of phenol/chloroform and one time with chloroform and precipitated by addition of 1/10 volume of 3M sodium acetate and two volumes of ethanol at −60° C. for 1 h. Samples were centrifuged 30 mm. and the nucleic acid pellets were washed with 70% cold ethanol and solubilized in 10 mM Tris-HCI, pH 8.0. These preparations were stored at −60° C. 
     PCR reaction mix (50 μl) consisted of 5 mM MgCl2, 50 mM Tris, pH 8.0, 15 mM (NH4)2SO4, 10 mM B-Mercaptoethanol, 500 μM dATP, dCTP, dGTP, and DTTP, 0.025% BSA, 1 μM of each outer primer, 1 U Taq polymerase (Roche Molecular biochemicals, Laval, Canada), and 5-10 μl nucleic acid sample. For the first round PCR, the denaturation, annealing, and elongation temperatures and times used were 95° C. for 30 s, 42° C. for 30 s, and 78° C. for 2 m, respectively, for 42 cycles. After the final cycle, the products were kept at 78° C. for 10 m. One μl of the PCR product was subjected to a second round PCR with the set of inner primers. Denaturation, annealing, and elongation temperatures and times used were 95° C. for 30 s, 47° C. for 30 s, and 78° C. for 1 m, respectively, for 42 cycles, followed by a single incubation at 78° C. for 10 m. After the first and second round PCR, 10 μl of PCR product was analyzed by gel electrophoresis using 1.5% agarose, stained with ethidium bromide, visualized under ultraviolet light and photographed. Visible bands with appropriate size were cut and sequenced using the inner primers (DNA Landmarks, St-Jean sur le Richelieu, Canada). Sequence homology search was performed using the BLAST program of the NIH web site. 
     Samples negative for the appropriate band by PCR were subjected to a first round RT-PCR followed by a second round PCR. RT-PCR reaction mix (50 μl) consisted of 5 mM MgCl2, 50 mM Tris, pH 8.0, 15 mM (NH4)2SO4, 10 mM B-Mercaptoethanol, 500 μM dATP, dCTP, dGTP, and DTTP, 0.025% BSA, 1 μM of each outer primer, and Titan enzyme mix (Roche Molecular biochemicals, Laval, Canada), and 5 μl nucleic acid sample. The reverse transcription step was performed at 42° C. for 30 m. The first and second rounds PCR were performed as described above. 
     The precautions addressed elsewhere (Kwok and Higuchi, 1989) were followed to minimize the risk of false-positive results caused by the carry-over of previously amplified DNA. For example, extraction of nucleic acids and preparation of PCR/RT-PCR mix were performed under a sterile flow bench, only aliquoted reagents and filter tips were used, and negative controls were incorporated into each run. 
     Results 
     All Samples: 
     First round PCR/RT-PCR: no detection of expected amplicon (˜1,200 bp). 
     Second round PCR (nested-PCR): all amplicon (˜450 bp) sequences related to bacterial 16S ribosomal RNA gene. 
     Patients&#39; Lymphocytes (18 Samples): 
     No 450 bp amplicon detected by nested-PCR from first round PCR. 
     All samples positive (450 bp) by nested-PCR from RT-PCR. 
     Therefore, bacteria are in an “RNA state”, and are referred to herein as “Nanoforms”. 
     Samples of Patients&#39; Plasma: 
     11 samples/12 positive for 450 bp amplicon by nested-PCR from first round PCR. 
     Discussion 
     Cell wall deficient pathogenic microorganisms, which may be  mycoplasma , so-called L-forms, or potentially other types, are difficult to detect. Therefore, their involvement in pathology may be vastly under-reported. 
     It has been found, however, that these organisms have a well-conserved RNA sequences, such as the 16S rRNA, even when corresponding DNA or RNA is undetectable by a traditional polymerase chain reaction (PCR) or reverse transcriptase PCR (RT-PCR) method, which may be detected by nested RT-PCR amplification, using primers according to the present invention. 
     The present invention therefore provides a sensitive and specific method for detecting bacterial forms, which may be called “Nanoforms”, even when traditional methods fail. This therefore allows diagnosis of pathogens previously unrecognized, and monitoring of treatment thereof.