Molecular Diagnostics - Technologies, Markets and Companies

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Molecular Diagnostics - Technologies, Markets and Companies Published on September 2011

Report Summary This report describes and evaluates the molecular diagnostics technologies that will play an important role in practice of medicine, public health, pharmaceutical industry, forensics and biological warfare in the 21st century. This includes several polymerase chain reaction (PCR)-based technologies, fluorescent in situ hybridization (FISH), peptide nucleic acids (PNA), electrochemical detection of DNA, biochips, nanotechnology and proteomic technologies.

Initial applications of molecular diagnostics were mostly for infections but are now increasing in the areas of genetic disorders, preimplantation screening and cancer. Genetic screening tests, despite some restrictions is a promising area for future expansion of in vitro diagnostic market. Molecular diagnostics is being combined with therapeutics and forms an important component of integrated healthcare. Molecular diagnostic technologies are also involved in development of personalized medicine based on pharmacogenetics and pharmacogenomics. Currently, there has been a considerable interest in developing rapid diagnostic methods for for point-of-care and biowarfare agents such as anthrax.

The number of companies involved in molecular diagnostics has increased remarkably during the past few years. More than 500 companies have been identified to be involved in developing molecular diagnostics and 312 of these are profiled in the report along with tabulation of 695 collaborations. Despite the strict regulation, most of the development in molecular diagnostics has taken place in the United States, which has the largest number of companies.

The markets for molecular diagnostics technologies are difficult to estimate. Molecular diagnostics markets overlap with markets for non-molecular diagnostic technologies in the in vitro diagnostic market and are less well defined than those for pharmaceuticals.

Molecular diagnostic markets are analyzed for 2010 according to technologies, applications and geographical regions. Forecasts are made up to 2020. A major portion of the molecular diagnostic market can be attributed to advances in genomics and proteomics.

Biochip and nanobiotechnology are expected to make a significant contribution to the growth of molecular diagnostics.

This report was first published as DNA Diagnostics in 1995 by PJB Publications, UK. It was updated in 1997 as Molecular Diagnostics and the next edition, Molecular Diagnostics II, was published by Decision Resources Inc in 1999. All the three versions of the reports were well accepted and sold widely.The report has been rewritten several times since then.

Table of Content TABLE OF CONTENTS 0. Executive Summary 23 1. Introduction 25 Definitions and scope of the subject 25 Historical evolution of molecular diagnostics 25 Molecular biology relevant to molecular diagnostics 26 DNA 26 DNA polymerases 26

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Restriction endonucleases 27 DNA methylation 27 RNA 28 RNA polymerases 28 Non-coding RNAs 28 DNA transcription 29 Chromosomes 29 Telomeres 30 Mitochondrial DNA 30 Genes 31 The genetic code 31 Gene expression 31 The human genome 32 Variations in the human genome 32 Variations in DNA sequences 32 Single nucleotide polymorphisms 33 Haplotyping 33 Copy number variations in the human genome 34 Genotype and haplotypes 35 Insertions and deletions in the human genome 35 Complex chromosomal rearrangements 36 Large scale variation in human genome 36 Structural variations in the human genome 37 Replication of the DNA helix 37 Transposons 38 Proteins 38 Proteomics 38 Monoclonal antibodies 39 Aptamers 39 Basics of molecular diagnostics 39 Tracking DNA: the Southern blot 39 Pulsed-field gel electrophoresis 40 DNA Probes 40 The polymerase chain reaction 41 Basic Principles of PCR 41 Target selection 41 Detection of amplified DNA 41 Impact of human genome project on molecular diagnostics 42 Mapping and sequencing of structural variation from human genomes 43 1000 Genomes Project 43 Human Variome Project 44 Role of bioinformatics in molecular diagnostics 45 Systems biology approach to molecular diagnostics 45 Biomarkers 45 Applications of molecular diagnostics 46 2. Molecular Diagnostic Technologies 49 Introduction 49 DNA sample collection and extraction 49

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Blood samples 49 Dried blood spots 49 Buccal swabs and saliva 50 Urine samples for transrenal DNA 50 Manual vs automated DNA extraction 50 Sample preparation 51 Pressure Cycling Technology 51 Membrane immobilization of nucleic acids 51 Automation of sample preparation in molecular diagnostics 52 ABI PRISM 6700 Automated Nucleic Acid Workstation 52 BioRobot technology 52 COBAS AmpliPrep System 52 GENESIS FE500 Workcell 53 GeneMole 53 PCR BioCube 53 QIAsymphony 53 Tigris instrument system 54 Techniques for sample preparation that are suitable for automation 54 Xtra Amp Genomic DNA Extraction 54 Extraction of DNA from paraffin sections 54 Dynabead technology 54 Pressure Cycling Technology 55 SamPrep 55 Use of magnetic particles for automation in genome analysis 55 Companies involved in nucleic acid isolation 56 Labeling and detection of nucleic acids 57 Novel PCR methods 57 Addressing limitations of PCR 57 CAST-PCR 58 Combined PCR-ELISA 58 Convection PCR 58 Digital PCR 59 Emulsion PCR 59 ExCyto PCR 60 Long and accurate PCR 60 Multiplex PCR 60 Real-time PCR systems 61 Dyes used in real-time PCR 62 Commercially available real-time PCR systems 62 LightCycler PCR system 63 LightUp probes based on real-time PCR 63 READ' real-time PCR method 63 StellARray' technology 64 7500 Fast Dx Real-Time PCR Instrument 64 Applications of real-time PCR 64 Limitations of real-time PCR 65 Quantitative PCR for accurate low level DNA analysis 65 Guidelines for real-time qPCR 65

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Future applications of real-time qPCR 66 Reverse transcriptase (RT)-PCR 66 Standardized reverse transcriptase PCR 67 Single cell PCR 67 LATE-PCR 67 COLD-PCR 68 AmpliGrid-System 69 Post-PCR genotyping methods 69 High-resolution melt analysis 69 Monitoring of gene amplification in molecular diagnostics 70 Non-PCR nucleic acid amplification methods 70 DNA probes with conjugated minor groove binder 70 Dynamic Flux Amplification 70 Isothermal reaction for amplification of oligonucleotides 71 ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids) 71 Linked Linear Amplification 71 Multiplex Ligation-Dependent Probe Amplification 72 Rapid analysis of gene expression 72 Rolling circle amplification technology 72 Gene-based diagnostics through RCAT 74 RCAT-immunodiagnostics 74 RCAT-pharmacogenomics 75 Circle-to-circle amplification 75 Ramification amplification method 75 Single Primer Isothermal Amplification 76 Transcription mediated amplification 76 WAVE nucleic acid fragment analysis system 76 Technologies for signal amplification 77 3 DNA dendrimer signal amplification 77 Hybridization signal amplification method 78 Signal mediated amplification of RNA technology 79 Invader assays 79 Hybrid Capture technology 81 Branched DNA test 82 Tyramide signal amplification 82 Non-enzymatic signal amplification technologies 82 Direct molecular analysis without amplification 83 Trilogy' Platform 83 Direct detection of dsDNA 84 Multiplex assays 85 Fluorescent in situ hybridization 85 FISH technique 86 Applications of FISH 86 Modifications of FISH 87 Direct visual in situ hybridization 87 Direct labeled Satellite FISH probes 87 Comparative genomic hybridization 88 Primed in situ labeling 88

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Interphase FISH 88 FISH with telomere-specific probes 89 Multicolor FISH 89 Simultaneous Ultrasensitive Subpopulation staining/Hybridization In situ 89 Automation of FISH 90 Companies involved in FISH diagnostics 90 RNA diagnostics 91 RNA isolation from tissue samples 91 Commercially available tests for mRNA detection and quantitation 91 Branched-chain DNA assay for measurement of RNA 92 Cycling probe technology 92 Invader RNA assays 93 Linear RNA amplification 93 Non-isotopic RNase cleavage assay 93 Nucleic acid sequence-based amplification 94 Q Beta replicase system 96 RNAScope 96 Solid Phase Transcription Chain Reaction 96 Transcriptome analysis 96 Visualization of mRNA expression in vivo 97 MicroRNA diagnostics 97 Real-time PCR for expression profiling of miRNAs 97 Microarray vs quantitative PCR for measuring miRNAs 98 Use of LNA to explore miRNA 98 Nuclease Protection Assay to measure miRNA expression 99 Microarrays for analysis of miRNA gene expression 99 Modification of in situ hybridization for detection of miRNAs 100 Whole genome amplification 100 Companies that provide technologies for whole genome amplification 100 QIAGEN's Repli-G system 101 GenomePlex' Whole Genome Amplification 101 DNA sequencing 102 Companies involved in sequencing 103 Applications of next generation sequencing in molecular diagnostics 104 Companies developing sequencing for molecular diagnostics 105 Genome-wide approach for chromatin mapping 106 Mitochondrial sequencing 106 Identification of unknown DNA sequences 106 Optical mapping 107 Gene expression analysis 107 Gene expression profiling on whole blood samples 108 Gene expression patterns of white blood cells 108 Gene expression profiling based on alternative RNA splicing 109 MAUI (MicroArray User Interface) hybridization 109 Monitoring in vivo gene expression by molecular imaging 110 Serial analysis of gene expression (SAGE) 110 Single-cell gene expression analysis 110 T cell receptor expression analysis 111

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Tangerine' expression profiling 111 Whole genome expression array 112 Ziplex' system 112 Companies involved in gene expression analysis 113 Peptide nucleic acid technology 114 Use of PNA with fluorescence in situ hybridization 114 PNA and PCR 115 Use of PNA with biosensors 115 PNA-based PD-loop technology 116 PNA-DNA hybrid quadruplexes 116 Companies involved in PNA diagnostics 116 Locked nucleic acids 117 Zip Nucleic Acids 117 Electrochemical detection of DNA 118 Mediated nucleic acid oxidation 118 Detection of hybridized nucleic acid with cyclic voltametry 119 Electrochemical detection based on Toshiba's CMOS technology 119 Concluding remarks on electrochemical DNA detection 119 Bead-based assay platforms 120 Scorpions' technology 121 The Scorpions reaction 121 Applications of Scorpions 121 Nucleic acid lateral flow molecular diagnostics 122 3. Biochips, Biosensors, and Molecular Labels 123 Introduction to biochip technology 123 Applications of biochips in diagnostics 123 GeneChip 124 GeneChip Human Genome Arrays 125 AmpliChip CYP450 125 Electronic detection of nucleic acids on microarrays 125 Microchip capillary electrophoresis 126 Strand displacement amplification on a biochip 126 Rolling circle amplification on microarrays 126 LiquiChip-RCAT 126 Fast PCR biochip 127 Multiplex microarray-enhanced PCR for DNA analysis 127 Multiplexed Molecular Profiling 127 Universal DNA microarray combining PCR and ligase detection reaction 128 Genomewide association scans 128 Whole genome chips/microarrays 129 Transposon insertion site profiling chip 129 Standardizing the microarrays 130 Companies involved in developing biochip technology for diagnostics 130 Future of biochip technology for molecular diagnostics 131 Microfluidic chips 132 Fish-on-chip 132 Lab-on-a-chip 132 LabCD 133

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Micronics' microfluidic technology 133 Microfluidic chips/arrays using PCR 133 Microfluidic automated DNA analysis using PCR 133 Digital PCR Array 133 Digital PCR on a SlipChip 134 Microfluidic chips integrated with RCAT 134 Microfluidic chips integrated with PET 134 Companies developing microfluidic technologies 135 Biosensor technologies 135 Classification of biosensor technologies 136 DNA-based biosensors 137 DNA hybridization biosensor chips 137 PCR-free DNA biosensor 137 DNA based biosensor to detects metallic ions 138 Genetically engineered B lymphocytes 138 Biosensors immunoassays 138 PNA (peptide nucleic acid)-based biosensors 139 Protein-based biosensors 139 Antibody biosensors 139 Cell-based biosensors (cytosensors) 139 Multicell biosensors 140 Microbial biosensors 140 Optical biosensors 141 Surface plasmon resonance technology 141 Label-free optical biosensor 142 Microsensors using with nano/microelectronic communications technology 142 Electrochemical sensors 142 Enzyme electrodes for biosensing 142 Conductometric sensors 143 Electrochemical genosensors 143 Electrochemical nanobiosensor 143 Bioelectronic sensors 144 Phototransistor biochip biosensor 144 Ribozyme-based sensors 144 RiboReporters 145 Concluding remarks and future prospects of biosensor technology 145 Companies developing biosensors for molecular diagnostics 146 Molecular labels and detection 147 Detection technologies for molecular labels 148 Fluorescence and chemiluminescence 148 Fluorescence technologies for label detection 149 Companies with fluorescence and chemiluminescence products 149 Molecular beacons 150 The Green fluorescent protein 151 Multiophoton detection radioimmunoassay 152 Multi-pixel photon counter 152 Enzyme labels and detection by fluorescence 152 Phase-sensitive flow cytometry 153

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Microtransponder-based DNA diagnostics 153 Laboratory Multiple Analyte Profile 154 Multiple labels 154 Protein-DNA chimeras for detection of small numbers of molecules 155 Single molecule detection 155 Atomic force microscopy 156 Capillary electrophoresis 156 Confocal laser scanning 156 nCounter Analysis System 156 Spectrally resolved fluorescence lifetime imaging microscopy 157 Molecular imaging 157 Basic research in molecular imaging 158 Devices for molecular imaging 158 Molecular imaging in clinical practice 158 Challenges and future prospects of molecular imaging 159 Companies involved in molecular imaging 159 Nanobiotechnology for molecular diagnostics 159 Magnetic nanoparticles 160 Gold nanoparticles 161 Quantum dot technology 162 Nanotechnology on a chip 163 Nanogen's NanoChip 163 Fullerene photodetectors for chemiluminescence detection on microfluidic chip 163 Diagnostics based on nanopore technology 164 Nanosensors 164 Detection of cocaine molecules by nanoparticle-labeled aptasensors 164 Nanosensors for glucose monitoring 165 PEBBLE nanosensors 165 Quartz nanobalance biosensor 165 Cantilever arrays 165 Resonance Light Scattering technology 166 DNA nanomachines for molecular diagnostics 167 Nanobarcodes technology for molecular diagnostics 167 Qdot nanobarcode for multiplexed gene expression profiling 167 Role of nanobiotechnology in improving molecular diagnostics 168 Companies involved in nanomolecular diagnostics 168 Concluding remarks about nanodiagnostics 171 Future prospects of nanodiagnostics 171 4. Proteomic Technologies for Molecular Diagnostics 173 Introduction 173 Proteomic technologies 173 Biomarkers of disease 173 Proteomic tools for biomarkers 173 Search for biomarkers in body fluids 174 Captamers with proximity extension assay for proteins 174 Cyclical amplification of proteins 174 Detection of misfolded proteins by ELISA with exponential signal amplification 175 Diagnostics based on designed repeat proteins 175

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Differential Peptide Display 175 Light-switching excimer probes 176 MALDI-TOF MS 176 Molecular beacon aptamer 177 Molecular beacon assay 177 Proteomic patterns 178 Real-time PCR for protein quantification 178 Protein biochip technologies 179 ProteinChip 180 LabChip for protein analysis 180 TRINECTIN proteome chip 181 Protein chips for antigen-antibody interactions molecular diagnostics 181 Microfluidic devices for proteomics-based diagnostics 181 Nanotechnology-based protein biochips/microarrays 182 Nanoparticle protein chip 182 Protein nanobiochip 182 Protein biochips based on fluorescence planar wave guide technology 182 New developments in protein chips/microarrays 183 Antibody microarrays 183 Aptamer-based protein biochip 184 Multiplexed Protein Profiling on Microarrays 184 Proteomic pattern analysis 184 Single molecule array 185 Viral protein chip 185 Commercial development of protein chips for molecular diagnostics 185 Proteome Identification Kit 187 Laser capture microdissection (LCM) 187 LCM technology 187 Applications of LCM in molecular diagnostics 188 Proteomic diagnosis of CNS disorders 188 Cerebrospinal fluids tests based on proteomics 188 Urine tests for CNS disorders based on proteins in urine 189 Diagnosis of CNS disorders by examination of proteins in the blood 189 Diagnosis of CNS disorders by examination of proteins in tears 190 Role of proteomics in the diagnosis of Alzheimer's disease 190 Role of proteomics in the diagnosis of Creutzfeldt-Jakob disease 191 Future prospects of use of proteomics for diagnosis of CNS disorders 191 Concluding remarks on the use of proteomics in diagnostics 191 5. Molecular Diagnosis of Genetic Disorders 193 Introduction 193 Cytogenetics 194 FISH with probes to the telomeres 194 Single copy FISH probes 194 Comparative genomic hybridization 195 Use of biochips in genetic disorders 195 Representational oligonucleotide microarray analysis 196 SignatureChip速-based diagnostics for cytogenetic abnormalities 196 Diagnosis of genomic rearrangements by multiplex PCR 196

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Quantitative fluorescent PCR 196 Mutation detection technologies 197 PCR-based methods for mutation detection 198 Cleavase Fragment Length Polymorphism 198 Direct dideoxy DNA sequencing 198 Digital Genetic Analysis (DGA) 198 Fluorescence-based directed termination PCR 199 Fluorescence melting curve analysis for multiplex mutation detection 199 Heteroduplex analysis 200 Restriction fragment length polymorphism 200 Single-stranded conformation polymorphism (SSCP) analysis 200 TaqMan real-time PCR 201 Non-PCR methods for mutation detection 201 Arrayed primer extension 201 BEAMing (beads, emulsion, amplification, and magnetics) 202 ELISA-protein truncation test 202 Enzymatic mutation detection 202 Specific anchor nucleotide incorporation 203 Conversion analysis for mutation detection 203 Biochip technologies for mutation detection 203 Combination of FISH and gene chips 204 Haplotype Specific Extraction 204 Technologies for SNP analysis 204 DNA sequencing 205 Electrochemical DNA probes 206 Use of NanoChip for detection of SNPs 206 Single base extension-tag array 206 Laboratory Multiple Analyte Profile 207 SNP genotyping with gold nanoparticle probes 207 PCR-CTPP (confronting two-pair primers) 207 Peptide nucleic acid probes for SNP detection 208 SNP genotyping on a genome-wide amplified DOP-PCR template 208 Pyrosequencing 208 Reversed enzyme activity DNA interrogation test 209 Smart amplification process version 2 210 Zinc finger proteins 210 UCAN method (Takara Biomedical) 210 Biochip and microarray-based detection of SNPs 210 SNP genotyping by MassARRAY 210 Electronic dot blot assay 211 Biochip combining BeadArray and ZipCode technologies 211 SNP-IT primer-extension technology 211 OmniScan SNP genotyping 212 Affymetrix SNP genotyping array 212 Concluding remarks on SNP genotyping 212 Limitations of SNP in genetic testing 213 Haplotyping versus SNP genotyping 213 Nanofluidics technology for high throughput SNP genotyping 213

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Companies involved in developing technologies/products for SNP analysis 214 Role of copy number variations in genetic diagnostic testing 215 CNVs in various diseases 215 CNVs in genetic epilepsy syndromes 215 CNVs associated with schizophrenia 216 Methods for determination of CNVs 216 Digital array for CNV detection 217 Wellcome Trust Case Control Consortium CNV typing array 217 CNVer algorithm for CNV detection 217 Study of rare variants in pinpointing disease-causing genes 218 Prenatal DNA diagnosis 218 Amniocentesis 218 Chorionic villus sampling 219 Separating fetal cells in maternal blood for genetic diagnosis 219 Antenatal screening for Down's syndrome 219 Fetal DNA in maternal blood 220 Molecular methods for prenatal diagnosis 220 aCGH for prenatal diagnosis 220 BAC HD Scan test 221 FISH for prenatal diagnosis 221 PCR for prenatal diagnosis 221 Plasma DNA sequencing to detect fetal chromosomal aneuploidies 222 In vivo gene expression analysis of the living human fetus 222 Noninvasive prenatal diagnosis of monogenic diseases 222 Digital relative mutation dosage 222 Massively parallel plasma DNA sequencing 223 Applications of prenatal diagnosis 223 Diagnosis of congenital infections 224 Diagnosis of congenital CMV 224 Diagnosis of eclampsia 225 Use of transrenal DNA for prenatal testing 225 Preimplantation genetic diagnosis 225 Technologies for preimplantation genetic diagnosis (PGD) 226 PCR for preimplantation genetic diagnosis 226 FISH for preimplantation genetic diagnosis 226 Microarrays for preimplantation genetic diagnosis 227 Conditions detected by preimplantation genetic diagnosis 227 The future of preimplantation genetic diagnosis 227 Companies involved in prenatal/preimplantation diagnosis 228 Cystic fibrosis 229 Detection of CFTR gene mutations 229 CFTR technologies of various companies 230 Genzyme's CF gene sequencing 231 CF Plus' Tag-It Cystic Fibrosis Kit 231 Asuragen's bead array test 231 The Ambry CF Test 232 Biochip for CF diagnosis 232 Identification of CF variants by PCR/Oligonucleotide Ligation Assay 232

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SensiGene (SEQUENOM) CF carrier screening test 233 Serum proteomic signature for CF using antibody microarrays 233 Guidelines for genetic screening for CF 233 Congenital adrenal hyperplasia 233 Primary immunodeficiencies 234 Hematological disorders 235 Hemoglobinopathies 235 Sickle cell anemia 235 Thalassemia 236 Paroxysmal nocturnal hemoglobinuria 236 Hemophilia 236 Hereditary hemochromatosis 237 Polycystic kidney disease 237 Hereditary metabolic disorders 237 Lesch-Nyhan Syndrome 237 Gaucher's Disease 238 Acute Intermittent Porphyria 238 Phenylketonuria 238 Hereditary periodic fever 239 Achondroplasia 239 Molecular diagnosis of cardiovascular disorders 239 Coronary heart disease 240 Genomics of coronary heart disease 241 Cardiomyopathy 242 Familial Hypertrophic Cardiomyopathy 242 Idiopathic dilated cardiomyopathy 242 Cardiac Arrhythmias 242 Long Q-T Syndrome 242 Familial atrial fibrillation 243 Idiopathic ventricular fibrillation 243 Congestive heart failure 243 Hypertension 244 Disturbances of blood lipids 244 Familial dyslipoproteinemias 244 Hypercholesterolemia 244 Thrombotic disorders 245 Factor V Leiden mutation 245 Pulmonary embolism 245 Companies developing molecular diagnostics for cardiovascular disorders 246 Molecular diagnosis of eye diseases 247 Molecular diagnosis of retinitis pigmentosa 247 Genetic screening for glaucoma 247 Role of molecular diagnostics in rheumatoid arthritis 248 Molecular diagnosis of neurogenetic disorders 248 Alzheimer's disease 250 Autism spectrum disorders 250 CNVs associated with autism 250 Charcot-Marie Tooth disease 251

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Down syndrome 252 Duchenne and Becker muscular dystrophy 252 eNOS gene polymorphisms as predictor of cerebral aneurysm rupture 253 Fragile X syndrome 253 Huntington disease 254 Hereditary neuropathy with liability to pressure palsies 254 Mitochondrial disorders affecting the nervous system 255 Parkinson's disease 255 Pompe's disease 256 Spinal muscular atrophy 256 Triple repeat disorders 256 Genetic testing for disease predisposition 257 Direct-to-consumer genetic tests 257 6. Molecular Diagnosis of Infections 261 Introduction 261 Molecular techniques for the diagnosis of infections 261 Antibody-enhanced microplate hybridization assays 262 Bacteriophage-based methods for detection of bacteria 262 Biosensors for detection of microorganisms 263 Ibis T5000' Biosensor System 263 DNA enzyme immunoassay 263 DNA biochip/microarray in diagnosis of infections 263 DNA-based typing methods 264 Restriction fragment length polymorphism analysis 264 Ribotyping 265 Random amplified polymorphic DNA 265 Combinatorial DNA melting assay 265 Electrochemical detection of pathogens 265 FISH for detection of infections 266 Helicase-dependent isothermal amplification for rapid detection of pathogens 266 High resolution melt analysis for diagnosis of infections 266 Ligase chain reaction 266 Mass spectrometry for microbial identification 267 Metagenomic pyrosequencing 267 Multiplex PCR for detection of infections 268 Dual priming oligonucleotide for multiplex PCR 269 LightCycler速 SeptiFast Test 269 Multiplex amplified nominal tandem repeat analysis 270 VYOO速 Sepsis Test 270 NASBA for detection of microorganisms 270 Nucleic acid probes 270 Neutrophil CD11b expression as a diagnostic marker 271 Optical Mapping 271 PNA-FISH for diagnosis of infections 271 QuantiFERON速 technology for pre-molecular diagnosis of infections 272 Quantitative reverse-transcription PCR for bacterial diagnostics 272 Rupture event scanning 272 Real-time single-molecule imaging of virus particles 273

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Single-strand conformational polymorphism 273 SmartGene platform for identifying pathogens based on genetic sequences 273 Tessera array technology 273 Applications, advantages and limitations of molecular diagnostics 274 Molecular diagnostics versus other microbial detection technologies 274 Advantages of nucleic acid-based diagnostics in infections 274 Drawbacks of nucleic acid-based diagnostics in infections 275 Nanotechnology for detection of infectious agents 275 Bacterial and fungal infections 276 Mycobacterium tuberculosis 277 Conventional diagnosis of tuberculosis 277 Microscopic Observation Drug Susceptible Assay for tuberculosis 278 Molecular diagnostics for tuberculosis 278 Combined tuberculin testing and ELISpotPLUS assay 280 Biomarkers for tuberculosis 280 Diagnosis of drug-resistant M. tuberculosis infection 280 Xpert MTB/RIF automated molecular test for MTB 281 Diagnosis of other mycobacteria 282 Chlamydial infections 282 Neisseria gonorrhoeae 284 Bacteria associated with bacterial vaginosis 284 Streptococcal infections 284 Group B Streptococci 284 Streptococcus pyogenes and Streptococcus dysgalactiae 285 Pseudomonas aeruginosa 285 Helicobacter pylori 285 Lyme disease 286 Mycoplasmas 287 Fungal infections 287 Aspergillus 288 Candida species 288 Viral infections 289 HIV/AIDS 289 Diagnosis of HIV 290 Neonatal screening of infants of HIV-positive mothers 290 Screening of cadaveric tissue donors 291 Detection of HIV provirus 291 Resolution of indeterminate Western blot 291 Global Surveillance of HIV-1 genetic variations 291 Genotyping for drug-resistance in HIV 292 Phenotyping as predictor of drug susceptibility/resistance in HIV 293 Tests used for quantification of HIV 293 Conclusions about HIV genotyping 294 Hepatitis viruses 295 Hepatitis A virus 295 Hepatitis B virus 295 Hepatitis C virus 296 Detection and quantification of HCV RNA 297

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Quantification of HCV RNA levels as a guide to antiviral therapy 297 Electrochemical DNA chip for diagnosis of HCV 298 HCV Genotyping as a guide to therapy 298 Enteroviruses 298 Adenoviruses 299 Rhinoviruses 300 Herpes viruses 300 Herpes simplex virus 300 Genital and neonatal herpes simplex 300 Human cytomegalovirus infections 301 Epstein-Barr virus 301 Human papilloma virus 302 Molecular diagnostics for HPV 302 Detection of encephalitis viruses 303 West Nile and St. Louis encephalitis 303 Venezuelan equine encephalitis virus 303 Protozoal infections 304 Amebiasis 304 Cryptosporidium parvum 304 Leishmaniasis 304 Malaria 304 Neurocysticercosis 305 Pneumocystis carinii 305 Toxoplasmosis 306 Infections of various systems 306 CNS infections 306 Molecular diagnosis in bacterial meningitis 306 Molecular diagnosis in herpes simplex encephalitis 306 Diagnosis of transmissible spongiform encephalopathies 307 Molecular diagnosis of respiratory viruses 308 SARS-associated coronavirus 308 Influenza viruses 310 Avian influenza 311 H1N1 influenza 315 Gastrointestinal infections 317 Periodontal infections 318 Diagnosis of urinary infections by a biosensor 319 Role of molecular diagnostics in septicemia 319 Limitations and needs of diagnostics for infections 320 Differentiation between live and antibiotic-killed bacteria 321 Cell-based methods for identifying pathogenic microorganisms 321 Cell-based virus assays 321 Cell-based detection of host response to infection 321 Role of molecular diagnostics in hospital acquired infections 322 Detection of hospital-acquired bacterial infections 322 Detection of methicillin-resistant S. aureus 322 Detection of vancomycin-resistant enterococci 323 Detection of hospital-acquired C. difficile 323

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Bacterial genome sequencing in antimicrobial resistance 324 Detection of hospital-acquired viral infections 325 Molecular diagnosis of BK virus 325 Diagnosis of hospital-acquired rotavirus gastroenteritis 325 Molecular diagnostics and the microbiome 326 Human Microbiome Project 326 Application of metagenomics to study of the microbiome 326 MicroBiome Analysis Center 327 Concluding remarks and future prospects of diagnosis of infections 327 Rapid point-of-care diagnosis of infection 328 Diagnosis of viruses using protein fingerprinting 330 QIAplex PCR multiplex technology 330 Companies involved in molecular diagnosis of infectious diseases 331 7. Molecular Diagnosis of Cancer 333 Introduction 333 Cancer genomics 333 Cancer genes 334 Oncogenes 334 Tumor Suppressor Genes 334 p53 335 p16 336 CNVs in cancer 336 Allele-specific copy number analysis of tumors 337 Viruses and cancer 337 Detecting viral agents in cancer 338 Conventional cancer diagnosis 339 Molecular techniques for cancer diagnosis 339 Genome analysis at the molecular level 341 Mutation detection at molecular level 341 Expression profiling of tumor cells sorted by flow cytometry 342 MicroRNA expression profiling for cancer diagnostics 342 Biomarkers in cancer 342 Circulating nucleosomes in serum of cancer patients 343 Detection of DNA methylation 343 eTag assay system for cancer biomarkers 345 HAAH as a biomarker for cancer 346 LigAmp for detection of gene mutations in cancer 346 Mitochondrial DNA as a cancer biomarker 346 Oncoproteins as biomarkers for cancer 347 Sequencing-based approaches for detection of cancer biomarkers 347 Molecular fingerprinting of cancer 348 Fluorescent in situ hybridization 348 Genetic analysis of cancer 349 Comparative genomic hybridization in cancer diagnostics 349 Loss of heterozygosity 349 Digital karyotyping 349 Gene expression profiles predict chromosomal instability in tumors 350 PCR Techniques 350

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Realtime quantitative PCR for diagnosis of cancer 351 Cold-PCR 351 Antibody-based diagnosis of cancer 351 Monoclonal antibodies for diagnosis of cancer 351 Recombinant antibodies as a novel approach to cancer diagnosis 352 Combined immunological and nucleic acid tests 352 Combination of MAbs and RT-PCR 352 Immunobead RT-PCR 352 Assays for determining susceptibility to cancer 353 Gene expression profiling in cancer 353 Microarrays for gene expression profiling in cancer 354 Serial analysis of gene expression (SAGE) 354 DNA tags for finding genes expressed in cancer 354 Suppression subtractive hybridization 355 Measurement of telomerase activity 355 Detection of circulating tumor cells in blood 356 Biochips/microfluidics for detection of CTCs 356 CellSearch 357 CellTracks速 AutoPrep速 System 357 CTCscope system for detection of CTCs 357 CTChip' 357 Fiber-optic array scanning technology 357 Lab-on-chip for the isolation and detection of CTCs 358 MagSweeper 358 Future prospects of detection of cancer cells in blood 358 Epithelial aggregate separation and isolation 359 Proteomic technologies for the molecular diagnosis of cancer 359 Proteomic technologies for tumor biomarkers 359 Affibodies as contrast agents for imaging in cancer 360 Aptamer-based technology for protein signatures of cancer cells 360 Aptamer probes for in vivo diagnosis of cancer 360 Aptamers for combined diagnosis and therapeutics of cancer 361 Automated image analysis of nuclear protein distribution 361 Laser capture microdissection in oncology 362 Layered expression scanning 362 Membrane-type serine protease-1 362 Survivin and molecular diagnosis of cancer 363 Biochip/microarrays for cancer diagnosis 363 Role of DNA microarrays in gene expression profiling 364 Biochip detection of FHIT gene 364 Nanobiotechnology for early detection of cancer 364 Detection of nanoparticle self assembly in tumors by MRI 365 Differentiation between normal and cancer cells by nanosensors 365 Magnetic nanoparticle probes 365 Quantum dots for early detection of cancer 365 Molecular imaging of cancer 366 In vivo tumor illumination by ade

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