Methods to Detect Microbes in the Environment Part 2Pathogen Detection by Biochemical MethodsMicroscopic and Imaging Detection of PathogensSlide 4Cryptosporidium parvum Differential Interference Contrast MicroscopySlide 6Microscopic Analysis of Bacteria Fluorescent In Situ Hybridization - FISHFISH: DAPI-stained Bacteria Incubated with INT (Tetrazolium Salt)MOLECULAR BIOLOGY TECHNIQUESMolecular Pathology and DiagnosticsNUCLEIC ACIDSBasesBase-pairingHydrogen BondsDNA: HelixComplementarityAntiparallel ChainsGeneNucleasesRestriction enzymesRestriction enzymes (cont’d)Detection of Pathogens by Detection of Nucleic AcidsHybridizationDenaturation - RenaturationProbesSolid Support HybridizationSouthern BlotsSouthern BlotSlide 29Slide 30Dot/Slot BlotsSlide 32Slide 33Slide 34Nucleic Acid Hybridization to Genotype F+ RNA ColiphagesAgarose Gel ElectrophoresisDirect Detection of Viruses and Other Microbes by Nucleic Acid AmplificationNucleic Acid Amplification - PCRExample: RT-PCR and Oligoprobe Detection of Enteroviruses in WaterReal-Time PCR and Quantitative Fluorogenic DetectionReal-Time, Multiplex RT-PCR: Hepatitis A Virus (HAV) and Enteroviruses (EV)Assessing DNA Polymorphisms to Detect and Characterize Specific BacteriaRestriction Endonucleases used in Molecular BiologyRestriction Fragment PolymorphismsExample: Macrorestriction Analysis of E. coli IsolatesRFLP Analysis ProcedureSlide 47RibotypingSlide 49Slide 50RFLP of Other GenesArbitrarily-Primed PCR (Randomly Amplified Polymorphic DNA or RAPID)Repetitive Element-PCR (Rep-PCR)Detecting Active or Viable Pathogens Using Nucleic Acid TargetsSlide 55Detecting Infectious Viruses by Direct Nucleic Acid Analysis - A Functional ApproachVirus Capture Plus RT-PCR to Detect Infectious Viruses - The sCAR SystemSlide 58Slide 59Slide 60Summary - Detecting and Quantifying Microbes in the EnvironmentFuture Directions in Microbial Detection in the EnvironmentMethods to Detect Microbes in the EnvironmentPart 2ENVR 421Mark D. Sobsey2Pathogen Detection by Biochemical Methods•Enzymatic activities unique to target microbe•Signature Biolipid Analysis:–Detection of unique biolipids by gas-chromatography, mass spectrometry and other advanced organic analytical methods•Extract and purify from cells•Analyze•Other biochemical markers unique to a specific pathogen or class of pathogens.3Microscopic and Imaging Detection of Pathogens•Still widely used for parasites and bacteria•Specific staining and advanced imaging to distinguish target from non-target organisms–Differential interference contrast microscopy–Confocal laser microscopy•Distinguish infectious from non-infectious organisms–Combine with infectivity, viability or activity assays•Overcome sample size limitation due to presence of non-target particles–Flow cytometry and other advanced imaging techniques–Advanced imaging methods require expensive hardware4Microscopic Detection of PathogensStill Widely Used in Clinical Diagnostic Microbiology•Preferred method for parasites–Example: Cryptosporidium parvum oocysts, ~5 um diam.–Acid fast stain of fecal preparation5Cryptosporidium parvumDifferential Interference Contrast MicroscopyImage courtesy of O.D. “Chip” Simmons, III6Cryptosporidium parvum: Microscopic Analysis of NC field isolateDifferential Interference ContrastDAPI stainImmunofluorescenceImages courtesy of O.D. “Chip” Simmons, III7Microscopic Analysis of BacteriaFluorescent In Situ Hybridization - FISH• Bacteria of the target group are red• Other bacteria are blue(artificial colors)8FISH: DAPI-stained Bacteria Incubated with INT (Tetrazolium Salt)Enhanced image with artificial colors. •Blue: DAPI stain •Red: INT grains; indicate respiratory active bacteria.9 MOLECULAR BIOLOGY TECHNIQUESUtilize DNA, RNA, and enzymes that interact with nucleic acids to identify, understand, characterize and quantify biological structures, process, phenomena and activities at a molecular leve10Molecular Pathology and Diagnostics•INHERITED DISEASES (GENETICS)–Cystic fibrosis–Sickle cell anemia–Predispositions to cancer•INFECTIOUS DISEASES–Bacteria–Viruses–Fungi11NUCLEIC ACIDS•Genetic material of all known organisms•DNA: deoxyribonucleic acid•RNA: ribonucleic acid (e.g., some viruses)•Consist of chemically linked sequences of nucleotides•Nitrogenous base•Pentose- 5-carbon sugar (ribose or deoxyribose)•Phosphate group•The sequence of bases provides the genetic information12Bases•Two types of bases•Purines are fused five- and six-membered rings•Adenine A DNA RNA•Guanine G DNA RNA•Pyrimidines are six-membered rings•Cytosine C DNA RNA•Thymine T DNA•Uracil U RNA13Base-pairing•Hydrogen bonds are relatively weak bonds compared to covalent bonds•Hydrogen bonds can form between a pyrimidine and a purine•Watson-Crick base-pairing rules•A T•G C14Hydrogen BondsHHHHOOHCCCCNNCThymineHNHHNCCCCNNHNCAdenineHONHCCCNNCCytosineHHHNCCCCNNHNCGuanineNHOH15DNA: Helix5’5’3’3’In general, DNA is double-stranded. Double-stranded (ds) DNA takes the form of a right handed helix with approximately 10 base pairs per turn of the helix.16Complementarity•In the DNA double helix, purines and pyrimidines face each other•The two polynucleotide chains in the double helix are connected by hydrogen bonds between the bases•Watson-Crick base-pairing rules•A T•G C•GC base pairs (bps)have more energy than AT bps•Since one strand of DNA is complementary to the other, genetic material can be accurately reproduced; each strand serves as the template for the synthesis of the other17Antiparallel Chains5’pOH3’3’OHp5’Two strands of the DNA double helix are antiparallel and complementary to each other18GenepromoterStructural geneflankflankupstreamdownstream5’3’•A gene is a unit of inheritance•Carries the information for a:-polypeptide-structural RNA molecule19NucleasesEndonuclease5’ Exonuclease 3’ Exonuclease20Restriction enzymes•Specific endonucleases•Recognize specific short sequences of DNA and cleave the DNA at or near the recognition sequence•Recognition sequences: usually 4 or 6 bases but there are some that are 5, 8, or longer•Recognition sequences are palindromes•Palindrome: sequence of DNA that is the same when one strand is read from left to right or the other strand is read from right to left– consists of adjacent inverted repeats21Restriction enzymes (cont’d)•Example of a