Methods to Detect Microbes in the Environment ENVR 133 – Part 2

1. Methods to Detect Microbes in the EnvironmentENVR 133 – Part 2 Mark D. Sobsey

2. Detection of Pathogens by Detection and Amplification of Nucleic Acids Nucleic Acid Hybridization: potentially very useful, but: (i)high detection limits (about 100-1000 genomic targets or more) (ii) large sample volumes; impractical for most hybridization protocols without further concentration (iii) hybridization reaction failures (false negatives) and ambiguities (false positives) due to sample-related interferences and non‑specific reactions, and (iv) uncertainties about whether positive reactions are truly indicative of infectious pathogens.

3. Some Methods for Molecular Genetic Detection & Typing of Microbes

4. Progress in Detection of Environmental Pathogens by Nucleic Acid Hybridization Cons: early 1990s • High detection limits (>1000 genomic targets) • Sample volumes too large without concentration • False (-) and false (+) due to sample interferences • Uncertain if positive reactions truly indicate infectious pathogens Pros: late 1990s • Confirm identity of PCR and RT-PCR products • Oligoprobe hybridization • Detect PCR products as they are generated • Labeled primers • Simultaneously genotype many gene targets with multiple probes • Reverse Line Blot Hybridization Assay (caliciviruses)

5. Agarose Gel Electrophoresis • Separate nucleic acid fragments in an agarose gel • Resolves small DNA molecules: 0.1 to 50 kb • % agarose determines resolution of DNA size: • 0.3% w/v: resolves 5 to 50 kb • 2% w/v resolves 0.1 to 2 kb • Resolving large molecules (up to 500 kb) requires specialized methods • Pulse-field gel electrophoresis (PFGE) DNA marker ladder Specific DNA fragment

6. Direct Detection of Viruses and Other Microbes by Nucleic Acid Amplification For viruses not growing in lab hosts: • Detect directly by in-vitro amplification of their nucleic acids • PCR (DNA viruses) or RT-PCR (RNA viruses) • Amplify nucleic acids (105-106 times) • Detect by oligoprobe hybridization OR: • Amplify nucleic acids and detect in real-time by fluorescent signal as primers are incorporated during amplification • Taqman PCR with LightCycler

7. Nucleic Acid Amplification - PCR

8. Example: RT-PCR and Oligoprobe Detection of Enteroviruses in Water • Filter • Elute • Precipitate • Extract RNA • RT-PCR • Oligoprobe • (10 ul sample)

9. Real-Time PCR and Quantitative Fluorogenic Detection • Molecular beacon. Several 5' bases form base pairs with several 3' bases; reporter and quencher in close proximity. • If reporter is excited by light, its emission is absorbed by quencher & no fluorescence is detected. • Detection of PCR product by molecular beacon. • Beacon binds to PCR product and fluoresces when excited by the appropriate  of light. • [Fluorescence] proportional to [PCR product amplified]

10. Real-Time, Multiplex RT-PCR:Hepatitis A Virus (HAV) and Enteroviruses (EV) • 1 2 3 4 5 6 7 8 • Fluorescent probes to simultaneously detect HAV and EV (CVB3). • HAV and EV primer pairs gave predicted 244 and 145-bp products. • Detect <10 genomic RNA copies • Evaluated for virus detection in spiked water concentrate. • Fluorogenic reporter probes (FAM- and ROX-labeled) specifically detected HAV or enterovirus, respectively. • No amplified products from viruses not belong to these group. • 1. Std, 100 bp fragments • 2. CVB3 , 145 bp • 3. negative control • 4. HAV, 244 bp • 5.negative control • 6. CVB3 and HAV • 7.negative control • 8. Std, 100 bp fragments

11. Assessing DNA Polymorphisms to Detect and Characterize Specific Bacteria • Molecular methods used to group or type bacteria based on genomic homogeniety or diversity • Identifies groups of closely-related isolates (presumed to arise from a common ancestor in the same chain of transmission) and divergent, epidemiologically unrelated isolates arising from independent sources. • Restriction fragment length polymorphisms: variable and distinct size fragments of DNA detected by cutting DNA at unique sites using specific restriction endonucleases • Macrorestriction analysis • Ribotyping: cutting DNA amplifies from 16S ribosomal RNA • Restriction analysis of virulence-associated genes • Arbitrary-primed PCR (Randomly Amplified Polymorphic DNA)

12. Restriction Endonucleases used in Molecular Biology

13. Restriction Fragment Polymorphisms • Variations in DNA sequences are manifest as changes in some recognition sites for specific restriction endonuclease enzymes • Alters size and number of DNA fragments obtained from restriction enzyme digestion of chromosomal DNA • Whole genomic DNA: macrorestriction analysis • Specific gene(s): ribotyping (rRNA operons)

14. Example: Macrorestriction Analysis of E. coli Isolates

15. RFLP Analysis Procedure • Isolate chromosomal DNA • Digest DNA with restriction endonuclease • Agarose gel electrophoresis • Macrorestriction analysis • Southern blotting and hybridization • Transfer DNA from gel to membrane (cellulose or nylon) • Hybridize with labeled probe to gene of interest • e.g., rDNA • Ribotype

16. DNA from electrophoresed gel (left) is transferred to membrane filter by contact and DNA on membrane is hybridized with specific probe(s) (right)

17. Ribotyping • Gene-specific RFLP for polymorphisms in rRNA genes (rDNA) • Identify rDNA fragments from electrophoresed chromosomal restriction digests by Southern hybridization • Use specific restriction enzymes with good discrimination abilities to generate restriction patterns from rDNA • rRNA is found in all bacteria • Some sequences are highly conserved and are common in broad groups (genera); can identify genus as first step with broad rRNA probe • rDNA has less but sufficient variability compared to other genes to type specific species and strains of related bacteria

20. RFLP of Other Genes • Species-specific genes as targets for RFLP • Virulence genes • Toxins • Pili • Flagellar genes • Outer membrane protein genes

21. Arbitrarily-Primed PCR (Randomly Amplified Polymorphic DNA or RAPID) • Identifies strain-specific variations in DNA • Use arbitrarily-chosen primers pairs (10- to 20-mers) to amplify chromosomal DNA under non-stringent conditions • Variations in DNA sequences of different strains will give differences in numbers and sizes of their PCR products • Provides a unique DNA fingerprint • Limited number of patterns or groups per species of bacterium • Problems in reproducability and interpretation have occurred

22. Repetitive Element-PCR (Rep-PCR) • PCR amplify specific fragments of chromosomal DNA lying between known repeat motifs of the chromosome • Use two outwardly directed primers for the repeat element at high stringency to generate unique DNA products that are strain-specific.

23. Detecting Active or Viable Pathogens Using Nucleic Acid Targets Detect short-lived nucleic acids present in only viable/infectious microbes: • ribosomal RNA • messenger RNA • genomic RNA of viruses (large amplicons) • Detect pathogen nucleic acid by fluorescent in-situ hybridization (FISH) • applied to bacteria, protozoan cysts and oocysts, as well as viruses in infected cell cultures • (see pictures in later slides)

24. Viruses (and other microbes) growing slowly or without visible signs of growth: Detect rapidly by amplification of nucleic acids produced in cells or by vial nucleic acids in host cells Integrated cell culture-PCR (or RT-PCR) for viruses mRNA in viable cells Infectious Microbe Detection by Nucleic Acid Amplification Target RNA (viral RNA or mRNA) Reverse transcribe Polymerase Chain Reaction Amplification (PCR) Nucleic acids in cells or in virus-infected infected cells

25. Detecting Infectious Viruses by Direct Nucleic Acid Analysis - A Functional Approach Infectious Non-infectious Nucleic acid • Direct nucleic acid analysis alone does not assure detection of infectious viruses • Nucleic acid still present in inactivated viruses or free in the sample (water, etc.) • Infectious viruses have intact surface chemistries (epitopes) that react with host cells to initiate virus infection • The presence of functional surface epitopes for binding to cell receptors is evidence of virus infectivity Cell Receptor In Out

26. Virus Capture Plus RT-PCR to Detect Infectious Viruses - The sCAR System • The cell receptor gene for Coxsackieviruses and Adenoviruses has been cloned and expressed, producing a soluble protein receptor, sCAR • Expressed, purified and bound sCAR to solid phases to capture infectious Coxsackieviruses from environmental samples • The nucleic acid of the sCAR-captured viruses is RT-PCR amplified for detection and quantitation

27. Application of sCAR with Para-Magnetic Beads for Virus Particle Capture and then RT-PCR sCAR purification Covalent coupling to paramagnetic beads Culture + media; :sCAR produced Blocking post-coupling (RT-) PCR : sCAR NA extraction Sample containing viruses : Virus Particle : Blocking protein Amine Terminated Support Magnetic Bead : BioSpheres(Biosource) Pre-coated to provide available amine groups for covalent coupling of proteins or other ligands by glutaraldehyde-mediated coupling method

28. 200bp SM 103 100 10 1 0.1 PFU 103 100 + Ligand Capture of CVB3 Followed by RT-PCR (Magnetic Bead-sCAR-CVB3) Ligand capture Bead control Ligand capture: capture of CVB3 with magnetic beads coupled with purified sCAR Bead control : Reaction of CVB3 with BSA coated magnetic bead Magnetic Bead : BioSpheres (Amine Terminated Support) Viral RNA extraction: QIAamp kit

29. Microbe Nucleic Acid Detection by DNA Microarrays or “Gene Chip” Technology Generate/obtain DNA complimentary to genes (sequences) of interest; • 1000s of different ones Apply tiny quantities of each different one onto solid surfaces at defined positions • “gene chip” or “DNA microarray” Isolate or amplify target NA of interest and label with a fluorescent probe Apply sample NA to the “gene chip” surface • Sample NA binds to specific DNA probes on chip surface; wash away unbound NA Detect bound DNA or RNA by fluorescence after laser excitation Analyze hybridization data using imaging systems and computer software Fluorescing Gene Chip or DNA Microarray

30. Microscopic Detection of Pathogens: Still Widely Used in Clinical Diagnostic Microbiology C. parvum oocysts ~5 um diam. Acid fast stain of fecal preparation

31. Microscopic 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 hardware

32. Fluorescent In Situ Hybridization - FISH • Bacteria of the target group are red • Other bacteria are blue • (artificial colors)

33. FISH: DAPI-stained Bacteria Incubated with INT (Tetrazolium Salt) • Enhanced image with artificial colors. • Blue: DAPI stain • Red: INT grains; indicate respiratory active bacteria.

34. Cryptosporidium parvumDifferential Interference Contrast Microscopy Image courtesy of O.D. “Chip” Simmons, III

35. Cryptosporidium parvum: Microscopic Analysis of NC field isolate Immunofluorescence Differential Interference Contrast DAPI stain Images courtesy of O.D. “Chip” Simmons, III

36. Pathogen 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.

37. Summary - Detecting and Quantifying Microbes in the Environment • Get representative samples • Recover the microbes from the samples • may have to separate, concentrate and purify • very low numbers lots of other similar objects and other stuff (interferences) • Analyze for the recovered microbes: • observe and count them - microscopy/imaging • culture them on media or in live hosts • detect them as antigens (immunoassays) • detect their genetic material (nucleic acid assays) • detect their unique or characteristic chemical properties or other properties (e.g., antibiotic resistance)

38. Future Directions in Microbial Detection in the Environment • Rapid and Sensitive Pathogen Detection Methods • Molecular detection for real-time or near real-time monitoring of pathogens (BT agents, too). • Real-time PCR • Couple with methods to selectively recover and detect potentially infectious microbes • Enrich for virulence genes of microbes in environmental media - early warning/alerts system • Nucleic acid microarrays (“gene chips”) for 1000s at a time • Culture plus molecular or immunodetection • Detect pathogen nucleic acids or antigens early in microbial proliferation in culture