1	Principles and applications of molecular tests in microbiology Steven Drews October 14, 2005 9:15-9:55
2	Objectives Introduce some basic molecular biology concepts Introduce principles of some common molecular diagnostic tests
3	Tests can be used indirectly or directly on patient specimens Indirectly-targets are specific nucleic acid sequences in microorganisms isolated from a patient specimen Directly-targets are nucleic acid from a pathogen in a clinical sample without isolation of the organisms
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5	The basic structural components of stored genetic information A sugar backbone with an outward-facing negatively charged phosphate An inward-facing base; either a purine (guanosine, adenine) or pyrimidine (cytosine, thymine, uracil) base
6	The bases
7	The purines
8	The purines
9	The pyrimidines
10	The pyrimidines
11	The pyrimidines
12	Characteristics of the DNA and RNA backbone C1 linked to a purine or pyrimidine base C2 position determines if we have a ribose or a deoxyribose RNA has a hydroxyl at C2 DNA has a hydrogen at C2 so become “deoxyribonucleic acid” C5 has a negatively charged phosphate group
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14	Polymerization of DNA and RNA requires template, enzymes and co-factors
15	Complementary new strand synthesis is due to basepairing and anti-parallel polymerization in 5’ to 3’ direction
16	Complementary new strand synthesis is due to basepairing and anti-parallel polymerization in 5’ to 3’ direction
17	DNA
18	RNA uses Uracil instead of Thymine to basepair to Adenine
19	How can I create ssDNA for polymerization of a new strand?
20	Break H bonds with heat
21	Break H bonds with an enzyme
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25	Example 1: Signal amplification
26	Versant® Hepatatis B virus branch chain DNA (bDNA) 3.0 Assay Direct identification of HBV DNA in blood or serum in range 2 x 10³ to 1 x 10⁸DNA copies/ml
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29	Release target DNA and denature dsDNA to ssDNA
30	Capture target ssDNA with a DNA probe
31	Target 2^nd probe against the trapped DNA target
32	Hybridize a DNA amplifier to 2^nd probe
33	Hybridize AP-labeled probes to DNA amplifier
34	Add dioxetane substrate
35	Measure light production with a luminometer
36	Signal amplification pros and cons Multiple probes and amplifiers has dropped limit of detection Large probes less susceptible to errors due to target sequence heterogeneity Not affected by enzyme inhibitors as are polymerases Not creating amplicons, so reduced concerns about false + due to x-contamination Non-specific hybridization May not work if target copy # is low
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38	Taqman Real-time Reverse transcriptase-PCR assay For West Nile Virus, Lanciotti et al, J. Clin. Micro. (2000), 38 (11) Home brew test for identification of WNV in with brain tissue and field-collected mosquitoes Primers and probes target regions in the capsid open reading frame (ORF), 3’-untranslated region, and envelope protein ORF
39	Release viral RNA from tissue
40	Complementary DNA primers bind to target viral RNA
41	Reverse transcriptase complexes with primer and target RNA
42	Reverse transcriptase recruits dNTPs to generate a new strand of cDNA
43	Reverse transcriptase recruits dNTPs to generate a new strand of cDNA
44	Reverse transcriptase recruits dNTPs to generate a new strand of cDNA
45	Reverse transcriptase degrades template RNA with its own RNAse H
46	Initiate polymerase chain reaction (PCR) cycle Need a thermocycler Melt ds DNA at 97°C Bind primers at 55-60°C DNA polymerase polymerizes a new DNA strand in 5’-3’ direction at 72°C Amplicons increase every cycle by 2ⁿ (where n=cycle #) Key point is that these step are cycled 25-30x…. So you need a special DNA polymerase
47	Taq DNA polymerase can withstand extreme temperature changes DNA polymerase from the bacterium Thermus aquaticus C-terminal 5'–3' polymerase domain 5'–3' exonuclease domain at its amino terminus
48	Single stranded “1^st strand” cDNA enters PCR reaction: Melt all secondary cDNA structure
49	Complementary DNA primers bind to cDNA target sequence
50	Taq DNA polymerase recruited to primer binding site
51	New DNA strand is polymerized
52	New DNA strand is polymerized
53	Generate ds cDNA strand
54	Fluorescent probes detect specific amplicons during the PCR cycle
55	Melt ds DNA
56	Quenched probes bind target DNA
57	Complementary primers bind target DNA
58	DNA polymerase recruited to primer binding site
59	DNA polymerase generates new DNA strand and 5’-exonuclease destroys cleaves the cleaves quencher from reporter
60	DNA polymerase 5’-exonuclease destroys probe and releases an unquenched reporter
61	Two new daughter DNA strands are generated and fluorescence from reporters is measured by detector
62	Amplicons increase every cycle by 2ⁿ
63	RT-PCR benefits and drawbacks Detection of small quantities of target RNA in clinical samples Real-time detection Use of probe adds specificity Chance of carry-over contamination Needs a thermocycler May still fail to pick-up low viremia in asymptomatic patients especially if use minipools
64	Example 3: Isothermic target amplification
65	Amplification of M. tuberculosis direct (AMTD-2) An FDA approved test for MTB in respiratory samples Smear +; sensitivity 98% vs culture and clinical criteria Smear -; sensitivity 77% vs culture and clinical criteria Isothermal test- so no thermocycling
66	What happens during transcription mediated amplification? Need a reverse transcriptase with inherent RNAse activity for reverse transcription of specific rRNA targets to DNA Need an T7 RNA polymerase for transcription of the DNA intermediate template to a billion RNA amplicon copies Detection of RNA amplicon by a probe hybridization protection assay
67	Promoter primer binds to rRNA target
68	Promoter primer binds to rRNA target
69	Reverse transcriptase creates a cDNA copy of the rRNA target
70	Reverse transcriptase creates a cDNA copy of the rRNA target
71	RNAseH activity of RT degrades the RNA target
72	Primer 2 binds to DNA and RT creates a new DNA copy with a promoter sequence
73	Primer 2 binds to DNA and RT creates a new DNA copy with a promoter sequence
74	Primer 2 binds to DNA and RT creates a new DNA copy
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76	RNA polymerase uses its helicase activity to unwind dsDNA and generate RNA
77	RNA polymerase generates 100-1000 RNA amplicons
78	RNA polymerase generates 100-1000 RNA amplicons
79	Primer 2 binds each RNA amplicon
80	Primer 2 binds each RNA amplicon
81	RT creates a new cDNA copy
82	RT creates a new cDNA copy
83	RNAse activity of RT destroys the RNA in the RNA:DNA duplex
84	Promoter-primer binds to DNA
85	Promoter-primer binds to DNA
86	RT creates a ds DNA
87	RT creates a ds DNA
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89	RNA polymerase uses its helicase activity to unwind dsDNA and generate RNA
90	RNA polymerase generates 100-1000 RNA amplicons for each DNA template
91	RNA polymerase cycle repeats with a billion-fold amplification
92	How do we detect all these new RNA amplicons?
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94	TMA pros and cons Transcription can produce 100-1000 RNA amplicons per DNA molecule Instability of RNA amplicons creates less likely chance of carry-over Isothermal Susceptible to inhibitors Data processing deals with relative light units
95	Commonalties for all technologies discussed today Targets within a pathogen’s genetic material are bound and recognized in a complementary manner with either probes or primers The problem of having very low #s of target genetic material is overcome by either probe-target signal amplification or the logarithmic creation of amplicons All systems described used a probe for either signal amplification, identification of amplicons or the maintenance of assay specificity
96	Conclusions There are a wide variety of molecular tests available Choice of test depends on resources available, clinical sample, and patient The test you pick will probably utilize probe technology Tests are complex and require equipment for detection