REAL-TIME PCR

1. REAL-TIME PCR

2. PCR • PCR is a widely used technique for detecting and quantifying DNA from organisms and has become an essential tool in research laboratories and diagnostic tool used in clinical research • However, the combination of existing detection assays for PCR suffer from long and tedious post PCR processes • Gel electrophoresis • Hybridization • Blocking, Washing and Rinsing • Cross contamination by subsequent steps • This led to kinetic studies of reactions to devise the real time PCR system

3. REAL-TIME PCR • A revolutionary method of specific DNA/RNA sequence identification and amplification that can simultaneously quantify the amount of each starting sample • Relies on the detection of specific sequence primers and quantization by fluorescent reporter probes in which the signal increases directly proportional to the amount of PCR product in a reaction • Records the amount of fluorescence emission at each cycle using a computer algorithmic program

4. APPLICATIONS • Quantization of gene expression • Viral quantization • Pathogen detection • Genotyping • Prenatal diagnosis • Allelic discrimination

5. ADVANTAGES • Maximizes power of traditional PCR applications • Eliminates post PCR processes • Low turn–around time • Cost effectiveness/practicality of supplies • Minimizes cross–contamination • High sensitivity • needs < 5 copies of DNA/RNA • Dynamic range of quantification • multiplexing • High throughput capacity • A lot of amplifications within a small time frame as opposed to other microbiological methods • Cell culturing • Viral growth

6. REAGENTS • Traditional PCR reagents • RNA/DNA sample • dH20 • DNA Polymerase • PCR buffer • Mg2+ • Salt • Tris-HCl base at specific pH • dNTPs • Forward and Reverse Primers • Additional real time PCR reagents • Fluorophore labeled probes • Internal control elements

7. PROBES • The probe is an oligonucleotide that has both a reporter fluorescent dye and a quencher dye attached and is sequence specific to the amplified target • As long as the quencher is in close proximity of the reporter dye the fluorescence emitted by the reporter is blocked • The distance separation of the reporter dye from the quencher allows the fluorescence emission to be emitted and detected by the real time PCR instrument

8. EXAMPLE OF FLUROPROBES • Taqman [Figure A] • Depend on the 5’ nuclease activity of DNA polymerase for PCR to degrade the hybridized fluroprobe, removing the reporter from the quencher • Molecular Beacon [Figure B] • adopts hairpin loop structure while in solution to bring the reporter dye and quencher dye in close proximity for FRET to occur • once the beacon hybridizes to the target sequence during the annealing step, the reporter dye is separated from the quencher dye and the reporter fluorescents

9. EXAMPLE OF FLUROPROBES • Scorpions • maintains stem–loop configuration when not hybridized • when the stem–loop is opened due to complementary binding of 3’, end a fluorescent signal is observed • SYBR Green • Binds dsDNA, indiscriminate of sequence specificity, and emits signal

10. INSTRUMENTATION • Thermocycler with optics for fluorescent excitation and emission collection • Computer connected to thermocycler instrument • Analysis software for data acquisition and calculation

11. DATA ACQUISITION • The computer measures the intensity of each well, individually, by using a sensitive camera to monitor the fluorescence at cyclic intervals during the PCR reaction in the thermocycler

12. DATA INTERPRETATION • Intensity (Rn) is the measure of fluorescence • this value indicates magnitude of the signal generated • Threshold is the average standard deviation of Rn for the early cycles (background) • Intersection between the amplification plot and the baseline is the cycle threshold or CT value • CT value is the cycle at which a significant increase in the change of Rn is first detected • It is at this cycle, that we can start to compare and analyze the samples relative to each other

13. DATA INTERPRETATION • A plateau phase represents the point at which DNA is no longer being amplified • It at this cycle that we must stop comparing and analyzing the samples relative to each other. • Exponential phase provides the most useful information about the reaction because it is here that the log-linear phase can be conferred [Figure 32B] • The slope of a log-linear phase (transcribe exponential plot into log-linear) is a direct reflection of the amplification efficiency

14. ACTUAL DATA READOUT

15. CRITICAL PARAMETERS • Specificity • Primers • Must flank the region of amplification • Must not be complementary • Avoid primer dimers • Probe • Specific to a target sequence within the amplified region or to dsDNA • Must carry a quencher and fluorolabeled probe • Sensitivity • Amplification of small amount of sample • Contamination free/Quality control procedure • Adequate PCR environment and reagents • Signal Amplification • For multiplexing dyes – insurance of distinguishing fluorescence emissions • Removal of other agents that give off similar fluorescence readings • Controls • positive control – controls for failure of PCR reagents • negative control – controls for presence of contaminant DNA • Efficiency of Curves • Threshold • Log-linear slopes

16. IDI-Strep B Real time PCR DNA-based diagnostic test for detecting Group B streptococcus

17. INTRODUCTION • IDI-Strep B is an assay that can rapidly detect Group B streptococcus (GBS) in pregnant women • Qualitative • Quantitative • Specific through DNA complementary binding • Sensitive (ideally needing only a single copy of DNA) • in vitro • Molecular diagnostic utility detecting DNA (as opposed to culture based testing)

18. GBS • Group B Streptococcus (GBS) has been recognized as the primary cause of bacterial infection in newborn babies, resulting in life threatening conditions within newborns. • 35-40% of women will carry the GBS bacteria in their urinary tract, digestive tract, and/or urinary tract. • During pregnancy and after delivery, GBS can cause serious illness in both mothers and newborns. • 15% to 20% of pregnant women have a different colonization status at delivery time than at 35-37 weeks • Due to the underdeveloped immune system, primarily in preterm births, GBS poses a leading cause of death within newborns • Life-threatening conditions, such as pneumonia and meningitis, as a few examples, are a result of GBS infections that occur early on in life of newborns (developing within a week) • About half of all infants born with GBS will die and the rest may develop in serious brain damage • 10% term babies and 40% preterm babies • Most cases of GBS infection in newborns can be prevented by giving certain pregnant women antibiotics during labor. • Antibiotic treatment before labor does not prevent GBS infection in newborns.

19. GBS • How is GBS infection diagnosed? • The current ‘gold standard’ screening test is a rectovaginal culture in selective medium, taken at 35-37 weeks (8.5 months) • Results are available within 2 days using culturing methods in selective medium • Problems: cannot accurately predict genital tract colonization at time of labor. • Risk factor method for diagnosing GBS targets treatments indiscriminately to women believed to be at great risk. • Problem: This is a guess! We would miss many colonized mothers and at risk infants. • Problem: We would economically and production-wise WASTE by giving medication unnecessarily!

20. GBS Detection • IDI-Strep B – real time PCR • Specifically designed diagnostic test to detect GBS in pregnant women in less than 1 hour of delivery! • Combination of diagnostic benefits • Speed: Less than 1 hour! • Sensitivity: 94% • Specificity: 96% • Simplicity • Uses normal vaginorectal swab techniques • Easy 3 step nucleic acid extraction • Simple real time PCR automated processing

21. IDI-Strep B • IDI-Strep B uses real time PCR for amplification of the specific cfb gene of GBS coupled simultaneously with fluorogenic target-specific hybridization for detection of amplified DNA • Test/Assay utilizes: • Polymerase chain reaction (PCR) Fluorogenic target-specific hybridization • Computer analysis programs to qualify, validate, and quantify results • Power of the Assay: • Highly specific and sensitive • Increases quantitative power of PCR by measuring fluorescence activity per cycle of amplification • Speed and efficiency of the results • Removal of post-PCR processes • Gel Electrophoresis • X-ray film

22. IDI-Strep B • Technical background • IDI-Strep B uses the molecular beacon method for probe/fluorescence targeting • 1. Cell Extraction of DNA • A vaginal/rectal specimen is collected • Swab is placed in a sample preparation buffer to elute the contents • An aliquot of the specimen is then lysed and added, as the sample, to the PCR reagents • Entire process takes 15 minutes! Whew, that’s fast!

23. IDI-Strep B • 2. Primers: Amplification of the GBS gene, cfb, using two GBS-specific primers. • Cfb is a 154bp region found only within GBS! • An internal control (IC) is also used to confirm the integrity of the assay reagents • Similar to the purpose of amplifying beta-globin gene we used in 14;18 translocation

24. IDI-Strep B • 3. Fluorescence by Hybridization probes • Amplified targets (cfb and IC) are detected with fluorescence labeled hybridization probes • There is a different probe for both the cfb and the IC • Each labeled probe is designed to be complementary to each other and form an arm • However, the intervening loop is complementary to the cfb (or IC) gene • In solution, they adopt a hairpin structure brining the fluorescent reporter dye and quencher dye together in close proximity quenching fluorescence.

25. IDI-Strep B • 3. Fluorescence by Hybridization probes • The presence of a complimentary target allows the flurolabled-probes to bind • Binding causes the quencher and the flurolabeld probe to be far enough that the fluorescence emission is no longer quenched and the reporter dye instead fluoresces • Each beacon-target hybrid fluoresces at a wavelength characteristic of the specific flurophore used • This means that the IC probe flurophore is different than the cfb probe, allowing measurement of different emissions simultaneously • Multiplexing!

26. IDI-Strep B • How fluorescence works in the PCR amplification real time • As the PCR reaction undergoes, the newly synthesizes PCR products are denatured by high temperatures • As each strand of the product is separated, the labeled probe is also denatured. • As the temperature cools for the next round of primer annealing, the molecular beacon is able to hybridize with the appropriate strand of the PCR product

27. IDI-Strep B • How fluorescence works in the PCR amplification real time • Any probes that don’t bind, reform into the hairpin structure and fluorescence is quenched • Those that DO BIND, remove the ability of the quencher to block fluorescence from the report dye. • Therefore, as PCR product accumulates, there is an exponential increase in fluorescence • The probe must rebind to the target in every cycle for signal measurement, as the probes are only designed to remain intact during amplification reaction.

28. IDI-Strep B • 4. Quantification • IDI-Strep B uses the Smart Cycler connected to a computer to amplify and measure sample products • The amount of fluorescence emitted by each labeled probe, at any given cycle, is measured and computed within the Smart Cycler • The amount of fluorescence at any given cycle is dependent on the amount of sample present at that time.

29. CULTURE TECHNIQUES Uses cell culturing in growth medium and then post-test into specific growth medium Time frame ranges 18-48 hours for culture techniques Specificity of culture techniques is 97% Sensitivity was a little more than 50% predicting colonization at labor and delivery Cannot be performed directly at time of labor IDI-STREP B Uses real time PCR and requires no post-processes Can be performed in less than one hour Specificities was 96% Sensitivities was a whopping 94% compared to culture techniques! Can be performed directly at time of labor Giving results within 45 minutes COMPARISONS

30. CONCLUSIONS • The IDI-Strep B system provides sensitivity, specificity, and speed performance characteristics for determining GBS colonization of pregnant women • Advantages of IDI-Strep B • Time: Samples obtained during labor and at time of delivery can be run quickly via real time PCR and determine if a mother has a GBS infection before her baby is born • If she does, a simple dose of antibiotics is given before the baby is born • This also reduces the chance of developing antimicrobial resistance in the women’s bacterial flora when administering antibiotics prematurely • Economics/Practicality: More than 750,000 women receive intravenous antibiotics at labor and delivery for no reason other than safety. Real time PCR eliminates these issues allowing safety and assurance • Speed + Specificity + Sensitivity + Flexibility= Efficiency • Decrease of False Positives due to imbedded validation/confirmation by hybridization probes

31. CONCLUSIONS • Disadvantages and Limitations • IDI-Strep B would yield false negative results in GBS mutants that carried a significant number of point mutations within the genome. • False negative results may occur due to any contamination product mistakenly input with PCR reagents • DNA synthesis inhibitors, proteases, restriction enzymes • Test is not designed to differentiate carriers of GBS from those with streptococcal infections • Essential laboratory care is needed in preparation of reagents and maintenance of contamination-free areas • As is with all highly sensitive assays!

32. SIGNIFICANCE • IDI Strep B assay offers significant benefit to women • Patient management for women who: • Deliver preterm • Do not have the advantage of prenatal care • Have a different colonization status at delivery than at 35-37 weeks • Do not have culture results available at time of delivery • Health benefits • Less infant mortality/morbidity • Fewer unnecessary antibiotic prescriptions • Economic Benefits • Simple and rapid PCR-based test is the most cost-effective strategy when both sensitivity and specificity reach at least 94%

33. FUTURE APPLICATIONS • The use of the real time PCR can be applied to a wide range of other applications including: • Real time confirmation of the presence of absence of organisms and virus • Monitoring the levels of specific gene activity as a result of growth under manipulated conditions • Host/environment dependent experiments • Altered viral entry or replication, caused by modification of target tissue • Inhibition experiments • Epidemiological studies have been improved in speed and scope through the use of real-time PCR because it can measure the amount of two nucleic acid targets within a single reaction

34. FUTURE APPLICATIONS • The use of the real time PCR can be applied to a wide range of other applications including: • Discrimination of multiple cellular and viral genotypes within a single reaction vessel • Alternative methods to detect morbidity and mortality analysis • Use for detecting efficiency of previous cellular extraction and DNA/RNA isolation steps, use of specific reverse transcriptase, and PCR reagent mixtures • Measure of viral loads over course of infections • Quick HIV titer analysis, as opposed to mAb detection • Assessment of viral gene therapy vectors prior to their use in clinical trials