REAL-TIME PCR REAL-TIME PCR

PCRPCR PCR is a widely used technique for detecting and PCR is a widely used technique for detecting and

quantifying DNA from organisms and has become quantifying DNA from organisms and has become an essential tool in research laboratories and an essential tool in research laboratories and diagnostic tool used in clinical researchdiagnostic tool used in clinical research

However, the combination of existing detection However, the combination of existing detection assays for PCR suffer from long and tedious post assays for PCR suffer from long and tedious post PCR processes PCR processes Gel electrophoresisGel electrophoresis HybridizationHybridization Blocking, Washing and RinsingBlocking, Washing and Rinsing Cross contamination by subsequent stepsCross contamination by subsequent steps

This led to kinetic studies of reactions to devise This led to kinetic studies of reactions to devise the real time PCR systemthe real time PCR system

REAL-TIME PCRREAL-TIME PCR

A revolutionary method of specific DNA/RNA A revolutionary method of specific DNA/RNA sequence identification and amplification that sequence identification and amplification that can simultaneously quantify the amount of can simultaneously quantify the amount of each starting sampleeach starting sample

Relies on the detection of specific sequence Relies on the detection of specific sequence primers and quantization by fluorescent primers and quantization by fluorescent reporter probes in which the signal increases reporter probes in which the signal increases directly proportional to the amount of PCR directly proportional to the amount of PCR product in a reaction product in a reaction

Records the amount of fluorescence emission Records the amount of fluorescence emission at at each cycleeach cycle using a computer algorithmic using a computer algorithmic program program

APPLICATIONSAPPLICATIONS

Quantization of Quantization of gene expressiongene expression

Viral quantizationViral quantization Pathogen detectionPathogen detection Genotyping Genotyping Prenatal diagnosis Prenatal diagnosis Allelic Allelic

discriminationdiscrimination

ADVANTAGESADVANTAGES Maximizes power of traditional PCR applicationsMaximizes power of traditional PCR applications Eliminates post PCR processesEliminates post PCR processes Low turn–around timeLow turn–around time Cost effectiveness/practicality of suppliesCost effectiveness/practicality of supplies Minimizes cross–contaminationMinimizes cross–contamination High sensitivityHigh sensitivity

needs < 5 copies of DNA/RNAneeds < 5 copies of DNA/RNA

Dynamic range of quantificationDynamic range of quantification multiplexingmultiplexing

High throughput capacityHigh throughput capacity A lot of amplifications within a small time frame as opposed to A lot of amplifications within a small time frame as opposed to

other microbiological methodsother microbiological methods Cell culturingCell culturing Viral growthViral growth

REAGENTSREAGENTS

Traditional PCR reagentsTraditional PCR reagents RNA/DNA sampleRNA/DNA sample dH20dH20 DNA PolymeraseDNA Polymerase PCR bufferPCR buffer

Mg2+Mg2+ SaltSalt Tris-HCl base at specific Tris-HCl base at specific

pHpH dNTPsdNTPs Forward and Reverse Forward and Reverse

PrimersPrimers Additional real time PCR Additional real time PCR

reagentsreagents Fluorophore labeled probesFluorophore labeled probes Internal control elementsInternal control elements

PROBESPROBES The probe is an The probe is an

oligonucleotide that has oligonucleotide that has both a reporter fluorescent both a reporter fluorescent dye and a quencher dye dye and a quencher dye attached and is sequence attached and is sequence specific to the amplified specific to the amplified targettarget

As long as the quencher is As long as the quencher is in close proximity of the in close proximity of the reporter dye the reporter dye the fluorescence emitted by fluorescence emitted by the reporter is blockedthe reporter is blocked

The distance separation of The distance separation of the reporter dye from the the reporter dye from the quencher allows the quencher allows the fluorescence emission to fluorescence emission to be emitted and detected be emitted and detected by the real time PCR by the real time PCR instrumentinstrument

EXAMPLE OF FLUROPROBESEXAMPLE OF FLUROPROBES

Taqman [Figure A]Taqman [Figure A] Depend on the 5’ nuclease Depend on the 5’ nuclease

activity of DNA polymerase activity of DNA polymerase for PCR to degrade the for PCR to degrade the hybridized fluroprobe, hybridized fluroprobe, removing the reporter from removing the reporter from the quencherthe quencher

Molecular Beacon [Figure B]Molecular Beacon [Figure B] adopts hairpin loop adopts hairpin loop

structure while in solution structure while in solution to bring the reporter dye to bring the reporter dye and quencher dye in close and quencher dye in close proximity for FRET to occurproximity for FRET to occur

once the beacon hybridizes once the beacon hybridizes to the target sequence to the target sequence during the annealing step, during the annealing step, the reporter dye is the reporter dye is separated from the separated from the quencher dye and the quencher dye and the reporter fluorescentsreporter fluorescents

EXAMPLE OF FLUROPROBESEXAMPLE OF FLUROPROBES

ScorpionsScorpions maintains stem–loop maintains stem–loop

configuration when not configuration when not hybridizedhybridized

when the stem–loop is when the stem–loop is opened due to opened due to complementary binding of complementary binding of 3’, end a fluorescent 3’, end a fluorescent signal is observedsignal is observed

SYBR GreenSYBR Green Binds dsDNA, Binds dsDNA,

indiscriminate of indiscriminate of sequence specificity, and sequence specificity, and emits signalemits signal

INSTRUMENTATIONINSTRUMENTATION

Thermocycler with Thermocycler with optics for fluorescent optics for fluorescent excitation and excitation and emission collectionemission collection

Computer connected Computer connected to thermocycler to thermocycler instrumentinstrument

Analysis software for Analysis software for data acquisition and data acquisition and calculationcalculation

DATA ACQUISITIONDATA ACQUISITION

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

DATA INTERPRETATIONDATA INTERPRETATION Intensity (Rn) is the measure Intensity (Rn) is the measure

of fluorescence of fluorescence this value indicates this value indicates

magnitude of the signal magnitude of the signal generatedgenerated

Threshold is the average Threshold is the average standard deviation of Rn for standard deviation of Rn for the early cycles the early cycles (background)(background)

Intersection between the Intersection between the amplification plot and the amplification plot and the baseline is the cycle baseline is the cycle threshold or CT valuethreshold or CT value

CT value is the CT value is the cycle cycle at at which a which a significant significant increase increase in the change of Rn is first in the change of Rn is first detecteddetected It is at this cycle, that we It is at this cycle, that we

can start to compare and can start to compare and analyze the samples relative analyze the samples relative to each otherto each other

DATA INTERPRETATIONDATA INTERPRETATION

A plateau phase represents the point at which DNA is no longer A plateau phase represents the point at which DNA is no longer being amplifiedbeing amplified

It at this cycle that we must stop comparing and analyzing the It at this cycle that we must stop comparing and analyzing the samples relative to each other.samples relative to each other.

Exponential phase provides the most useful information about the Exponential phase provides the most useful information about the reaction because it is here that the log-linear phase can be reaction because it is here that the log-linear phase can be conferred [Figure 32B]conferred [Figure 32B]

The slope of a log-linear phase (transcribe exponential plot into The slope of a log-linear phase (transcribe exponential plot into log-linear) is a direct reflection of the amplification efficiencylog-linear) is a direct reflection of the amplification efficiency

ACTUAL DATA READOUTACTUAL DATA READOUT

CRITICAL PARAMETERSCRITICAL PARAMETERS SpecificitySpecificity

PrimersPrimers Must flank the region of amplificationMust flank the region of amplification Must not be complementaryMust not be complementary

Avoid primer dimersAvoid primer dimers ProbeProbe

Specific to a target sequence within the amplified region or to dsDNASpecific to a target sequence within the amplified region or to dsDNA Must carry a quencher and fluorolabeled probeMust carry a quencher and fluorolabeled probe

SensitivitySensitivity Amplification of small amount of sampleAmplification of small amount of sample

Contamination free/Quality control procedureContamination free/Quality control procedure Adequate PCR environment and reagentsAdequate PCR environment and reagents

Signal AmplificationSignal Amplification For multiplexing dyes – insurance of distinguishing fluorescence emissionsFor multiplexing dyes – insurance of distinguishing fluorescence emissions Removal of other agents that give off similar fluorescence readingsRemoval of other agents that give off similar fluorescence readings

ControlsControls positive control – controls for failure of PCR reagentspositive control – controls for failure of PCR reagents negative control – controls for presence of contaminant DNAnegative control – controls for presence of contaminant DNA Efficiency of CurvesEfficiency of Curves

ThresholdThreshold Log-linear slopesLog-linear slopes

IDI-Strep BIDI-Strep B

Real time PCR DNA-based Real time PCR DNA-based diagnostic test for detecting diagnostic test for detecting

Group B streptococcusGroup B streptococcus

INTRODUCTIONINTRODUCTION IDI-Strep B is an assay IDI-Strep B is an assay

that can rapidly detect that can rapidly detect Group B streptococcus Group B streptococcus (GBS) in pregnant (GBS) in pregnant womenwomen

QualitativeQualitative QuantitativeQuantitative Specific through DNA Specific through DNA

complementary bindingcomplementary binding Sensitive (ideally Sensitive (ideally

needing only a single needing only a single copy of DNA)copy of DNA)

in vitroin vitro Molecular diagnostic Molecular diagnostic

utility detecting DNA (as utility detecting DNA (as opposed to culture opposed to culture based testing)based testing)

GBSGBS

Group B Streptococcus (GBS) has been recognized as the primary Group B Streptococcus (GBS) has been recognized as the primary cause of bacterial infection in newborn babies, resulting in life cause of bacterial infection in newborn babies, resulting in life threatening conditions within newborns.threatening conditions within newborns.

35-40% of women will carry the GBS bacteria in their urinary tract, 35-40% of women will carry the GBS bacteria in their urinary tract, digestive tract, and/or urinary tract.digestive tract, and/or urinary tract.

During pregnancy and after delivery, GBS can cause serious illness in During pregnancy and after delivery, GBS can cause serious illness in both mothers and newborns.both mothers and newborns.

15% to 20% of pregnant women have a different colonization status at 15% to 20% of pregnant women have a different colonization status at delivery time than at 35-37 weeksdelivery time than at 35-37 weeks

Due to the underdeveloped immune system, primarily in preterm Due to the underdeveloped immune system, primarily in preterm births, GBS poses a leading cause of death within newbornsbirths, GBS poses a leading cause of death within newborns

Life-threatening conditions, such as pneumonia and meningitis, as a Life-threatening conditions, such as pneumonia and meningitis, as a few examples, are a result of GBS infections that occur early on in life few examples, are a result of GBS infections that occur early on in life of newborns (developing within a week)of newborns (developing within a week)

About half of all infants born with GBS will die and the rest may About half of all infants born with GBS will die and the rest may develop in serious brain damagedevelop in serious brain damage

10% term babies and 40% preterm babies10% term babies and 40% preterm babies Most cases of GBS infection in newborns can be prevented by Most cases of GBS infection in newborns can be prevented by

giving certain pregnant women antibiotics during labor. giving certain pregnant women antibiotics during labor. Antibiotic treatment before labor does not prevent GBS infection in Antibiotic treatment before labor does not prevent GBS infection in

newborns.newborns.

GBSGBS

How is GBS infection diagnosed?How is GBS infection diagnosed? The current ‘gold standard’ The current ‘gold standard’

screening test is a rectovaginal screening test is a rectovaginal culture in selective medium, taken culture in selective medium, taken at 35-37 weeks (8.5 months)at 35-37 weeks (8.5 months)

Results are available within Results are available within 2 2 days days using culturing methods in using culturing methods in selective mediumselective medium

Problems: cannot accurately Problems: cannot accurately predict genital tract predict genital tract colonization at time of laborcolonization at time of labor..

Risk factor method for diagnosing Risk factor method for diagnosing GBS targets treatments GBS targets treatments indiscriminately to women indiscriminately to women believed to be at great riskbelieved to be at great risk..

Problem: This is a guess! We Problem: This is a guess! We would miss many colonized would miss many colonized mothers and at risk infants.mothers and at risk infants.

Problem: We would economically Problem: We would economically and production-wise and production-wise WASTEWASTE by by giving medication unnecessarily!giving medication unnecessarily!

GBS DetectionGBS Detection

IDI-Strep B – real time PCRIDI-Strep B – real time PCR Specifically designed Specifically designed

diagnostic test to detect diagnostic test to detect GBS in pregnant women in GBS in pregnant women in less than 1 hour of delivery!less than 1 hour of delivery!

Combination of diagnostic Combination of diagnostic benefitsbenefits

Speed: Less than 1 hour!Speed: Less than 1 hour! Sensitivity: 94%Sensitivity: 94% Specificity: 96%Specificity: 96% SimplicitySimplicity

Uses normal Uses normal vaginorectal swab vaginorectal swab techniquestechniques

Easy 3 step nucleic Easy 3 step nucleic acid extractionacid extraction

Simple real time PCR Simple real time PCR automatedautomated processing processing

IDI-Strep BIDI-Strep B IDI-Strep B uses IDI-Strep B uses real time PCRreal time PCR

for amplification of the specific for amplification of the specific cfb cfb gene of GBS gene of GBS coupled coupled simultaneouslysimultaneously with with fluorogenic fluorogenic target-specific hybridization target-specific hybridization for detection of amplified DNAfor detection of amplified DNA

Test/Assay utilizes:Test/Assay utilizes: Polymerase chain reaction (PCR) Polymerase chain reaction (PCR)

Fluorogenic target-specific Fluorogenic target-specific hybridization hybridization

Computer analysis programs to Computer analysis programs to qualify, validate, and qualify, validate, and quantify quantify resultsresults

Power of the AssayPower of the Assay:: Highly specific and sensitiveHighly specific and sensitive Increases quantitative power of Increases quantitative power of

PCR by measuring fluorescence PCR by measuring fluorescence activity per cycle of amplificationactivity per cycle of amplification

Speed and efficiency of the Speed and efficiency of the resultsresults

Removal of post-PCR processesRemoval of post-PCR processes Gel ElectrophoresisGel Electrophoresis X-ray filmX-ray film

IDI-Strep BIDI-Strep B

Technical backgroundTechnical background IDI-Strep B uses the IDI-Strep B uses the

molecular beacon method molecular beacon method for probe/fluorescence for probe/fluorescence targetingtargeting

1. Cell Extraction of DNA 1. Cell Extraction of DNA A vaginal/rectal specimen is A vaginal/rectal specimen is

collectedcollected Swab is placed in a sample Swab is placed in a sample

preparation buffer to elute preparation buffer to elute the contentsthe contents

An aliquot of the specimen An aliquot of the specimen is then lysed and added, as is then lysed and added, as the sample, to the PCR the sample, to the PCR reagentsreagents

Entire process takes 15 Entire process takes 15 minutes! Whew, that’s minutes! Whew, that’s fast!fast!

IDI-Strep BIDI-Strep B

2. Primers: 2. Primers: Amplification of the GBS Amplification of the GBS gene, gene, cfb,cfb, using two using two GBS-specific primers.GBS-specific primers.

Cfb Cfb is a 154bp region is a 154bp region found only within GBS!found only within GBS!

An internal control (IC) An internal control (IC) is also used to confirm is also used to confirm the integrity of the the integrity of the assay reagentsassay reagents

Similar to the purpose Similar to the purpose of amplifying beta-of amplifying beta-globin gene we used globin gene we used in 14;18 translocation in 14;18 translocation

IDI-Strep BIDI-Strep B

3. Fluorescence by 3. Fluorescence by Hybridization probesHybridization probes

Amplified targets (Amplified targets (cfb cfb and and IC) are detected with IC) are detected with fluorescence labeled fluorescence labeled hybridization probeshybridization probes

There is a different There is a different probe for both the probe for both the cfb cfb and the ICand the IC

Each labeled probe is Each labeled probe is designed to be designed to be complementary to each complementary to each other and form an armother and form an arm

However, the intervening However, the intervening loop is complementary to loop is complementary to the the cfb cfb (or IC) gene(or IC) gene

In solution, they adopt a In solution, they adopt a hairpin structure brining hairpin structure brining the fluorescent reporter the fluorescent reporter dye and quencher dye dye and quencher dye together in close together in close proximity quenching proximity quenching fluorescence.fluorescence.

IDI-Strep BIDI-Strep B

3. Fluorescence by 3. Fluorescence by Hybridization probesHybridization probes

The presence of a The presence of a complimentary target allows complimentary target allows the flurolabled-probes to bindthe flurolabled-probes to bind

Binding causes the quencher Binding causes the quencher and the flurolabeld probe to and the flurolabeld probe to be far enough that the be far enough that the fluorescence emission is no fluorescence emission is no longer quenched and the longer quenched and the reporter dye instead reporter dye instead fluorescesfluoresces

Each beacon-target hybrid Each beacon-target hybrid fluoresces at a wavelength fluoresces at a wavelength characteristic of the specific characteristic of the specific flurophore usedflurophore used

This means that the IC probe This means that the IC probe flurophore is different than flurophore is different than the the cfbcfb probe, allowing probe, allowing measurement of different measurement of different emissions simultaneouslyemissions simultaneously

Multiplexing!Multiplexing!

IDI-Strep BIDI-Strep B

How fluorescence works in How fluorescence works in the PCR amplification real the PCR amplification real timetime

As the PCR reaction As the PCR reaction undergoes, the newly undergoes, the newly synthesizes PCR products synthesizes PCR products are denatured by high are denatured by high temperaturestemperatures

As each strand of the As each strand of the product is separated, the product is separated, the labeled probe labeled probe is also is also denatured.denatured.

As the temperature cools As the temperature cools for the next round of for the next round of primer annealing, the primer annealing, the molecular beacon is able molecular beacon is able to hybridize with the to hybridize with the appropriate strand of the appropriate strand of the PCR productPCR product

IDI-Strep BIDI-Strep B

How fluorescence works in How fluorescence works in the PCR amplification real the PCR amplification real timetime

Any probes that don’t bind, Any probes that don’t bind, reformreform into the hairpin into the hairpin structure and fluorescence structure and fluorescence is quenchedis quenched

Those that DO BIND, Those that DO BIND, remove the ability of the remove the ability of the quencher to block quencher to block fluorescence from the fluorescence from the report dye.report dye.

Therefore, as PCR product Therefore, as PCR product accumulates, there is an accumulates, there is an exponential increase in exponential increase in fluorescencefluorescence

The probe must rebind to The probe must rebind to the target in every cycle for the target in every cycle for signal measurement, as signal measurement, as the probes are only the probes are only designed to remain intact designed to remain intact during amplification during amplification reaction.reaction.

IDI-Strep BIDI-Strep B

4. Quantification4. Quantification IDI-Strep B uses the IDI-Strep B uses the

Smart Cycler connected Smart Cycler connected to a computer to amplify to a computer to amplify and measure sample and measure sample productsproducts

The amount of The amount of fluorescence emitted by fluorescence emitted by each labeled probe, at each labeled probe, at any given cycle, is any given cycle, is measured and computed measured and computed within the Smart Cyclerwithin the Smart Cycler

The amount of The amount of fluorescence at any fluorescence at any given cycle is given cycle is dependentdependent on the on the amount of sample amount of sample present at that time.present at that time.

COMPARISONSCOMPARISONS

CULTURE TECHNIQUESCULTURE TECHNIQUES Uses cell culturing in Uses cell culturing in

growth medium and then growth medium and then post-test into specific post-test into specific growth mediumgrowth medium

Time frame ranges 18-48 Time frame ranges 18-48 hours for culture hours for culture techniquestechniques

Specificity of culture Specificity of culture techniques is 97%techniques is 97%

Sensitivity was a little Sensitivity was a little more more than 50%than 50% predicting predicting colonization at labor and colonization at labor and deliverydelivery

Cannot be performed Cannot be performed directly at time of labordirectly at time of labor

IDI-STREP BIDI-STREP B Uses real time PCR and Uses real time PCR and

requires no post-processesrequires no post-processes Can be performed Can be performed in lessin less

than one hourthan one hour Specificities was 96% Specificities was 96% Sensitivities was a Sensitivities was a

whopping 94% compared whopping 94% compared to culture techniques!to culture techniques!

Can be performed directly Can be performed directly at time of laborat time of labor

Giving results within 45 Giving results within 45 minutesminutes

CONCLUSIONSCONCLUSIONS The IDI-Strep B system provides sensitivity, The IDI-Strep B system provides sensitivity,

specificity, and speed performance characteristics specificity, and speed performance characteristics for determining GBS colonization of pregnant for determining GBS colonization of pregnant womenwomen Advantages of IDI-Strep BAdvantages of IDI-Strep B

Time: Time: Samples obtained during labor and at time of delivery Samples obtained during labor and at time of delivery can be run quickly via real time PCR and determine if a can be run quickly via real time PCR and determine if a mother has a GBS infection before her baby is bornmother has a GBS infection before her baby is born

If she does, a simple dose of antibiotics is given before the baby If she does, a simple dose of antibiotics is given before the baby is bornis born

This also reduces the chance of developing antimicrobial This also reduces the chance of developing antimicrobial resistance in the women’s bacterial flora when administering resistance in the women’s bacterial flora when administering antibiotics prematurelyantibiotics prematurely

Economics/Practicality: Economics/Practicality: More than 750,000 women receive More than 750,000 women receive intravenous antibiotics at labor and delivery for no reason intravenous antibiotics at labor and delivery for no reason other than safety. Real time PCR eliminates these issues other than safety. Real time PCR eliminates these issues allowing safety and assuranceallowing safety and assurance

Speed + Specificity + Sensitivity + Flexibility= Speed + Specificity + Sensitivity + Flexibility= EfficiencyEfficiency

Decrease of False PositivesDecrease of False Positives due to imbedded due to imbedded validation/confirmation by hybridization probesvalidation/confirmation by hybridization probes

CONCLUSIONSCONCLUSIONS

Disadvantages and LimitationsDisadvantages and Limitations IDI-Strep B would yield false negative results in IDI-Strep B would yield false negative results in

GBS mutants that carried a significant number of GBS mutants that carried a significant number of point mutations within the genome.point mutations within the genome.

False negative results may occur due to any False negative results may occur due to any contamination product mistakenly input with PCR contamination product mistakenly input with PCR reagentsreagents

DNA synthesis inhibitors, proteases, restriction enzymesDNA synthesis inhibitors, proteases, restriction enzymes Test is not designed to differentiate carriers of GBS Test is not designed to differentiate carriers of GBS

from those with streptococcal infectionsfrom those with streptococcal infections Essential laboratory care is needed in preparation Essential laboratory care is needed in preparation

of reagents and maintenance of contamination-free of reagents and maintenance of contamination-free areasareas

As is with all highly sensitive assays!As is with all highly sensitive assays!

SIGNIFICANCESIGNIFICANCE IDI Strep B assay offers IDI Strep B assay offers

significant benefit to womensignificant benefit to women Patient management for Patient management for

women who:women who: Deliver pretermDeliver preterm Do not have the advantage of Do not have the advantage of

prenatal careprenatal care Have a different colonization Have a different colonization

status at delivery than at 35-status at delivery than at 35-37 weeks37 weeks

Do not have culture results Do not have culture results available at time of deliveryavailable at time of delivery

Health benefitsHealth benefits Less infant Less infant

mortality/morbidity mortality/morbidity Fewer unnecessary antibiotic Fewer unnecessary antibiotic

prescriptionsprescriptions Economic BenefitsEconomic Benefits

Simple and rapid PCR-based Simple and rapid PCR-based test is the most cost-effective test is the most cost-effective strategy when both sensitivity strategy when both sensitivity and specificity reach at least and specificity reach at least 94%94%

FUTURE APPLICATIONSFUTURE APPLICATIONS

The use of the real time PCR can be applied to a The use of the real time PCR can be applied to a wide range of other applications including:wide range of other applications including: Real time confirmation of the presence of absence of Real time confirmation of the presence of absence of

organisms and virusorganisms and virus Monitoring the levels of specific gene activity as a result of Monitoring the levels of specific gene activity as a result of

growth under manipulated conditionsgrowth under manipulated conditions Host/environment dependent experimentsHost/environment dependent experiments

Altered viral entry or replication, caused by modification of Altered viral entry or replication, caused by modification of target tissuetarget tissue

Inhibition experiments Inhibition experiments Epidemiological studies have been improved in speed and Epidemiological studies have been improved in speed and

scope through the use of real-time PCR because it can scope through the use of real-time PCR because it can measure the amount of two nucleic acid targets within a measure the amount of two nucleic acid targets within a single reactionsingle reaction

FUTURE APPLICATIONSFUTURE APPLICATIONS

The use of the real time PCR can be applied to The use of the real time PCR can be applied to a wide range of other applications including:a wide range of other applications including:

Discrimination of multiple cellular and viral genotypes Discrimination of multiple cellular and viral genotypes within a single reaction vesselwithin a single reaction vessel

Alternative methods to detect morbidity and mortality Alternative methods to detect morbidity and mortality analysisanalysis

Use for detecting efficiency of previous cellular extraction Use for detecting efficiency of previous cellular extraction and DNA/RNA isolation steps, use of specific reverse and DNA/RNA isolation steps, use of specific reverse transcriptase, and PCR reagent mixturestranscriptase, and PCR reagent mixtures

Measure of viral loads over course of infectionsMeasure of viral loads over course of infections Quick HIV titer analysis, as opposed to mAb detectionQuick HIV titer analysis, as opposed to mAb detection

Assessment of viral gene therapy vectors prior to their use Assessment of viral gene therapy vectors prior to their use in clinical trialsin clinical trials