Ligase Chain Reaction for the Ligase Chain Reaction for the Detection of Detection of Chlamydia Chlamydia trachomatistrachomatis

Marysol GarciaKevin Nielson

BIOL 413 - Molecular Diagnostics

Chlamydia trachomatisChlamydia trachomatis Chlamydia trachomatis Chlamydia trachomatis is one of the is one of the

most common sexually transmitted most common sexually transmitted pathogens in developed countries.pathogens in developed countries.

It is a bacterial infection, easily curable It is a bacterial infection, easily curable if detected in time.if detected in time.

Its asymptomatic nature gives rise to Its asymptomatic nature gives rise to the development of more serious the development of more serious complications. complications.

Chlamydia trachomatisChlamydia trachomatis When undetected causes pelvic When undetected causes pelvic

Inflammatory Disease (PID), and Inflammatory Disease (PID), and ectopic pregnancy in women.ectopic pregnancy in women.

Newborns exposed to infected mothers Newborns exposed to infected mothers can develop neonatal pneumonia.can develop neonatal pneumonia.

In males it causes epididymitis and In males it causes epididymitis and urethritis, predominantly; can cause urethritis, predominantly; can cause infertility if untreated.infertility if untreated.

Chlamydia trachomatisChlamydia trachomatis When symptoms are common they When symptoms are common they

include:include:MALE MALE burning sensation during urination burning sensation during urination discharge from the penis discharge from the penis testicular tenderness or pain testicular tenderness or pain rectal discharge or painrectal discharge or pain

Chlamydia trachomatisChlamydia trachomatis Symptoms in FemaleSymptoms in Female

vaginal discharge vaginal discharge burning sensation during urination burning sensation during urination sexual intercourse, painful sexual intercourse, painful symptoms of PID, salpingitis, perihepatitis symptoms of PID, salpingitis, perihepatitis

(liver inflammation similar to hepatitis) (liver inflammation similar to hepatitis) rectal pain or dischargerectal pain or discharge

Chlamydia trachomatisChlamydia trachomatis

Chlamydia trachomatis Life CycleChlamydia trachomatis Life Cycle

Previous Screening MethodsPrevious Screening Methods

Culture PlatesCulture Plates A swab from a A swab from a

potentially infected potentially infected area is cultured on a area is cultured on a growth mediumgrowth medium

ELISAELISA Amplified product Amplified product

bound to antibody bound to antibody and visualized, and visualized, usually via HRPusually via HRP

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SEM image of chlamydia infected liver cells.

Titer plates used in ELISA screening

Previous Methods: Pros/ConsPrevious Methods: Pros/ConsCultureCultureNear 100% specificityNear 100% specificityAutomated procedureAutomated procedureInexpensiveInexpensive

ELISAELISAHighly specific assayHighly specific assayUseful for screening large Useful for screening large sample sizes quicklysample sizes quickly

But…But…Highly variable sensitivityHighly variable sensitivityInvasive sample collectionInvasive sample collection

But…But…ExpensiveExpensiveHighly variable sensitivityHighly variable sensitivity

Ligase Chain ReactionLigase Chain Reaction

Denaturing of target DNA

Cooling to bind probe

DNA Pol to fill gaps DNA

ligase to join

Amplified product to detect

Incubation to bind to microparticle

Conjugate binds to complex

Substrate is added

Fluorescence occurs

Ligase Chain ReactionLigase Chain Reaction Is a probe based amplification system.Is a probe based amplification system. It amplifies the probe rather than the It amplifies the probe rather than the

target region.target region. Two probes are used for each strand Two probes are used for each strand

and ligated to form single probe.and ligated to form single probe. LCR uses two enzymes, a DNA LCR uses two enzymes, a DNA

polymerase and a DNA ligase, to drive polymerase and a DNA ligase, to drive reaction. reaction.

Ligase Chain ReactionLigase Chain Reaction Like PCR, LCR requires a thermal Like PCR, LCR requires a thermal

cycler.cycler. Each cycle results in doubling of the Each cycle results in doubling of the

target nucleic acid molecule.target nucleic acid molecule. The reaction is completed in about The reaction is completed in about

ninety minutes.ninety minutes. It uses antibody-antigen reaction to It uses antibody-antigen reaction to

detect the probes.detect the probes.

C. trachomatisC. trachomatis Detection Assay Detection Assay Sample Preparation from urethral, endocervical or Sample Preparation from urethral, endocervical or

urineurine Transport Buffer contains ≥50mM MgClTransport Buffer contains ≥50mM MgCl22 and Sodium Azide and Sodium Azide

as preservative.as preservative.

Separating DNA strands to make accessible to enzymes.Separating DNA strands to make accessible to enzymes. Heat specimens at 97°C in LCx Dry Bath for 15 minutes (± Heat specimens at 97°C in LCx Dry Bath for 15 minutes (±

1minute). 1minute). (failure to reach 97°C will give false negative)(failure to reach 97°C will give false negative)

Allow specimens to cool at room temperature for 15 min (± 5 Allow specimens to cool at room temperature for 15 min (± 5 min).min).

Preparation from urine sample is non-invasive and rapid Preparation from urine sample is non-invasive and rapid (Ideal for at-risk populations, i.e. teenagers)(Ideal for at-risk populations, i.e. teenagers)

LCx AmplificationLCx Amplification Four oligonucleotide probes at > 10Four oligonucleotide probes at > 101010

molecules per reaction.molecules per reaction. Enzymes ≥1 unit of thermostable DNA Enzymes ≥1 unit of thermostable DNA

polymerase and ≥10,000 units of polymerase and ≥10,000 units of thermostable DNA ligase.thermostable DNA ligase.

33µM each of the dNTPs, ≥20M each of the dNTPs, ≥20µM NADM NAD Stabilizers in buffer solutionStabilizers in buffer solution Sodium Azide as preservativeSodium Azide as preservative

LCx AmplificationLCx Amplification

Chlamydia Negative ControlChlamydia Negative Control ≥ ≥ 1.81.8µg/mL Salmon Testes DNA in buffer g/mL Salmon Testes DNA in buffer

solution.solution. Chlamydia Positive ControlChlamydia Positive Control

Extracted DNA from inactivated Extracted DNA from inactivated C. C. trachomatis trachomatis at ~20 IFU/mLat ~20 IFU/mL in buffer in buffer solution.solution.

LCx AmplificationLCx Amplification Four oligonucleotide probe pairs hybridize to Four oligonucleotide probe pairs hybridize to

complementary ss complementary ss C.trachomatis C.trachomatis target target sequence.sequence.

After hybridization a gap is left which is filled After hybridization a gap is left which is filled by polymerase and ligase joins them.by polymerase and ligase joins them.

Temp is raised to dissociate from target Temp is raised to dissociate from target sequence.sequence.

Lowered to hybridize to available targets.Lowered to hybridize to available targets.

DetectionDetection

Alkaline-phosphatase labelled Alkaline-phosphatase labelled antibodies bound to productantibodies bound to product

Amplification vials placed into Amplification vials placed into reaction cells with two negative reaction cells with two negative controls (cells 1, 2) and two controls (cells 1, 2) and two calibrators (cells 3, 4)calibrators (cells 3, 4)

Reaction mixtures 1-4 added Reaction mixtures 1-4 added ordinallyordinally

Run using LCx analyzer Run using LCx analyzer (approximately 90 min.)(approximately 90 min.)

Schematic of detection mechanism

InterpretationInterpretation Results invalidated by errant negative Results invalidated by errant negative

controls or calibrators (these samples controls or calibrators (these samples must be placed in the correct positions!)must be placed in the correct positions!)

S/CO (sample/cutoff value) > 1.00 is S/CO (sample/cutoff value) > 1.00 is positivepositive

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S/CO < 0.80 S/CO < 0.80 negativenegative0.80 < S/CO < 0.99 0.80 < S/CO < 0.99 warrants a retest. If warrants a retest. If retest S/CO < 1.00, retest S/CO < 1.00, then test is negative.then test is negative.

Interior of analyzer showing carousel.

Limitations of ResultsLimitations of Results Cannot detect plasmid-free variants of Cannot detect plasmid-free variants of

chlamydiachlamydia Cannot monitor treatment success since Cannot monitor treatment success since

chlamydia nucleic acids may remain even chlamydia nucleic acids may remain even after successful treatmentafter successful treatment

Non-ideal samples cause inhibition of assayNon-ideal samples cause inhibition of assay

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Diagnostic print-out from LCx Analyzer

Cannot quantitatively evaluate Cannot quantitatively evaluate presence of chlamydiapresence of chlamydia

Factors affecting…Factors affecting…

SensitivitySensitivity Preparation of Preparation of

negatives and negatives and calibratorscalibrators

Blood or mucous Blood or mucous in sample (or in sample (or other LCR other LCR inhibitors)inhibitors)

SpecificitySpecificityPreparation of negatives Preparation of negatives and calibratorsand calibratorsCross-contaminationCross-contaminationPoor WashingPoor Washing

LCx Amplification ParametersLCx Amplification Parameters

Cycle fileCycle file Segment 1 - 93°C for 1 secondSegment 1 - 93°C for 1 second Segment 2 - 59°C for 1 secondSegment 2 - 59°C for 1 second Segment 3 - 62°Cfor 1min 10 secondsSegment 3 - 62°Cfor 1min 10 seconds

Repeat for 40 cycles Repeat for 40 cycles (up to a billion-fold amp)(up to a billion-fold amp)

Amplification lasts approximately 90 Amplification lasts approximately 90 minutesminutes

Assay Sensitivity and SpecificityAssay Sensitivity and Specificity SpecificitySpecificity

Highly specific probe sequenceHighly specific probe sequence Careful washing and stringent detection criteriaCareful washing and stringent detection criteria

SensitivitySensitivity Enzymatic visualizationEnzymatic visualization Highly efficient LCR amplificationHighly efficient LCR amplification Careful assay protocol prevents contamination Careful assay protocol prevents contamination

(high signal to noise)(high signal to noise) Separation of sample preparation and Separation of sample preparation and

amplification/detection improves both amplification/detection improves both sensitivity and specificitysensitivity and specificity

LCx Assay LimitationsLCx Assay Limitations Threat of carryover contamination, Threat of carryover contamination,

leading to false-positives.leading to false-positives. Difficult or impossible to verify + Difficult or impossible to verify +

amplification in clinical lab.amplification in clinical lab. Susceptibility to inhibition of the Susceptibility to inhibition of the

enzymes resulting in false-negatives.enzymes resulting in false-negatives. Increased labor requirements and high Increased labor requirements and high

cost.cost.

Further Development of LCRFurther Development of LCR Target Capture: Attachment of amplified Target Capture: Attachment of amplified

product to magnetic particles to remove product to magnetic particles to remove inhibiting substances —> Improved sensitivity inhibiting substances —> Improved sensitivity and specificityand specificity

Dual-Kinetic Assay: Detecting and Dual-Kinetic Assay: Detecting and differentiating two products simultaneouslydifferentiating two products simultaneously

TIGRIS: Highly automated apparatus for TIGRIS: Highly automated apparatus for processing 2000 samples/dayprocessing 2000 samples/day

LCnext: Adding new protocols for the LCnext: Adding new protocols for the detection of new diseases such as TB, HIVdetection of new diseases such as TB, HIV