1. Ligase Chain Reaction for the Detection of Chlamydia trachomatis Marysol Garcia Kevin Nielson BIOL 413 - Molecular Diagnostics

2. Chlamydia trachomatis Chlamydia trachomatis is one of the most common sexually transmitted pathogens in developed countries. Chlamydia trachomatis is one of the most common sexually transmitted pathogens in developed countries. It is a bacterial infection, easily curable if detected in time. It is a bacterial infection, easily curable if detected in time. Its asymptomatic nature gives rise to the development of more serious complications. Its asymptomatic nature gives rise to the development of more serious complications.

3. Chlamydia trachomatis When undetected causes pelvic Inflammatory Disease (PID), and ectopic pregnancy in women. When undetected causes pelvic Inflammatory Disease (PID), and ectopic pregnancy in women. Newborns exposed to infected mothers can develop neonatal pneumonia. Newborns exposed to infected mothers can develop neonatal pneumonia. In males it causes epididymitis and urethritis, predominantly; can cause infertility if untreated. In males it causes epididymitis and urethritis, predominantly; can cause infertility if untreated.

4. Chlamydia trachomatis When symptoms are common they include: When symptoms are common they include: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 pain rectal discharge or pain

5. Chlamydia trachomatis Symptoms in Female Symptoms 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 (liver inflammation similar to hepatitis) symptoms of PID, salpingitis, perihepatitis (liver inflammation similar to hepatitis) rectal pain or discharge rectal pain or discharge

6. Chlamydia trachomatis

7. Chlamydia trachomatis Life Cycle

8. Previous Screening Methods Culture Plates A swab from a potentially infected area is cultured on a growth medium A swab from a potentially infected area is cultured on a growth medium ELISA Amplified product bound to antibody and visualized, usually via HRP SEM image of chlamydia infected liver cells. Titer plates used in ELISA screening

9. Previous Methods: Pros/Cons Culture Near 100% specificity Automated procedure InexpensiveELISA Highly specific assay Useful for screening large sample sizes quickly But… Highly variable sensitivity Invasive sample collection But…Expensive Highly variable sensitivity

10. Ligase 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

11. Ligase Chain Reaction Is a probe based amplification system. Is a probe based amplification system. It amplifies the probe rather than the target region. It amplifies the probe rather than the target region. Two probes are used for each strand and ligated to form single probe. Two probes are used for each strand and ligated to form single probe. LCR uses two enzymes, a DNA polymerase and a DNA ligase, to drive reaction. LCR uses two enzymes, a DNA polymerase and a DNA ligase, to drive reaction.

12. Ligase Chain Reaction Like PCR, LCR requires a thermal cycler. Like PCR, LCR requires a thermal cycler. Each cycle results in doubling of the target nucleic acid molecule. Each cycle results in doubling of the target nucleic acid molecule. The reaction is completed in about ninety minutes. The reaction is completed in about ninety minutes. It uses antibody-antigen reaction to detect the probes. It uses antibody-antigen reaction to detect the probes.

13. C. trachomatis Detection Assay Sample Preparation from urethral, endocervical or urine Sample Preparation from urethral, endocervical or urine Transport Buffer contains ≥50mM MgCl 2 and Sodium Azide as preservative. Transport Buffer contains ≥50mM MgCl 2 and Sodium Azide 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 (± 1minute). (failure to reach 97°C will give false negative) Heat specimens at 97°C in LCx Dry Bath for 15 minutes (± 1minute). (failure to reach 97°C will give false negative) Allow specimens to cool at room temperature for 15 min (± 5 min). Allow specimens to cool at room temperature for 15 min (± 5 min). Preparation from urine sample is non-invasive and rapid (Ideal for at-risk populations, i.e. teenagers) Preparation from urine sample is non-invasive and rapid (Ideal for at-risk populations, i.e. teenagers)

14. LCx Amplification Four oligonucleotide probes at > 10 10 molecules per reaction. Four oligonucleotide probes at > 10 10 molecules per reaction. Enzymes ≥1 unit of thermostable DNA polymerase and ≥10,000 units of thermostable DNA ligase. Enzymes ≥1 unit of thermostable DNA polymerase and ≥10,000 units of thermostable DNA ligase. 3M each of the dNTPs, ≥20M NAD 3 µ M each of the dNTPs, ≥20 µ M NAD Stabilizers in buffer solution Stabilizers in buffer solution Sodium Azide as preservative Sodium Azide as preservative

15. LCx Amplification Chlamydia Negative Control Chlamydia Negative Control ≥ 1.8g/mL Salmon Testes DNA in buffer solution. ≥ 1.8 µ g/mL Salmon Testes DNA in buffer solution. Chlamydia Positive Control Chlamydia Positive Control Extracted DNA from inactivated C. trachomatis at ~20 IFU/mL in buffer solution. Extracted DNA from inactivated C. trachomatis at ~20 IFU/mL in buffer solution.

16. LCx Amplification Four oligonucleotide probe pairs hybridize to complementary ss C.trachomatis target sequence. Four oligonucleotide probe pairs hybridize to complementary ss C.trachomatis target sequence. After hybridization a gap is left which is filled by polymerase and ligase joins them. After hybridization a gap is left which is filled by polymerase and ligase joins them. Temp is raised to dissociate from target sequence. Temp is raised to dissociate from target sequence. Lowered to hybridize to available targets. Lowered to hybridize to available targets.

17. Detection Alkaline-phosphatase labelled antibodies bound to product Alkaline-phosphatase labelled antibodies bound to product Amplification vials placed into reaction cells with two negative controls (cells 1, 2) and two calibrators (cells 3, 4) Amplification vials placed into reaction cells with two negative controls (cells 1, 2) and two calibrators (cells 3, 4) Reaction mixtures 1-4 added ordinally Reaction mixtures 1-4 added ordinally Run using LCx analyzer (approximately 90 min.) Run using LCx analyzer (approximately 90 min.) Schematic of detection mechanism

18. Interpretation Results invalidated by errant negative controls or calibrators (these samples must be placed in the correct positions!) Results invalidated by errant negative controls or calibrators (these samples must be placed in the correct positions!) S/CO (sample/cutoff value) > 1.00 is positive S/CO (sample/cutoff value) > 1.00 is positive S/CO < 0.80 negative 0.80 < S/CO < 0.99 warrants a retest. If retest S/CO < 1.00, then test is negative. Interior of analyzer showing carousel.

19. Limitations of Results Cannot detect plasmid-free variants of chlamydia Cannot detect plasmid-free variants of chlamydia Cannot monitor treatment success since chlamydia nucleic acids may remain even after successful treatment Cannot monitor treatment success since chlamydia nucleic acids may remain even after successful treatment Non-ideal samples cause inhibition of assay Non-ideal samples cause inhibition of assay Diagnostic print-out from LCx Analyzer Cannot quantitatively evaluate presence of chlamydia

20. Factors affecting… Sensitivity Preparation of negatives and calibrators Preparation of negatives and calibrators Blood or mucous in sample (or other LCR inhibitors) Blood or mucous in sample (or other LCR inhibitors)Specificity Preparation of negatives and calibrators Cross-contamination Poor Washing

21. LCx Amplification Parameters Cycle file Cycle file Segment 1 - 93°C for 1 second Segment 1 - 93°C for 1 second Segment 2 - 59°C for 1 second Segment 2 - 59°C for 1 second Segment 3 - 62°Cfor 1min 10 seconds Segment 3 - 62°Cfor 1min 10 seconds Repeat for 40 cycles (up to a billion-fold amp) Repeat for 40 cycles (up to a billion-fold amp) Amplification lasts approximately 90 minutes Amplification lasts approximately 90 minutes

22. Assay Sensitivity and Specificity Specificity Specificity Highly specific probe sequence Highly specific probe sequence Careful washing and stringent detection criteria Careful washing and stringent detection criteria Sensitivity Sensitivity Enzymatic visualization Enzymatic visualization Highly efficient LCR amplification Highly efficient LCR amplification Careful assay protocol prevents contamination (high signal to noise) Careful assay protocol prevents contamination (high signal to noise) Separation of sample preparation and amplification/detection improves both sensitivity and specificity Separation of sample preparation and amplification/detection improves both sensitivity and specificity

23. LCx Assay Limitations Threat of carryover contamination, leading to false-positives. Threat of carryover contamination, leading to false-positives. Difficult or impossible to verify + amplification in clinical lab. Difficult or impossible to verify + amplification in clinical lab. Susceptibility to inhibition of the enzymes resulting in false-negatives. Susceptibility to inhibition of the enzymes resulting in false-negatives. Increased labor requirements and high cost. Increased labor requirements and high cost.

24. Further Development of LCR Target Capture: Attachment of amplified product to magnetic particles to remove inhibiting substances —> Improved sensitivity and specificity Target Capture: Attachment of amplified product to magnetic particles to remove inhibiting substances —> Improved sensitivity and specificity Dual-Kinetic Assay: Detecting and differentiating two products simultaneously Dual-Kinetic Assay: Detecting and differentiating two products simultaneously TIGRIS: Highly automated apparatus for processing 2000 samples/day TIGRIS: Highly automated apparatus for processing 2000 samples/day LCnext: Adding new protocols for the detection of new diseases such as TB, HIV LCnext: Adding new protocols for the detection of new diseases such as TB, HIV