Ligase Chain Reaction for the Detection of Chlamydia trachomatis

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. • It is a bacterial infection, easily curable if detected in time. • 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. • Newborns exposed to infected mothers can develop neonatal pneumonia. • In males it causes epididymitis and urethritis, predominantly; can cause infertility if untreated.

4. Chlamydia trachomatis • When symptoms are common they include: MALE • burning sensation during urination • discharge from the penis • testicular tenderness or pain • rectal discharge or pain

5. Chlamydia trachomatis • Symptoms in Female • vaginal discharge • burning sensation during urination • sexual intercourse, painful • symptoms of PID, salpingitis, perihepatitis (liver inflammation similar to hepatitis) • rectal pain or discharge

6. Chlamydia trachomatis

7. Chlamydia trachomatis Life Cycle

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

9. Previous Methods: Pros/Cons Culture Near 100% specificity Automated procedure Inexpensive ELISA 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 Cooling to bind probe Denaturing of target DNA DNA Pol to fill gaps DNA ligase to join Amplified product to detect Incubation to bind to microparticle Substrate is added Conjugate binds to complex Fluorescence occurs

11. Ligase Chain Reaction • Is a probe based amplification system. • It amplifies the probe rather than the target region. • 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.

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

13. C. trachomatis Detection Assay • Sample Preparation from urethral, endocervical or urine • Transport Buffer contains ≥50mM MgCl2and Sodium Azide as preservative. • 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) • 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)

14. LCx Amplification • Four oligonucleotide probes at > 1010molecules per reaction. • Enzymes ≥1 unit of thermostable DNA polymerase and ≥10,000 units of thermostable DNA ligase. • 3µM each of the dNTPs, ≥20µM NAD • Stabilizers in buffer solution • Sodium Azide as preservative

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

16. LCx Amplification • 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. • Temp is raised to dissociate from target sequence. • Lowered to hybridize to available targets.

17. Detection • 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) • Reaction mixtures 1-4 added ordinally • Run using LCx analyzer (approximately 90 min.) Schematic of detection mechanism

18. 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 Interpretation 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 monitor treatment success since chlamydia nucleic acids may remain even after successful treatment • Non-ideal samples cause inhibition of assay Cannot quantitatively evaluate presence of chlamydia Diagnostic print-out from LCx Analyzer

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

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

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

23. LCx Assay Limitations • Threat of carryover contamination, leading to false-positives. • Difficult or impossible to verify + amplification in clinical lab. • Susceptibility to inhibition of the enzymes resulting in false-negatives. • 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 • Dual-Kinetic Assay: Detecting and differentiating two products simultaneously • TIGRIS: Highly automated apparatus for processing 2000 samples/day • LCnext: Adding new protocols for the detection of new diseases such as TB, HIV