Ligase Chain Reaction

Introduction

The ligase chain reaction (LCR) is one of many techniques developed in recent years to detect specific nucleic acid sequences by amplification of nucleic acid targets. The LCR has been used for genotyping studies to detect tumors and identify the presence of specific genetic disorders such as sickle cell disease caused by known nucleotide changes that occur as a result of point mutations and has now become widely used in infectious disease detection, both in the diagnostic and research settings, primarily focusing on infections caused by microbes that have proven difficult to detect by traditional culture techniques. The LCR is now recognized as the method of choice for detection of urogenital infections due to Chlamydia trachomatis because of its greater sensitivity as compared to traditional cell culture or nonamplified DNA probes or antigen-detection assays. When used for detection of infectious diseases, amplification tests such as the LCR have the additional advantages in that they do not require viable organisms in a specimen, a single specimen can be used to detect multiple different pathogens, provided suitable primers are available, and easily obtained specimens such as urine can be used for diagnostic purposes, making screening of large numbers of persons practical, as well as facilitating research to better understand the epidemiology of specific diseases.

History

Ligation of adjacent oligonucleotides while hybridized to a template was first investigated by Besmer et al. in 1972. The first use of ligases to join oligonucleotides as a means to differentiate sequence variants was reported in 1988 and the first “real” LCR utilizing cyclical denaturation hybridization and ligation of two pairs of oligomers was described in 1989. Thermostable ligases that eliminate the necessity of adding ligase after each denaturation step was introduced by Barany in 1991. Simple LCR consists of two complementary oligonucleotide pairs (four oligonucleotides 20–35 nucleotides each) that are homologous to adjacent sequences on the target DNA, as opposed to two used in the polymerase chain reaction (PCR) assay. The adjacent pairs are ligated when they hybridize to the complementary sequence next to each other in a 32 to 52 orientation on the same strand of the target DNA. The 52 nucleotide of the ends of the primers to be ligated must be phosphorylated. Newly ligated oligonucleotides become targets in subsequent cycles so logarithmic amplification occurs. The two complementary pairs can also, at low frequency, be blunt-end ligated to each other and serve as a template for amplification even though no target sequence was present in the original sample. Early work showed at least 10-fold greater efficiency of ligation of perfectly matched compared to a mismatched nucleotide at the ligation junction.

Advantages

LCR-based systems have some advantages over the PCR-based amplification systems. Because there is no newly synthesized DNA, misincorporated nucleotides are not replicated in the product allowing amplification of a different sequence than that found in the target nucleic acid. The LCR reactions are also more specific for the 3′ nucleotide allowing for higher discriminatory power against mismatches at a single chosen site. Thus, LCR is very useful for determining the nucleotide at a specific site such as a single base change, e.g., single-nucleotide polymorphisms (SNPs) used in mapping complex genomes. The LCR cycle has only two short steps allowing for shorter amplification times. The usually small target of LCR, 36 to 60 nucleotides, does not require high quality large fragment nucleic acids. There are several modifications to the simple LCR that have been used to overcome some of the problems that are inherent in this method. Owing to the specificity of the ligase reaction for perfect matches that is complicated by the blunt end ligation of double-stranded probes PCR, the Ligase Detection Reaction (LDR) was developed. The amplification step for LDR is PCR using outside primers, then only one pair of adjacent primers is used to detect the proper sequence in the PCR product. Because there are no double-stranded oligonucleotides to blunt end ligate, the background is very low and the linear detection of the PCR amplified sequence can be detected using ligation of oligonucleotides. Gap LCR utilizes a few base pair gaps between the two pair of oligonucleotides on the primer, thus requiring a thermostable polymerase (Taq polymerase) to fill in the gap before ligation of adjacent primers can occur. The Gap LCR prevents amplification of the blunt end ligated primers because the oligonucleotide pairs cannot be amplified on blunt-end ligated products.

Ligase

There are four thermostable ligases that can be used in LCR reactions. Taq ligase, Pfu which is advertised as having higher ligation specificity and lower background than Tth that is marketed by Abbott Diagnostics, Amplicase is sold by Epicenter Technologies.

Probe Design

The most successful probes are 18 to 30 bases in length. Probes with high GC content or those that form stable secondary structures or dimers should be avoided if possible. It is difficult to predict activity, so empiric testing is still necessary. The 5′ nucleotide of the adjacent end must be phosphorylated to promote ligation. The probe candidates must be tested for sensitivity by determining the lower level of detection of samples containing organisms of interest or their DNA. Specificity must be determined by testing related organisms, as well as other organisms likely to be found in specimens to be tested.

Target Sequence Selection

The sequence should be unique to the organism to be detected and multiple copies per organism increases sensitivity. If the ligase reaction is to be used to discriminate between single base pairs, the ultimate 3′ base is most sensitive, whereas the 5′ base is also relatively sensitive. Allele-specific PCRs used to detect single nucleotide polymorphisms are often not discriminating enough to differentiate between SNPs, whereas the ligase reactions are more discriminating against mismatches, especially on either side of the ligation site. All of the possible mismatches are discriminated against, but G-T and T-T less well, 1.5% as efficient as the matching nucleotide, while other mismatches were <0.4% using Taq ligase. All possible combinations of single base mismatches both on the 5′ and 3′ side of the ligation site have been tested for fidelity with Tth. T-G and G-T were less efficiently discriminated against (~2%). when on the 3′ side, whereas all perfect matches were >80%. When the mismatch was on the 5′ side the previous two and A-C, A-A, C-A, G-A, and T-T, all were all less discriminated against than when on the 3′ side, same patterns as were found with T4 ligase. Intentionally introducing a mismatch in the third site from the 3′ end of the probes increased the discriminating power. Nucleotide analogs in the probes in the 2 and 3 location from the 3′ end also increased discriminatory power. Site-directed mutagenesis was used on Tth and mutants that increased discriminating power 4- and 11-fold were found.

 Detection of Amplification Products

Multiplex LCR using a mixture of probes differing by the 3′ nucleotide involved in the ligation, that are labeled by being one or two extra bases on the nonligating 5′ end, allows polyacrylamide gel electrophoresis (PAGE) differentiation of the one or two base changes by differences in migration in PAGE. The probes can also be labeled with several different, easily detected labels such as 32P, fluorescent labels, immunologically detectable haptens (digoxigenen), and after amplification and electrophoresis the signal is detected by Southern blot to determine if ligation has taken place. Ligated products can also be detected by having immuno-capture of one of the probe ends and after washing, detecting the second probe with an enzyme conjugate. The only ligated product will be captured and also have the end with the ligand for which the enzyme-conjugated antibody is specific.

Contamination Control

One problem with LCR is that the target is amplified, resulting in a contamination risk. The method commonly used to inactivate PCR products does not work because of the small size of the amplicon in LCR. The potential for contamination requires strict adherence to physical separation of setup and detection areas and other containment methods such as bleach treatment of lab benches, Ultraviolet irradiation of setup areas, unit premixes for setup, as well as the use of aerosol barrier pipet tips. The commercial LCx instrument injects a binary inactivating agent that is capable of inactivating small amplicons by a factor of up to 109.

Inhibitors

LCR is less prone to problems with inhibitors from urogenital specimens than PCR for detection of Chlamydia. Freeze-thawing and dilution decreased the false negative rate of PCR. Possible inhibitors of LCR were removed by a mildly acid wash to remove CaHPO4 from concentrated specimens being tested for the presence of acid-fast bacteria. The potential effect of inhibitors on amplification results mandates rigorous quality control for all steps of the procedure as described in the technical procedures presented here.

Materials

Equipment

  1. Microcentrifuge capable of speeds of ≥9000g.
  2. Vortex mixer.
  3. 20°C freezer for sample storage if not processed immediately.
  4. LCx Analyzer or other detection systems with appropriately labeled reagents specific for the system employed.
  5. LCx Thermocycler.
  6. LCx dry bath capable of heating from 60° to 100°C.

Supplies and Reagents

  1. LCx kit.
  2. Taq ligase with appropriate buffers from the same supplier.
  3. Custom probes.
  4. Proteinase K (final concentration 200 μg/ml).
  5. Mineral oil.
  6. Specimen or analyte.
  7. Specimen Collection and Transport System (Uriprobe) containing 0.5 ml transport buffer.
  8. Sterile, preservative-free plastic screw-top containers for collection and transport of specimen.
  9. 100 μL aerosol barrier pipet tips and pipets.

Methods

 Procedural Precautions

  1. Work in a laboratory using DNA amplification methods should always flow in a one-way direction beginning in the specimen preparation and processing area (Area 1), then move to the amplification and detection area (Area 2). Do not bring any materials or equipment from Area 2 into Area 1.
  2. Surface cleaning using a 1% (v/v) sodium hypochlorite solution followed by 70% (v/v) ethanol should be performed on bench tops and pipets prior to beginning the LCR Assay. 3. Chlorine solutions may pit equipment and metal. Use sufficient amounts or repeated applications of 70% ethanol until chlorine residue is no longer visible.

The type of materials required for LCR assays varies greatly according to the ligase, amplification, and detection systems used, as well as the primers,  required that must be specific for the desired target.

Prototype LCR Technique for Detection of Neisseria gonorrhoeae

These methods were originally developed by Birkenmeyer and Armstrong and described in 1992. though this procedure was developed specifically for the detection of N. gonorrhoeae DNA, the basic principle can be adapted for the detection of other microbial agents, provided specific oligonucleotide primers are designed.

 Primer Selection

  1. Primers should be designed to be homologous to conserved sequences in N. gonorrhoeae that have mismatches with N. meningitidis, the species most homologous to N. gonorrhoeae. Because of the empiric nature of developing LCR, multiple primer sites should be developed. The site demonstrating the best sensitivity and specificity should be chosen and this must be validated. In the example given, three sites were chosen corresponding to sequences immediately upstream of several of the Opa gene family (Opa-2 and Opa-3) and a site downstream of several of the pil gene family (Pilin-2).
  2. The primers for this technique are designed to have a gap of 4 to 5 Gs that would be filled by adding the single deoxynucleotide dGTP and Taq polymerase that would not add more nucleotides than are needed to fill the gap. The Pilin-2 locus has 6 bp of nontargeted DNA added to one end that are complementary to the related primer.
  3. The ends of the primers are labeled with fluorescein on one pair of homologous primers (one 5′ one 3′) and biotin on the other nonligating ends of the other two primers. The sequences of the primers are described in the original publication describing this technique.
  4. The ligating 5′ ends are phosphorylated to facilitate ligation after the gap is filled.
  5. All of the modifications of the oligonucleotides can be ordered from numerous companies that will custom synthesize oligonucleotides for specific purposes and targets.

Clinical Specimens

  1. The type of specimen should be chosen according to the site that the organism is most likely to be detected in association with disease. For N. gonorrhoeae, urethral or endocervical swabs are collected and placed into 500 μl of specimen buffer containing 50 mM EPPS buffer [N-(2-hydroxyethyl)piperazine-N2-(3-propane sulfonic acid)] and 5 mM EDTA (pH 7.8). The tube can be maintained up to 24 to 48 h at room temperature if necessary for transport to the laboratory. The swabs are vigorously vortexed prior to removal and the sample is then frozen at -20°C until DNA extraction.
  2. Organism lysis to release DNA is accomplished by adding proteinase K to a final concentration of 200 μg/ml and incubating for 1 h at 60°C.
  3. Boiling for 10 min inactivates the proteinase K, further lyses the bacteria and denatures the DNA.
  4. The cell debris is pelleted by centrifuging for 10 min at 4°C at 13,000g.
  5. The supernatants can be stored at –20°C until used.

Amplification and Detection

  1. Each 50 μl amplification mixture will contain 4 μl of sample, and a final concentration of: 20 mM Tris-HCl (pH 7.6 @25°C) 25 mM potassium acetate, 10 mM magnesium acetate, 10 mM DTT, and 1 mM NAD and 0.1% Triton X-100. This buffer supplied as 10X the concentrations needed can be purchased commercially along with the Taq ligase.
  2. Each of the four oligonucleotides within a set should be diluted in advance and the appropriate volumes of each added to obtain the 830 fmol of each of the four in a set in each reaction tube.
  3. This mixture is overlaid with sterile mineral oil and heated for 3 min in a boiling water bath to ensure complete denaturation. After cooling, 1 unit of Amplitaq DNA polymerase and 15 units of Thermus aquaticus (Taq) DNA ligase is added.
  4. A positive control consisting of 270 cell equivalents of N. gonorrhoeae DNA in 80 ng/μl human placental DNA and a negative control must be included in each run.
  5. Gap LCR is performed in a thermocycler. The prototype technique for N. gonorrhoeae used 27 cycles (Opa-2), 33 cycles (Opa-3) or 31 cycles (Pilin-2). Each cycle consists of a denaturation step 85°C for 30 s and a hybridization, gap filling, ligation step of 60°C for Opa-2 and Pilin-2 and 53°C for Opa-3) for 1 min. When developing a new LCR procedure it is necessary to empirically test different numbers of cycles to determine the optimum for detection and specificity for each primer pair. 6. Detection is performed on 40 μl of each LCR reaction in the automated Abbott Imx, which uses anti-fluorescein-coated microparticles to capture the products of the gap LCR. If ligation has taken place, the biotin on the other end of the product is detected by anti-biotin conjugated to alkaline phosphatase. Alkaline phosphatase is detected by adding methylumbelliferone phosphate that, when the phosphatase acts on the phosphate, yields a fluorescent product that is detected by the IMx. The amount of fluorescence produced is proportional to the ligated oligonucleotides.
  6. Of the many methods used to detect ligation products, automated methods such as the IMx are the most labor efficient. Details and requirements regarding the actual use of automated systems for LCR detection are instrument-specific and must be performed by the manufacturers in order to ensure the validity and accuracy of the results.

Abbott LCx Commercial Automated Neisseria gonorrhoeae Detection System

The materials listed here and the procedures described in the subsequent section are based on what is needed for the performance of the commercially sold Abbott LCx assay for detection of N. gonorrhoeae from urogenital swabs or voided urine from men or women. Due to the proprietary nature of the LCx technology, use of these specific materials, including specimen transport systems and oligonucleotide primers that must be purchased from the manufacturer in kit form is necessary for the successful performance of this type of LCR assay. Strict adherence to the manufacturer’s instructions for sample collection, storage, and processing is necessary for satisfactory results. The LCx kits for C. trachomatis and M. tuberculosis follow the same general principles. Some modifications are necessary for the M. tuberculosis assay due to the nature of the different specimen types (respiratory secretions vs urogenital swabs or urine). It is possible to perform the LCR assay using other commercially available ligases and methods for detection of amplicons as described earlier, but the principles of the Abbott LCx and the notes regarding its performance are generally relevant for any type of LCR assay.

Specimen Collection

  1. 15–20 mL of first-void urine should be collected into a sterile plastic, preservative-free container. It is desirable to obtain specimens from patients who have not urinated within 1 h prior to collection.
  2. Swab specimens should be collected using the LCx swab collection and transport kit. For endocervical specimens in females, excess cervical mucus should be removed prior to sampling using the large-tipped cleaning swab provided in the collection system. When sampling the cervix, the small-tipped swab should be inserted into the endocervix and rotated for 15–30 s to ensure adequate sampling. In males, the small-tipped swab should be inserted 2–4 mm into the urethral meatus and rotated for 3 to 5 s. Swabs are then inoculated into the transport tube, broken off at the score line and then the cap is screwed securely onto the tube.

Specimen Storage

  1. Time and temperature conditions must be adhered to for storage and transport of specimens. Swabs in the transport system can be stored at 2–30°C. If more than 24 h will elapse before processing, the swabs should ideally be refrigerated at 2–8°C or frozen at –20°C or below.
  2. Urine specimens should be refrigerated immediately at 2–8°C and can be held at this temperature for up to 4 d before processing. If longer storage is necessary, swab or urine specimens can be frozen at –20°C or below for up to 60 d. Do not thaw urine until ready for testing.

Urine Specimen Preparation and Processing

  1. Allow urine specimen to completely thaw if frozen. Mix urine in the urine collection cup by swirling to resuspend any settled material.
  2. Using a pipet with aerosol barrier pipet tips, transfer 1 mL of mixed urine into the Urine Specimen Microfuge Tube from the Urine Specimen Preparation Kit.
  3. Centrifuge at >9000g for 15 min in a microcentrifuge.
  4. Using a fine-tipped, plastic disposable pipet, gently aspirate all of the urine supernatants. Be cautious not to contact or dislodge the pellet, which may be translucent. The time between centrifugation and removal of supernatant must not exceed 15 min.
  5. Using a pipettor with aerosol barrier pipet tips, add 1 mL of LCx Urine Specimen Resuspension Buffer. Close the lid of microfuge tube and resuspend the pellet by vortexing until the pellet is resuspended.
  6. Secure tube closure with a cap lock until it clicks into place.
  7. Insert specimen tubes in preheated dry bath wells. After the temperature of the heat block is stabilized at 97°C, heat specimens for 15 min.
  8. Remove the specimen from the dry bath and allow to cool at room temperature for 15 min. Remove cap lock and discard.
  9. Pulse-centrifuge the processed urine specimen in a microcentrifuge for a minimum of 10–15 s immediately before inoculating the LCx amplification vials.
  10. The amplification reagent level in the LCx amplification vial should measure approx two-thirds of the conical part of the vial. If necessary, the vial may be pulse centrifuged in a microcentrifuge for 10–15 s.
  11. Using a pipet with aerosol barrier pipet tips, add 100 μL of each processed urine specimen to the appropriately labeled LCx Amplification Vial, making sure each vial is securely closed and that only one at a time is open. The vial can now be transferred to Area 2 and placed immediately in the Thermal Cycler for amplification.

Swab Specimen Preparation

  1. Allow specimen to completely thaw if frozen.
  2. Insert specimen tubes in preheated dry bath wells. After the temperature of the heat block is stabilized at 97°C, heat specimens for 15 min. Failure to reach 97 + 2°C could limit the release of the DNA in the specimen causing false negative results.
  3. Remove the specimen from the dry bath and allow to cool at room temperature for 15 min.
  4. Unscrew cap and express swab along the side of the tube so that liquid drains back into the solution at the bottom of the tube. Discard swab and original closure, replacing with a new swab tube closure that is screwed on until it clicks into place.
  5. The amplification reagent level in the LCx amplification vial should measure approx two-thirds of the conical part of the vial.
  6. Using a pipet with aerosol barrier pipet tips, add 100 μL of each processed specimen to the appropriately labeled LCx Amplification Vial, making sure each vial is securely closed and that only one at a time is open. The vial can now be transferred to Area 2 and placed immediately in the Thermal Cycler for amplification.
  7. The LCx negative control and calibrator must be prepared in conjunction with specimens to be tested and run in duplicate with each carousel of clinical specimens.

Quality Control Procedures

  1. A negative control and calibrator preparations must take place in the dedicated Specimen Preparation Area (Area 1).
  2. The LCx procedure requires that the negative control and the calibrator be run in duplicate with each carousel of the specimen.
  3. The negative control and calibrator are activated by the addition of 100 μL of LCx activation reagent. It is important to make sure correct volume is added or the run may be invalid. After addition, the contents of the bottles are then recapped and vortexed for 20 s. Each bottle of activated negative control or calibrator is designed to be used up to 48 h if stored at 2–8°C.
  4. A positive control that monitors the entire assay procedure including the specimen processing step should be tested. A known positive urine specimen can be processed in parallel and tested with unknown specimens. The positive control should give a positive assay value (S/CO ratio >1.00). Each laboratory should establish a target value and limits from each control batch using statistical control rules. These target values and limits should be maintained throughout storage. Alternative choices for a positive control are type strains of the microorganism targeted in the assay, e.g., N. gonorrhoeae.

LCx Amplification Procedure

  1. Turn the LCx Thermal Cycler on for at least 15 min prior to use.
  2. Collect all LCx amplification vials containing samples, negative control and calibrator from Area 1 and transfer to Area 2 for thermal cycling.
  3. LCx thermal cycling conditions should be edited to the following amplification parameters: 93°C for 1 s, 59°C for 1 s, 62°C for 1 min, 10 s for 40 cycles.
  4. Place the amplification vials into the thermal cycler, and initiate run. After completion of the thermal cycler run, the amplification product may remain at 15–30°C for up to 72 h prior to LCx detection.

Detection of Amplification Product

  1. Refer to the LCx Analyzer Operations Manual for detailed instrument operation procedures. Before running the LCx Analyzer, check to see that LCx Inactivation Diluent (1) contains a minimum of 100 mL and the LCx System Diluent (2) contains a minimum of 250 mL. Remove the LCx Amplification Vials from the LCx Thermal Cycler.
  2. Place LCx Reaction Cells into a Carousel; lock the carousel.
  3. Pulse-centrifuge the LCx amplification vials in a microcentrifuge for 10–15 s before placing into the LCx reaction cells.
  4. Place the amplification vials into the LCx reaction cells in the following order: negative controls in positions 1 and 2, calibrators in positions 3 and 4, and specimens in the remaining positions.
  5. Place the carousel into the LCx Analyzer.
  6. Lock the amplification vial Retainer.
  7. Remove the LCx detection reagent Pack from 2–8°C storage, gently invert it five times and open the reagent pack bottles in the numeric order: 1, 2, 3, 4.
  8. Look for any film that may have formed over the opening of the reagent bottles. If present burst the bubble.
  9. Place the LCx detection reagent pack into the LCx Analyzer.
  10. Press Assay, then sample management to log in samples for the run. Press RUN on the LCx Analyzer control panel. Final assay results will be printed in approx 60 min.
  11. Store the detection reagent pack at 2–8°C in original packaging, decontaminated with 1% v/v hypochlorite or separate from unopened LCx kits.
  12. Remove the Carousel, individually remove the LCx reaction cells, and dispose appropriately. Rinse the carousel with water after each use.

Calculation of Results

  1. N. gonorrhoeae. The LCx Assay uses MEIA detection on the LCx Analyzer to detect DNA. All calculations are performed automatically.
  2. The presence or absence is determined by relating the LCx Assay results for the specimen to the Cutoff value. The Cutoff value is the mean RATE (c/s/s) of the LCx calibrator duplicates multiplied by 0.25.
  3. Calculation of the Cutoff value:

Cutoff value = 0.25 *(Mean of LCx Gonorrhea Calibrator RATES)

The S/CO value is determined by calculating a ratio of the sample RATE to the Cutoff value

S/CO=Sample rate/Cutoff value

Interpretation of Results

  1. gonorrhoeae

If S/CO Ratio

>1.20,LCx positive,N. gonorrhoeae DNA detected, and positive for N. gonorrhoeae by LCR amplification and MEIA detection.

<0.80,LCx negative,N. gonorrhoeae DNA not detected and presumed negative for N. gonorrhoeae by LCR amplification and MEIA detection. A presumed negative result may be caused by possible inhibition of the LCx method, collection variables or other factors

0.80–1.20, LCx equivocal, Repeat LCx test. If repeat test S/CO ratio is greater than or equal to 1.20, N. gonorrhoeae DNA detected, and positive for N. gonorrhoeae by LCR amplification and MEIA detection. If the repeat test is less than S/CO ratio 1.20, N. gonorrhoeae DNA not detected and presumed negative for N. gonorrhoeae by LCR amplification and MEIA detection.