Infectious Disease Testing by LCR

Abstract

Ligase Chain Reaction (LCR) is a highly sensitive method for detecting nucleic-acid sequences. The LCR uses two pairs of oligonucleotide probes to hybridize to denatured target DNA strands. The positive-strand probe pairs are designed to bind immediately adjacent to each other on the target molecule. The negative-strand probe pairs bind similarly on the negative strand of the target DNA. A thermostable DNA ligase is used to seal the nick between the annealed probes on each strand. Denaturation and annealing is achieved by varying the reaction temperature (thermocycling), and after strand separation, the newly ligated probe serves as a target template for subsequent reactions. Gapped LCR (G LCR) involves the use of DNA polymerase and DNA ligase to fill and seal a short gap between the annealed probes. In gapped LCR, the positive-sense probe pair binds to the target sequence, but they are separated by a few bases consisting of three or fewer of the four possible nucleotide bases. The negative-sense probe pairs bind similarly on the opposite-sense target strand. The thermostable DNA polymerase, provided with only a specific subset of the four possible nucleotides, extends the probe to a defined length until it is immediately adjacent to its downstream probe partner. The resulting nick between the extended probe and its partner is then ligated to complete the process. Similar to LCR, the newly ligated probe serves as a target template for subsequent reactions. GLCR has been commercialized for several microorganisms. The process can be qualitative and quantitative and has been shown to be more sensitive and specific than nonamplified assays for Chlamydia trachomatis and Neisseria gonorrhoeae in traditional swab specimens and noninvasive clinical specimens such as urine. LCR assays have also been developed for other organisms such as Mycobacterium tuberculosis , human immunodeficiency virus DNA sequences, orthopoxviruses, human papillomavirus, herpes simplex virus, and hepatitis C virus RNA. Keywords: Amplification Inhibitors; DNA Polymerase; Gap; Ligation; Microparticles; Probe Amplification