Polynucleotides for the amplification and detection of chlamydia trachomatis
Inventors
Lee, Matthew B. • Maamar, Hedia
Assignees
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Publication Number
US-12275999-B2
Publication Date
2025-04-15
Expiration Date
Abstract
The invention provides methods and compositions for the detection of Chlamydia trachomatis in a test sample. Its presence or absence in the sample is determined by nucleic acid based testing methods using primers and/or probes and or molecular beacons that bind to the 16S or 23S ribosomal genes or gene transcripts.
Core Innovation
A method is provided for detecting Chlamydia trachomatis in a test sample. The method includes extracting nucleic acid from the test sample, amplifying a target sequence, and detecting the presence or absence of an amplified product indicative of Chlamydia trachomatis in the test sample.
The amplification is performed by reacting the extracted nucleic acid with a reaction mixture comprising a strand displacement DNA polymerase and a sequence-specific primer set. The sequence-specific primer set is identified as Set-1 and comprises SEQ ID NO: 3 and SEQ ID NO: 4.
Detection is carried out for the amplified product, and the described detection compositions include probes and molecular beacons that bind to Chlamydia trachomatis ribosomal genes such as 16S or 23S. The document also describes fluorophore and quencher labeling for probes and molecular beacons used to detect amplification products.
Claims Coverage
The document centers on one independent claim covering nucleic acid extraction, strand-displacement amplification with Set-1 primers, and detection of an amplified product for indicating Chlamydia trachomatis. Dependent claims refine the amplification and detection by adding constraints on amplification time and temperature, optionally adding reverse transcriptase, specifying molecular beacons as probes, and adding concentration thresholds expressed in IFU/ml.
Nucleic acid extraction and indicative amplification detection
Extracting nucleic acid from the test sample; amplifying a target sequence with a strand displacement DNA polymerase and a sequence-specific primer set; detecting the presence or absence of an amplified product wherein the presence is indicative of Chlamydia trachomatis in the test sample.
Set-1 primer set defined by SEQ ID NO: 3 and SEQ ID NO: 4
Using a sequence-specific primer set comprising Set-1, wherein Set-1 comprises SEQ ID NO: 3 and SEQ ID NO: 4.
Strand-displacement amplification performed at about 60°C to about 67°C for less than 30 minutes
Performing the amplification of the target sequence at about 60°C to about 67°C for less than 30 minutes.
Short amplification duration of less than nine minutes
Performing the amplification step for less than nine minutes.
Adding reverse transcriptase to the reaction mixture
Adding reverse transcriptase to the reaction mixture.
Using a molecular beacon probe
Performing detection with a probe that is a molecular beacon.
Detection threshold for samples with concentration of ≤ 5 IFU/ml
Providing the method for a test sample that contains Chlamydia trachomatis at a concentration of ≤ 5 IFU/ml.
Overall claim coverage focuses on a nucleic-acid-extraction, strand-displacement DNA polymerase amplification workflow using Set-1 primers, with detection of amplified product indicative of Chlamydia trachomatis. Dependent claim refinements add amplification time and temperature constraints, optionally reverse transcriptase, molecular beacon probe detection, and low concentration thresholds expressed in IFU/ml.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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