Polynucleotides for the amplification and detection of chlamydia trachomatis
Inventors
DeDent, Andrea • Lee, Matt • Ma, Shuyuan • Maamar, Hedia
Assignees
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Publication Number
US-11326214-B2
Publication Date
2022-05-10
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 23S ribosomal genes or gene transcripts.
Core Innovation
The document describes a method of detecting Chlamydia trachomatis in a test sample by extracting nucleic acid and amplifying a target sequence using strand displacement DNA polymerase and a sequence-specific primer set. The primer set includes a forward inner primer comprising the nucleotide sequence of SEQ ID NO:83 and a backward inner primer comprising the nucleotide sequence of SEQ ID NO:84. The amplified product is generated from the nucleic acid extracted from the test sample.
Detection of amplification is performed by hybridizing the amplified product with a molecular beacon. The molecular beacon comprises a fluorophore, a quencher, and a polynucleotide sequence selected from SEQ ID NOs:97-101 and 103-130, including SEQ ID NO:115, with fluorophore/quencher pairs including FAM/BHQ1 and combinations such as ATTO 565 or Alexa 594 with BHQ1/BHQ2.
The document frames performance attributes including specificity, with molecular-beacon hybridization used to reduce off-target amplification and cross-reactivity, and sensitivity, including amplification detection at low copy numbers using specific primer set and molecular-beacon combinations. The assay is described as being tested in urine matrix, including evaluation of time-to-positive kinetics.
Claims Coverage
The independent claim includes three main inventive aspects: amplification of a Chlamydia trachomatis target sequence with strand displacement DNA polymerase using a specific inner primer pair, detection of the amplified product via molecular-beacon hybridization using defined molecular-beacon polynucleotide sequences, and use of the detection result as an indicator of the presence of Chlamydia trachomatis.
Strand displacement DNA polymerase amplification with defined inner primers
A method that extracts nucleic acid from a test sample and amplifies a target sequence by reacting the nucleic acid with a reaction mixture comprising a strand displacement DNA polymerase and a sequence-specific primer set comprising a forward inner primer of SEQ ID NO:83 and a backward inner primer of SEQ ID NO:84.
Molecular beacon hybridization detection with defined beacon sequences
Detecting the presence or absence of an amplified product by hybridizing the amplified product with a molecular beacon comprising a fluorophore, a quencher, and a polynucleotide sequence selected from SEQ ID NOs:97-101 and 103-130.
Presence indication of Chlamydia trachomatis from amplified-product detection
Providing that the presence of the amplification product is indicative of the presence of Chlamydia trachomatis in the test sample.
Across the independent claim, the inventive combination is an extraction-and-amplification workflow using strand displacement DNA polymerase with defined inner primers, followed by molecular-beacon hybridization detection using molecular-beacon polynucleotide sequences selected from SEQ ID NOs:97-101 and 103-130, where amplified-product presence indicates Chlamydia trachomatis.
Stated Advantages
Molecular beacons reduce off-target amplification and cross-reactivity.
Assay specificity against closely related species and other STI-associated organisms is demonstrated.
Sensitivity is demonstrated, including detection at low copy numbers.
Fast time-to-positive kinetics are described in urine matrix.
Documented Applications
Detecting Chlamydia trachomatis in a test sample, including testing in urine matrix.
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