Abstract
Nucleic acid amplification tests have become a common method for diagnosis of STIs due to their improved sensitivity over immunoassays and traditional culture-based methods. Isothermal nucleic acid amplification methods offer significant advantages over polymerase chain reaction (PCR) because they do not require sophisticated instruments needed for thermal cycling of PCR. We recently reported a novel isothermal nucleic acid amplification method, Strand Invasion-Based Amplification (SIBA), which exhibited high analytical sensitivity and specificity for amplification of DNA. However, because the reactions were detected using an intercalating dye, this method was only suitable for amplifying a single genomic target. Here, we report the development of multiplexed SIBA (mSIBA) that allows simultaneous detection of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and an internal control in the same reaction tube. SIBA is compatible with probes, allowing the detection of multiple DNA targets in the same reaction tube. The IC was developed to assess the quality of the isolated DNA and the integrity of the enzyme system, as well as to test oligonucleotides. The mSIBA assay retained high analytical sensitivity and specificity for the detection of CT and NG. The development of mSIBA enables rapid screening for CT and NG within point-of-care or central laboratory settings.
Highlights
Nucleic acid amplification tests have become a common method for diagnosis of STIs due to their improved sensitivity over immunoassays and traditional culture-based methods
We demonstrate the use of this multiplexed method in the simultaneous detection of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and internal control (IC) in a single reaction tube
Strand Invasion-Based Amplification (SIBA) assays that detected a specific sequence from the CT cryptic plasmid or NG-porA, and compared these assays with previously published loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays to detect the same targets
Summary
Nucleic acid amplification tests have become a common method for diagnosis of STIs due to their improved sensitivity over immunoassays and traditional culture-based methods. We recently reported a novel isothermal nucleic acid amplification method, Strand Invasion-Based Amplification (SIBA), which exhibited high analytical sensitivity and specificity for amplification of DNA. We report the development of multiplexed SIBA (mSIBA) that allows simultaneous detection of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and an internal control in the same reaction tube. In an attempt to reduce the sample processing time and overall cost of NAATs, it is often desirable to perform multiplexed tests to simultaneously detect two or more genomic targets or organisms in a single reaction tube. Several commercially available multiplexed NAATs can simultaneously detect of CT and NG, which are frequently comorbid These tests include an internal control (IC) for assessing potential sample-related inhibition. We previously described a novel isothermal nucleic acid amplification method, Strand Invasion-Based Amplification (SIBA), with high analytical sensitivity and specificity[6]. Non-target bacterial mix SIBA* LAMP* PCR¶ Cryptic Cryptic Cryptic plasmid plasmid plasmid performance of SIBA to those of two existing DNA amplification methods, real-time PCR and loop-mediated isothermal amplification (LAMP)
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