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Volume 69, Issue 12

Editor's Choice A novel peptide nucleic acid- and loop-mediated isothermal amplification assay for the detection of mutations in the 23S rRNA gene of Free

Abstract

Macrolides could be a potential alternative treatment for infections in patients; however, macrolide-resistant is spreading rapidly worldwide.

. There are presently no alternatives to serological tests for syphilis that can be used to evaluate therapeutic effects due to the fact that cannot be cultured .

In this study, we constructed a method for rapidly identifying and confirming macrolide resistance by using loop-mediated isothermal amplification (LAMP) with peptide nucleic acids (PNAs).

A set of LAMP primers was designed to span nucleotide positions 2058 and 2059 in 23S rRNA. A PNA clamping probe was also designed to be complementary to the wild-type sequence (A2058/A2059) and positioned to interfere with both the annealing of the 3′ end of the backward inner primer and the concomitant extension. Prior to the LAMP assay, swab samples from suspected syphilitic lesions were boiled for DNA extraction.

The assay had an equivalent detection limit of 1.0×10 copies/reaction and showed specificity against 38 pathogens. In the presence of a 4 µM PNA probe, LAMP amplified up to 1.0×101 copies/reaction using plasmids harbouring the complementary mutant sequences (A2058G or A2059G), whereas amplification was completely blocked for the wild-type sequence up to a concentration of 1.0×10 copies/reaction. For the 66 PCR-positive clinical specimens, the overall detection rate via LAMP was 93.9 % (62/66). Amplification was successful for all 53 mutant samples and was incomplete for all nine WT samples by the PNA-mediated LAMP assays.

We developed a PNA-mediated LAMP method that enabled us to rapidly identify and determine its macrolide susceptibility via a culture-independent protocol.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): 23S rRNA , loop-mediated isothermal amplification , macrolide , peptide nucleic acid and Treponema pallidum
Funding
This study was supported by the:
  • Japan Agency for Medical Research and Development (Award JP19fk0108091)
    • Principle Award Recipient: Norihito Tarumoto
  • Japan Society for the Promotion of Science (Award JP18K08446)
    • Principle Award Recipient: Norihito Tarumoto
© 2020 The Authors
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2020-11-12
2024-04-30
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A novel peptide nucleic acid- and loop-mediated isothermal amplification assay for the detection of mutations in the 23S rRNA gene of Treponema pallidum
J. Med. Microbiol. 69, 1339 (2020); https://doi.org/10.1099/jmm.0.001275
/content/journal/jmm/10.1099/jmm.0.001275
/content/journal/jmm/10.1099/jmm.0.001275
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