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Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae.
Shiluli, Clement; Kamath, Shwetha; Kanoi, Bernard N; Kimani, Racheal; Maina, Michael; Waweru, Harrison; Kamita, Moses; Ndirangu, Ibrahim; Abkallo, Hussein M; Oduor, Bernard; Pamme, Nicole; Dupaty, Joshua; Klapperich, Catherine M; Lolabattu, Srinivasa Raju; Gitaka, Jesse.
Afiliación
  • Shiluli C; Centre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, Kenya.
  • Kamath S; Division of Research and Development, Jigsaw Bio Solutions Private Limited, Bangalore, India.
  • Kanoi BN; Centre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, Kenya.
  • Kimani R; Centre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, Kenya.
  • Maina M; Centre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, Kenya.
  • Waweru H; Centre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, Kenya.
  • Kamita M; Centre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, Kenya.
  • Ndirangu I; Centre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, Kenya.
  • Abkallo HM; Animal and Human Health Program, International Livestock Research Institute, Nairobi, Kenya.
  • Oduor B; Animal and Human Health Program, International Livestock Research Institute, Nairobi, Kenya.
  • Pamme N; Department of Materials and Environmental Chemistry, Stockholm University, Sweden.
  • Dupaty J; Department of Biomedical Engineering, Boston University, Boston, MA, USA.
  • Klapperich CM; Department of Biomedical Engineering, Boston University, Boston, MA, USA.
  • Lolabattu SR; Division of Research and Development, Jigsaw Bio Solutions Private Limited, Bangalore, India.
  • Gitaka J; Centre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, Kenya.
Heliyon ; 10(6): e27344, 2024 Mar 30.
Article en En | MEDLINE | ID: mdl-38533083
ABSTRACT

Background:

Curable sexually transmitted infections (STIs), such as Neisseria gonorrhoeae (N. gonorrhoeae), are a major cause of poor pregnancy outcomes. The infection is often asymptomatic in pregnant women, and a syndrome-based approach of testing leads to a missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard nucleic acid amplification tests require advanced infrastructure settings, whereas point-of-care tests are limited to immunoassays with sensitivities and specificities insufficient to accurately diagnose asymptomatic cases. This necessitates the development and validation of assays that are fit for purpose.

Methods:

We identified new diagnostic target biomarker regions for N. gonorrhoeae using an algorithm for genome mining of identical multi-repeat sequences (IMRS). These were then developed as DNA amplification primers to design better diagnostic assays. To test the primer pair, genomic DNA was 10-fold serially diluted (100 pg/µL to 1 × 10-3 pg/µL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and both assay products were resolved on 1% agarose gel.

Results:

Our newly developed N. gonorrhoeae IMRS-PCR assay had an analytical sensitivity of 6 fg/µL representing better sensitivity than the 16S rRNA PCR assay with an analytical sensitivity of 4.3096 pg/µL. The assay was also successfully validated using clinical urethral swab samples. We further advanced this technique by developing an isothermal IMRS, which was both reliable and sensitive for detecting cultured N. gonorrhoeae isolates at a concentration of 38 ng/µL. Combining isothermal IMRS with a low-cost lateral flow assay, we were able to detect N. gonorrhoeae amplicons at a starting concentration of 100 pg/µL.

Conclusion:

Therefore, there is a potential to implement this concept within miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for highly reliable point-of-care testing.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Heliyon Año: 2024 Tipo del documento: Article País de afiliación: Kenia Pais de publicación: Reino Unido
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Heliyon Año: 2024 Tipo del documento: Article País de afiliación: Kenia Pais de publicación: Reino Unido