Enhanced Blastocystis subtyping from stool samples using semi-nested barcode PCR: validation with an NGS-based approach

Autores organización

• 

  Liliana Morales De La Pava

  Autor

• Jacqueline Chaparro Olaya

  Autor

Autores

• Nieto-Clavijo C.
• Delgado-Aldana A.
• Hernández P.C.
• Torres-Molina I.
• Gonzalez-Cuiza A.
• Cortés-Muñoz F.

Unidades de investigación

• Laboratorio de Parasitología Molecular

  Investigador

  Jacqueline Chaparro Olaya

Resumen

In 2006, a PCR method was introduced to subtype Blastocystis by Sanger sequencing of an ˜610 bp amplicon of the 18S rRNA gene. This method, known as barcoding-PCR, has become widespread, although the primer pair used can amplify non-Blastocystis sequences, which can result in false positives. Barcoding-PCR is most effective with DNA extracted from Blastocystis cultures, limiting its sensitivity when used directly with stool samples. As a result, barcoding-PCR can sometimes yield negative results for stool samples confirmed as Blastocystis-positive by microscopy. To improve subtyping from stool-derived DNA, we developed a Semi-Nested barcode PCR that amplifies the barcoding region in a second reaction. Our study shows that this Semi-Nested approach outperforms classical barcoding-PCR, detecting Blastocystis more reliably from stool samples with stronger gel signals and no false positives. This was confirmed by near-complete concordance (68/70 samples) with the Santin-PCR coupled to Next-Generation Sequencing (NGS) as reference standard for Blastocystis subtyping. Of particular interest, one amplicon matched the only previous report of ST35, marking this as the second global detection of ST35 and the first in Colombia. Overall, Semi-Nested barcoded PCR offers a more robust and sensitive alternative compared to classical barcoding-PCR for subtyping Blastocystis directly from stool samples. © 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

Keywords

• Blastocystis subtyping; next-generation sequencing; Santin PCR; semi-nested barcode PCR; ST35;Blastocystis; Blastocystis Infections; DNA Barcoding, Taxonomic; DNA, Protozoan; Feces; High-Throughput Nucleotide Sequencing; Humans; Polymerase Chain Reaction; RNA, Ribosomal, 18S; protozoal DNA; RNA 18S; protozoal DNA; RNA 18S; amplicon; Article; Blastocystis; Colombia; contamination; controlled study; DNA barcoding; DNA extraction; false negative result; false positive result; feces analysis; genotyping; high throughput sequencing; intermethod comparison; microorganism detection; nonhuman; process development; Sanger sequencing; semi-nested polymerase chain reaction; validation study; blastocystosis; classification; diagnosis; feces; genetics; human; isolation and purification; parasitology; polymerase chain reaction; procedures