Enhanced Blastocystis subtyping from stool samples using semi-nested barcode PCR: validation with an NGS-based approach
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. Barcod...
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Taylor & Francis Group
2024-12-01
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| Series: | BioTechniques |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/07366205.2024.2442835 |
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| author | Carlos Nieto-Clavijo Liliana Morales Andrés Delgado-Aldana Paula C. Hernández Isabel Torres-Molina Amanda Gonzalez-Cuiza Fabián Cortés-Muñoz Jacqueline Chaparro-Olaya |
| author_facet | Carlos Nieto-Clavijo Liliana Morales Andrés Delgado-Aldana Paula C. Hernández Isabel Torres-Molina Amanda Gonzalez-Cuiza Fabián Cortés-Muñoz Jacqueline Chaparro-Olaya |
| author_sort | Carlos Nieto-Clavijo |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-fa8177187fd244018676388b11c5d5d5 |
| institution | Kabale University |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-fa8177187fd244018676388b11c5d5d52025-02-06T15:38:41ZengTaylor & Francis GroupBioTechniques0736-62051940-98182024-12-01761258159110.1080/07366205.2024.2442835Enhanced Blastocystis subtyping from stool samples using semi-nested barcode PCR: validation with an NGS-based approachCarlos Nieto-Clavijo0Liliana Morales1Andrés Delgado-Aldana2Paula C. Hernández3Isabel Torres-Molina4Amanda Gonzalez-Cuiza5Fabián Cortés-Muñoz6Jacqueline Chaparro-Olaya7Laboratorio de Parasitología Molecular, Vicerrectoría de Investigaciones, Universidad El Bosque, Bogotá, ColombiaLaboratorio de Parasitología Molecular, Vicerrectoría de Investigaciones, Universidad El Bosque, Bogotá, ColombiaLaboratorio de Parasitología Molecular, Vicerrectoría de Investigaciones, Universidad El Bosque, Bogotá, ColombiaLaboratorio de Parasitología Molecular, Vicerrectoría de Investigaciones, Universidad El Bosque, Bogotá, ColombiaFundación Clínica Shaio, Bogotá, ColombiaFundación Clínica Shaio, Bogotá, ColombiaFundación Clínica Shaio, Bogotá, ColombiaLaboratorio de Parasitología Molecular, Vicerrectoría de Investigaciones, Universidad El Bosque, Bogotá, ColombiaIn 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.https://www.tandfonline.com/doi/10.1080/07366205.2024.2442835Blastocystis subtypingsemi-nested barcode PCRSantin PCRnext-generation sequencingST35 |
| spellingShingle | Carlos Nieto-Clavijo Liliana Morales Andrés Delgado-Aldana Paula C. Hernández Isabel Torres-Molina Amanda Gonzalez-Cuiza Fabián Cortés-Muñoz Jacqueline Chaparro-Olaya Enhanced Blastocystis subtyping from stool samples using semi-nested barcode PCR: validation with an NGS-based approach BioTechniques Blastocystis subtyping semi-nested barcode PCR Santin PCR next-generation sequencing ST35 |
| title | Enhanced Blastocystis subtyping from stool samples using semi-nested barcode PCR: validation with an NGS-based approach |
| title_full | Enhanced Blastocystis subtyping from stool samples using semi-nested barcode PCR: validation with an NGS-based approach |
| title_fullStr | Enhanced Blastocystis subtyping from stool samples using semi-nested barcode PCR: validation with an NGS-based approach |
| title_full_unstemmed | Enhanced Blastocystis subtyping from stool samples using semi-nested barcode PCR: validation with an NGS-based approach |
| title_short | Enhanced Blastocystis subtyping from stool samples using semi-nested barcode PCR: validation with an NGS-based approach |
| title_sort | enhanced blastocystis subtyping from stool samples using semi nested barcode pcr validation with an ngs based approach |
| topic | Blastocystis subtyping semi-nested barcode PCR Santin PCR next-generation sequencing ST35 |
| url | https://www.tandfonline.com/doi/10.1080/07366205.2024.2442835 |
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