Enhanced <i>Blastocystis</i> subtyping from stool samples using semi-nested barcode PCR: validation with an NGS-based approach
Creators: name: Nieto-Clavijo, Carlos
name: Morales, Liliana
name: Delgado-Aldana, Andrés
name: Hernández, Paula C.
name: Torres-Molina, Isabel
name: Gonzalez-Cuiza, Amanda
name: Cortés-Muñoz, Fabián
name: Chaparro-Olaya, Jacqueline
Publisher: name: Taylor & Francis
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Biotechnology
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Science Policy
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Plant Biology

description: In 2006, a PCR method was introduced to subtype <i>Blastocystis</i> 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-<i>Blastocystis</i> sequences, which can result in false positives. Barcoding-PCR is most effective with DNA extracted from <i>Blastocystis</i> 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 <i>Blastocystis</i>-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 <i>Blastocystis</i> 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 <i>Blastocystis</i> 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 <i>Blastocystis</i> directly from stool samples. DNA extracted from stool samples identified as <i>Blastocystis</i>-positive by microscopy was analyzed using both classical barcoding-PCR and Semi-Nested barcode PCR for genotyping. Semi-nested barcoding PCR produced more distinct amplicons on gels (83% versus 50%), leading to an evaluation of the potential for false-positive signals due to contamination. Therefore, Semi-Nested barcode PCR products were subjected to Sanger sequencing, and the resulting subtypes were compared with those identified using Santin-PCR combined with NGS (Illumina MiSeq). The results showed 98% concordance, demonstrating that, compared to classical barcoding-PCR, Semi-Nested barcode PCR enhances robustness and sensitivity for the direct analysis of stool samples. Development of barcoding-PCR (2006). Originally designed for <i>Blastocystis</i> subtyping by sequencing a 610 bp PCR product from the 18S rRNA gene.Limitations of barcoding-PCR. Sensitivity is limited with stool samples, which can occasionally result in false negatives even though the presence of <i>Blastocystis</i> has been confirmed by microscopy.Semi-Nested barcode PCR Development. Developed to improve sensitivity and robustness for stool sample analysis, eliminating the need for culture-based DNA.Semi-Nested Approach. Utilizes a second reaction that amplifies the barcoding region without increasing the risk of contamination.NGS validation. Subtypes established with the Semi-Nested barcode PCR were validated by Santin-PCR coupled to NGS.Improved Detection. Semi-Nested barcode PCR demonstrated higher detection rates and more intense gel signals compared to classical barcoding-PCR.High Accuracy. Semi-Nested barcode PCR achieved near-complete concordance (68/70) with the high-sensitivity Santin-PCR coupled to NGS, confirming specificity.Discovery of a new subtype in Colombia. A subtype was identified with 99.25% identity to the only known ST35 record worldwide, marking the first detection of ST35 in Colombia.Enhanced sensitivity and high reliability: Semi-Nested barcode PCR provides a robust, sensitive alternative to classical barcode PCR for subtyping <i>Blastocystis</i> directly from stool samples, with no contamination-related false positives, as confirmed by NGS validation.PubMLST Database Limitation. PubMLST failed to identify ST35, which was detected using a BLAST search, underscoring the need for alternative validation methods for detecting rare subtypes. Development of barcoding-PCR (2006). Originally designed for <i>Blastocystis</i> subtyping by sequencing a 610 bp PCR product from the 18S rRNA gene. Limitations of barcoding-PCR. Sensitivity is limited with stool samples, which can occasionally result in false negatives even though the presence of <i>Blastocystis</i> has been confirmed by microscopy. Semi-Nested barcode PCR Development. Developed to improve sensitivity and robustness for stool sample analysis, eliminating the need for culture-based DNA. Semi-Nested Approach. Utilizes a second reaction that amplifies the barcoding region without increasing the risk of contamination. NGS validation. Subtypes established with the Semi-Nested barcode PCR were validated by Santin-PCR coupled to NGS. Improved Detection. Semi-Nested barcode PCR demonstrated higher detection rates and more intense gel signals compared to classical barcoding-PCR. High Accuracy. Semi-Nested barcode PCR achieved near-complete concordance (68/70) with the high-sensitivity Santin-PCR coupled to NGS, confirming specificity. Discovery of a new subtype in Colombia. A subtype was identified with 99.25% identity to the only known ST35 record worldwide, marking the first detection of ST35 in Colombia. Enhanced sensitivity and high reliability: Semi-Nested barcode PCR provides a robust, sensitive alternative to classical barcode PCR for subtyping <i>Blastocystis</i> directly from stool samples, with no contamination-related false positives, as confirmed by NGS validation. PubMLST Database Limitation. PubMLST failed to identify ST35, which was detected using a BLAST search, underscoring the need for alternative validation methods for detecting rare subtypes.