A Two-Step Single Plex PCR Method for Evaluating Key Colonic Microbiota Markers in Young Mexicans with Autism Spectrum Disorders: Protocol and Pilot Epidemiological Application

Abstract

Download PDFsettingsOrder Article Reprints Open AccessArticle A Two-Step Single Plex PCR Method for Evaluating Key Colonic Microbiota Markers in Young Mexicans with Autism Spectrum Disorders: Protocol and Pilot Epidemiological Application by Julián Herrera-Mejía 1ORCID,Rocío Campos-Vega 2,Abraham Wall-Medrano 1,*ORCID andFlorinda Jiménez-Vega 1,*ORCID 1 Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Anillo Envolvente del PRONAF y Estocolmo s/n, Ciudad Juárez 32310, Chihuahua, Mexico 2 Programa de Posgrado en Alimentos del Centro de la República (PROPAC), Research and Graduate Studies in Food Science, School of Chemistry, Universidad Autónoma de Querétaro, Santiago de Querétaro 76010, Querétaro, Mexico * Authors to whom correspondence should be addressed. Diagnostics 2023, 13(14), 2387; https://doi.org/10.3390/diagnostics13142387 Submission received: 26 May 2023 / Revised: 9 July 2023 / Accepted: 14 July 2023 / Published: 17 July 2023 (This article belongs to the Section Point-of-Care Diagnostics and Devices) Downloadkeyboard_arrow_down Browse Figures Versions Notes Abstract Many neurological disorders have a distinctive colonic microbiome (CM) signature. Particularly, children with autism spectrum disorders (ASD) exhibit a very dissimilar CM when compared to neurotypical (NT) ones, mostly at the species level. Thus far, knowledge on this matter comes from high-throughput (yet very expensive and time-consuming) analytical platforms, such as massive high-throughput sequencing of bacterial 16S rRNA. Here, pure (260/280 nm, ~1.85) stool DNA samples (200 ng.µL−1) from 48 participants [39 ASD, 9 NT; 3–13 y] were used to amplify four candidate differential CM markers [Bacteroides fragilis (BF), Faecalibacterium prausnitzii (FP), Desulfovibrio vulgaris (DV), Akkermansia muciniphila (AM)], using micro-organism-specific oligonucleotide primers [265 bp (BF), 198 bp (FP), 196 bp (DV), 327 bp (AM)] and a standardized two-step [low (step 1: °Tm—5 °C) to high (stage 2: °Tm—0 °C) astringent annealing] PCR protocol (2S-PCR). The method was sensitive enough to differentiate all CM biomarkers in the studied stool donors [↑ abundance: NT (BF, FP, AM), ASD (DV)], and phylogenetic analysis confirmed the primers’ specificity. Keywords: Faecalibacterium prausnitzii; Bacteroides fragilis; Desulfovibrio vulgaris; Akkermansia muciniphila; microbiota; two-step PCR; 16S rRNA; autism; ASD