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.

Diagnostics (Basel)

Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Anillo Envolvente del PRONAF y Estocolmo s/n, Ciudad Juárez 32310, Chihuahua, Mexico.

Published: July 2023

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) from 48 participants [39 ASD, 9 NT; 3-13 y] were used to amplify four candidate differential CM markers [ (BF), (FP), (DV), (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: °-5 °C) to high (stage 2: °-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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10377852PMC
http://dx.doi.org/10.3390/diagnostics13142387DOI Listing

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