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.

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