Background The symbiotic relationship between gut microbiota and their fish hosts has fuelled extensive research into microbial distribution besides their active role in host body metabolisms and paving the way for the sustainable aquaculture. This study aims to optimize and evaluate DNA extraction techniques for characterizing the gut microbiota of fish with diverse feeding habits: Hilsa (planktivorous), Catla (zooplankton feeder), Rohu (herbivorous), and Mrigal (illiophagus). Methods and results Microbial genomic DNA was extracted using five traditional methods—PLICKS A, B, C, and CTAB (Methods D and E)—and three commercial kits (MN® Microbial, MN® Soil, and MN® Faecal), each with modifications. The efficacy of these methods was assessed based on DNA yield (traditional: 74–3070 ng/µL; commercial: 8.8–224 ng/µL), purity (traditional: A260/280: 1.38–1.92, A260/230: 1.03–2.21; commercial: A260/280: 1.30–3.25, A260/230: 0.5–2.0), and successful PCR amplification, a key step for downstream 16 S rRNA gene sequencing. Among traditional methods, PLICKS A (Catla), PLICKS C (Hilsa), CTAB (Mrigal and Catla), and PLICKS B (Catla, Rohu, Hilsa, Mrigal) delivered the highest DNA recovery (342–2080 ng/µL) and purity across different species. Similarly, among commercial kits, the MN® Microbial Modified Kit (Catla, Hilsa), MN® Soil Kit (Hilsa), MN® Soil Modified Kit (Catla, Rohu), MN® Faecal Kit (Catla), and MN® Modified Faecal Kit excelled, achieving optimal DNA recovery (108–224 ng/µL) and purity across various feeding habits. Overall, among traditional methods, PLICKS B proved to be the most effective, delivering high DNA yields (342–2080 ng/µL) with excellent purity (A260/280: 1.77–1.92; A260/230: 1.67–2.21) and enabling successful PCR amplification across fish species with diverse feeding habits. Similarly, among commercial kits, the MN Modified Faecal Kit achieved the highest DNA recovery (108–224 ng/µL) and purity (A260/280: 1.74–1.90; A260/230: 1.78–2.01), consistently supporting reliable amplification. Conclusions These findings highlight effective DNA extraction methods tailored to fish with different feeding habits. Careful selection and optimization of extraction protocols are therefore essential for the accurate characterization of fish gut microbiota.

Comparative performance of traditional and commercial DNA extraction methods for fish gut microbiota analysis

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发表日期：

2026年4月17日

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Amrita Mohanty,
Annam Pavan-Kumar,
Aparna Chaudhari,
Kavita Kumari,
Prem Kumar,
Praveen Maurye

摘要

Background

The symbiotic relationship between gut microbiota and their fish hosts has fuelled extensive research into microbial distribution besides their active role in host body metabolisms and paving the way for the sustainable aquaculture. This study aims to optimize and evaluate DNA extraction techniques for characterizing the gut microbiota of fish with diverse feeding habits: Hilsa (planktivorous), Catla (zooplankton feeder), Rohu (herbivorous), and Mrigal (illiophagus).

Methods and results

Microbial genomic DNA was extracted using five traditional methods—PLICKS A, B, C, and CTAB (Methods D and E)—and three commercial kits (MN^® Microbial, MN^® Soil, and MN^® Faecal), each with modifications. The efficacy of these methods was assessed based on DNA yield (traditional: 74–3070 ng/µL; commercial: 8.8–224 ng/µL), purity (traditional: A260/280: 1.38–1.92, A260/230: 1.03–2.21; commercial: A260/280: 1.30–3.25, A260/230: 0.5–2.0), and successful PCR amplification, a key step for downstream 16 S rRNA gene sequencing. Among traditional methods, PLICKS A (Catla), PLICKS C (Hilsa), CTAB (Mrigal and Catla), and PLICKS B (Catla, Rohu, Hilsa, Mrigal) delivered the highest DNA recovery (342–2080 ng/µL) and purity across different species. Similarly, among commercial kits, the MN^® Microbial Modified Kit (Catla, Hilsa), MN^® Soil Kit (Hilsa), MN^® Soil Modified Kit (Catla, Rohu), MN^® Faecal Kit (Catla), and MN^® Modified Faecal Kit excelled, achieving optimal DNA recovery (108–224 ng/µL) and purity across various feeding habits. Overall, among traditional methods, PLICKS B proved to be the most effective, delivering high DNA yields (342–2080 ng/µL) with excellent purity (A_260/280: 1.77–1.92; A_260/230: 1.67–2.21) and enabling successful PCR amplification across fish species with diverse feeding habits. Similarly, among commercial kits, the MN Modified Faecal Kit achieved the highest DNA recovery (108–224 ng/µL) and purity (A_260/280: 1.74–1.90; A_260/230: 1.78–2.01), consistently supporting reliable amplification.

Conclusions

These findings highlight effective DNA extraction methods tailored to fish with different feeding habits. Careful selection and optimization of extraction protocols are therefore essential for the accurate characterization of fish gut microbiota.