A Comparison of Five Methods for Effective Extraction of Bacterial Metagenomic DNA from Stools of Children Suffer from Diarrhea with Unknown Cause

Author: Thi Quy Nguyen, Thi Bich Thuy Phung, Thu Thao Ngoc and Thi Huyen Do

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Abstract

Diarrhea is common disease in children and in the most severity can cause mortality. Besides the identified-cause diarrhea, significant percent cases (accounted for 47.38% in Vietnam) having bacterial infection symptoms were unidentified reason. The typical properties of the diarrheal feces are the presence of many inhibitors against polymerase activity to amplify the indicator genes of pathogenic agents. Thus, investigation of methods for extracting bacterial metagenomic DNA with high quality and quantity from diarrheal feces for further analysis is necessary. In this research, two stool samples from children under two years old, one with persistent diarrhea of unknown cause and another of normal health, were used for bacterial metagenomic DNA extraction. Four commercial DNA extraction kits (GeneJET Genomic DNA purification kit, QIAamp Fast DNA Stool Mini Kit, E.Z.N.A Stool DNA kit, TopPURE stool DNA extraction kit), and conventional biochemical technique were used for bacterial metagenomic DNA extraction. The result showed that, from an equal amount of feces, bacterial mass in normal stool was higher than one in diarrheal feces. The bacterial metagenomic DNA from normal samples was successfully extracted with all the investigated methods at acceptable quality (the ratio A260/280 was in a range of 1.7-2.2) for next generation sequencing. However the E.Z.N.A gave very low total DNA yield, TopPURE kit generated DNA containing inhibitor impact PCR reaction. Whereas the metagenomic DNAs from the stool of diarrhea patient were only well extracted by GeneJET Genomic DNA Purification kit and biochemical method. The biochemical method gave higher DNA yield but lower DNA quality exhibited by DNA degradation. On the contrary, GeneJET Genomic DNA Purification kit generated lower DNA yield but with higher quality. All the metagenomic DNA samples extracted by two these methods did not inhibit polymerase activity in PCR for 16S rDNA amplification. Thus, the GeneJET Genomic DNA Purification kit and biochemical method will be improved for extraction of bacterial metagenomic DNA from diarrheal feces for whole metagenome sequencing.

Keywords

Bacteria, biochemical method, children, diarrhea, genomic DNA extraction, kit, stool

Conclusion

Metagenomic DNA derived from a normal and diarrheal feces were extracted by the four commercial kits and a biochemical method. The biochemical method and GeneJET Genomic DNA Purification kit gave the best DNA concentration, yield and quality. The DNA templates up to 50 ng/reaction did not inhibit PCR for 16S rDNA amplification. Biochemical method and GeneJET Genomic DNA Purification kit should be chosen for improvement to extract metagenomic DNA from diarrhea stool samples for further analysis.

References

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How to cite this article

Thi Quy Nguyen, Thi Bich Thuy Phung, Thu Thao Ngoc and Thi Huyen Do (2023). A Comparison of Five Methods for Effective Extraction of Bacterial Metagenomic DNA from Stools of Children Suffer from Diarrhea with Unknown Cause. Biological Forum – An International Journal, 15(8a): 01-09.