Modifications and optimization of manual methods for polymerase chain reaction and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta

Abstract

Background and Aim:A critical prerequisite for studying rumen microbial community by high throughput molecular biology methods is good quality community DNA. Current methods of extraction use kits designed for samples from the different origin for rumen. This puts stress on the development of a relevant manual method for DNA extraction. The objective of this study was to modify the existing methods of community DNA extraction and thereby systematic comparison of their efficiency based on DNA yield, purity, 16S rRNA gene sequencing, and identification to determine the optimal DNA extraction methods whose DNA products reflect targeted bacterial communities special to rumen.Materials and Methods:Enzymatic method, Chemical method, Enzymatic + Chemical method, and Enzymatic + Chemical + Physical method were modified toward evaluation of community DNA extraction from solid, squeezed, and liquid fractions of goat rumen digesta. Each method was assessed critically for nucleic acid yield and its quality. The methods resulting in high nucleic acid yield, optimal purity ratios with intact band on agarose gel electrophoresis were optimized further. Optimized methods were studied using standard polymerase chain reaction (PCR) with universal bacterial primers and 16S rRNA primers of targeted rumen bacteria. Methods denoting the presence of targeted rumen bacteria were assessed further with 16S rRNA gene sequencing and identification studies. It led toward methods efficacy estimation for molecular biology applications. Effect of rumen sample preservation on community DNA extraction was also studied. Their mean standard deviation values were calculated to understand sampling criticality.Results:Modified Chemical method (Cetrimonium bromide) and Enzymatic+Chemical+Physical (ECP) method (Lysozyme-Cetrimonium bromide-Sodium Dodecyl Sulfate-freeze-thaw) could extract 835 ng/µl and 161 ng/µl community DNA from 1.5 g solid and 2 ml squeezed rumen digesta with purity ratios of 1.8 (A260 nm/A280 nm) and 2.3 (A260 nm/A230 nm) respectively. Comparative analysis showed the better efficiency of ECP method and chemical method toward freshly squeezed rumen digesta and solid rumen digesta. However, sample preservation at −80°C for 1.5 months drastically affected the yield and purity ratios of community DNA. New protocol revealed targeted microbial community having Gram-positive as well as Gram-negative bacteria such as Prevotella ruminicola, Streptococcus lutetiensis, Ruminococcus flavefaciens, Fibrobacter succinogenes, and Selenomonas ruminantium.Conclusion:To date, this is the first report of modified methods wherein least chemicals and steps lead toward PCR and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta. Detection of targeted rumen bacteria in solid and squeezed rumen digesta proves their strongest association with rumen fiber mat. It also marks the presence of distinct microbial communities in solid and squeezed rumen fractions that in turn differs the performance of each different method employed and...

A Novel Method of Extraction and Purification of Bacterial DNA for PCR Detection of MecA, 16SrRNA, VanB

“…As a result, it is quick, simple, and inexpensive, and it may be used to isolate high-yield analytical grade DNA from gram-positive and gram-negative bacteria (Glenn, 2011). The author (Durgadevi A., 2018) there is a previous study on a modified method using fewer chemicals and techniques leading to PCR and the sequencing of 16S rRNA genes quality Isolation of DNA from Goat Rumen Digesta (Ali et al, 2021;Aphale et al, 2018;James et al, 2021). Following that, population DNA extracted using the boiling process approach could provide standard PCR amplification with general bacterial primers and specific bacterial 16S rRNA gene primers (Ahmed et al, 2017), demonstrating its suitability for a wide range of biological applications.…”

Diversity and Antimicrobial Resistance in the Streptococcus bovis/Streptococcus equinus Complex (SBSEC) Isolated from Korean Domestic Ruminants

“…Although it is difficult to directly compare the presence of SBSEC members in a single animal species due to the difficulties encountered over the years in the correct identification of SBSEC strains to the species (or subspecies) level by phenotypic and genotypic methods, the potential presence of S. lutetiensis (Previously classified as S. infantarius subsp. coli ) in domestic ruminants have been only addressed in caprine and their food products [ 57 , 58 ]. In this study, we also confirmed the presence of S. lutetiensis only in the rumen of Korean goats by direct isolation and sodA gene-based identification rather that from bovine, thus hypothesizing that the species might be one of the distinguished important commensal SBSEC in caprine.…”

Genetic characterization and phylogenetic study of Indonesian cuscuses from Maluku and Papua Island based on 16S rRNA gene

“…These mitochondrial DNA sequences are unique and can be used as genetic markers for species identification. The 16S rRNA gene has been widely used for phylogenetic study and detection in other species [19][20][21][22]. It is expected that the nucleotide diversity of each cuscus species can be used as genetic markers and can determine the phylogenetic relationship of the cuscuses from Maluku and Papua.…”