Optimization of the Cetyltrimethylammonium bromide (CTAB) DNA extraction protocol using forest elephant dung samples

Abstract

Among non-invasive biological samples, feces offer an important source of DNA and can easily be collected. However, working with fecal DNA from highly vegetarians species such as elephant is more challenging because plant secondary compounds have an inhibitory effect on PCR reactions. Working with forest elephant dung samples, we tested and adapted a protocol of DNA extraction developed on plants based on the Cetyltrimethylammonium bromide (CTAB) protocol. The protocol is relatively simple and yields a high DNA concentration. It is five-time less expensive compared to the methods of Benbouza et al. The extracted DNA is of good quality and easily amplified by PCR. The high-amplification percentage of mitochondrial genes in fecal DNA and subsequent sequencing of PCR products indicate that the proposed optimized method is reliable for molecular analysis of forest elephant dung samples.•Our optimized CTAB protocol has been adjusted by the addition of Sodium Dodecyl Sulfate (SDS) and proteinase K during the lysis phase. The combined effect of these reagents was capable of lysing cell walls and removing proteins efficiently.•Moreover, the prolonged time of incubation (overnight incubation at room temperature followed by 3 hours of incubation in a water bath) enhanced the increase of DNA yield but make the optimized protocol more time-consuming.

Keywords: DNA quality and quantity; Fecal sample; Improved CTAB method; Non-invasive sample; PCR success.

Graphical abstract

Fig. 1

Boxplot of the quantity () of the DNA extract obtained based on the method of Benbouza et al. and the optimized method using forest elephant dung samples stored in 70 % ethanol.

Fig. 2

Percentile curve of DNA concentration obtained from samples of forest elephant dung stored in 70 % ethanol and extracted with the old method of Benbouza et al. and the new optimized method.

Fig. 3

DO ratio of DNA samples extraction using the method of Benbouza et al. .

Fig. 4

DO ratio of DNA samples extraction using the optimized methods.

Fig. 5

Graph of 260/280 absorbance ratios (DNA quality parameters) for the optimized methods of DNA extraction.

Fig. 6

a. PCR amplification of the mitochondrial DNA D-loop region using total DNA extracted from forest elephant dung sample using the new optimized method. Lane 1: molecular weight marker; Lane NC: negative control; Lane 1,2,4-9,12-21 and 23: positive PCR using primer pair LafCR1_F/LafCR2_R b. PCR amplification of the mitochondrial DNA D-loop region using total DNA extracted from forest elephant dung sample using an old CTAB DNA extraction method. Lane 1: molecular weight marker; Lane NC: negative control; Lane 1 to 16: negative PCR showing LafCR1_F/LafCR2_R primer dimer

Fig. 7

Chromatogram of a fragment of the mtDNA D-loop control region extracted from forest elephant dung sample using the optimized CTAB DNA extraction method.