Abstract
The applications of conventional culture-dependent assays to quantify bacteria populations are limited by their dependence on the inconsistent success of the different culture-steps involved. In addition, some bacteria can be pathogenic or a source of endotoxins and pose a health risk to the researchers. Bacterial quantification based on the real-time PCR method can overcome the above-mentioned problems. However, the quantification of bacteria using this approach is commonly expressed as absolute quantities even though the composition of samples (like those of digesta) can vary widely; thus, the final results may be affected if the samples are not properly homogenized, especially when multiple samples are to be pooled together before DNA extraction. The objective of this study was to determine the correlation coefficients between four different methods of expressing the output data of real-time PCR-based bacterial quantification. The four methods were: (i) the common absolute method expressed as the cell number of specific bacteria per gram of digesta; (ii) the Livak and Schmittgen, ΔΔCt method; (iii) the Pfaffl equation; and (iv) a simple relative method based on the ratio of cell number of specific bacteria to the total bacterial cells. Because of the effect on total bacteria population in the results obtained using ΔCt-based methods (ΔΔCt and Pfaffl), these methods lack the acceptable consistency to be used as valid and reliable methods in real-time PCR-based bacterial quantification studies. On the other hand, because of the variable compositions of digesta samples, a simple ratio of cell number of specific bacteria to the corresponding total bacterial cells of the same sample can be a more accurate method to quantify the population.
Highlights
The gastrointestinal tract (GIT) of chicken contains an array of bacterial populations that are essential for the growth and health of the host animal by influencing intestinal morphology, digestion and absorption processes as well as protecting it against infection [1,2]
Most previous studies in broiler chickens used culture-dependent approaches and have shown that the culturable bacteria in the GIT mostly belong to the Lactobacilli, Enterococci, and Enterobacteria, while Lactobacilli, Enterococci, Bacteroides, and Clostridia are the predominant bacteria of the cecum [14,15,16,17]
In a pre-experiment, we examined the data on DNA extraction from digesta samples based on differing moisture contents and found that bacterial loads of digesta samples are mainly in the liquid phase of the digesta and that the best absolute unit was expressed as bacterial colony-forming unit (CFU) per mL of digesta liquid content rather than on a dry matter basis [25]
Summary
The gastrointestinal tract (GIT) of chicken contains an array of bacterial populations that are essential for the growth and health of the host animal by influencing intestinal morphology, digestion and absorption processes as well as protecting it against infection [1,2]. Feng et al [28] used the Livak and Schmittgen ΔΔCt method [29] to calculate the relative quantity of different bacterial population relative to the total bacteria mass as a reference (like the role of housekeeping genes in gene expression studies). This methodology was originally developed to calculate relative gene expression in reverse transcription-real-time PCR methods, and there are some obvious differences between bacterial population quantification and gene expression studies. The effects of bacterial types and dietary treatments on the correlations of the three methods were determined
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