Comparison of DNA-extraction methods and Selective Enrichment broths on the detection of Salmonella Typhimurium in swine feces by polymerase chain reaction (PCR)

Abstract

The aim of this study was to compare different DNA-extraction methods and selective enrichment broths for their effectiveness to detect Salmonella Typhimurium in artificially inoculated swine feces samples (100 CFU/ g) by polymerase chain reaction. After enrichment in Rappaport-Vassiliadis, selenite cystine or Müller-Kauffmann tetrathionate, aliquots were used for DNA extraction by three different methods: boilingcentrifugation, phenol-chloroform and salting-out. Aliquots of extracted DNA were then used as template in PCR. The selective enrichment broths had no effect on the efficiency of PCR when boiling-centrifugation and salting-out were used. On the other hand, phenol-chloroform was superior (P<0.05) when combined to Rappaport-Vassiliadis. Considering cost and efficiency parameters, we encourage the use of Müller-Kauffmann tetrathionate broth in combination with boiling-centrifugation DNA-extraction procedure.

Application of real-time PCR for detection of Lawsonia intracellularis and Brachyspira hyodysenteriae in fecal samples from pigs

“…These findings are in agreement with our experience (data not shown) that bacterial DNA isolated by silica membrane-based methods is better purified and of higher quality. Finally, according to studies performed by Freschi et al (2005) the efficiency of PCR performed on samples subjected to the boiling-centrifugation DNA extraction method was very similar to those achieved by using the commercial kit. However, the speed and low cost of the boiling-centrifugation technique must be emphasized.…”

“…Although average Ct values for both analyzed variants of extractions were similar and statistically no differences were observed, a commercially available Genomic Mini kit, based on silica-membrane, was chosen for the further analysis. The choice was supported by the results obtained by other authors (Song et al 2004, Freschi et al 2005, Nelson et al 2010. Pilot studies performed by Song et al (2004) have confirmed that the use of commercial kit for DNA extraction produces high-quality DNA free of PCR-inhibiting substances.…”

Application of real-time PCR for detection of Lawsonia intracellularis and Brachyspira hyodysenteriae in fecal samples from pigs

“…These findings are in agreement with our experience (data not shown) that bacterial DNA isolated by silica membrane-based methods is better purified and of higher quality. Finally, according to studies performed by Freschi et al (2005) the efficiency of PCR performed on samples subjected to the boiling-centrifugation DNA extraction method was very similar to those achieved by using the commercial kit. However, the speed and low cost of the boiling-centrifugation technique must be emphasized.…”

“…Although average Ct values for both analyzed variants of extractions were similar and statistically no differences were observed, a commercially available Genomic Mini kit, based on silica-membrane, was chosen for the further analysis. The choice was supported by the results obtained by other authors (Song et al 2004, Freschi et al 2005, Nelson et al 2010. Pilot studies performed by Song et al (2004) have confirmed that the use of commercial kit for DNA extraction produces high-quality DNA free of PCR-inhibiting substances.…”

In Vitro Probiotic Potential of Hemophilin-producing Strains of Haemophilus haemolyticus

“…Given the high volume of samples generated from growth experiments, a cheap and high-throughput DNA extraction method was required to reliably distinguish and quantify NTHi and Hh in co-culture. Extraction utilizing thermal lysis has previously been shown to be an efficient and cost-effective method to harvest bacterial DNA for quantitative real-time PCR from suspensions of several bacterial species in a range of sample matrices (36–41). Crude DNA extraction methods are also prone to contamination with PCR inhibitors originating from sample matrices (39, 42).…”

Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification

“…To ensure the cleaner PCR product and lower background, a lower concentration of primer (0.4 µM) than the one used so far (0.8 µM) was tested. Despite the typical PCR primer concentrations range from 0.1 to 1 µM, in general, the optimal primer concentrations were from 0.1 to 0.5 µM (Innis and Gelfand 1990) and this range of primer concentration has been used with a relative frequency (Maneu et al 2000;Andrighetto et al 2000Freschi et al 2005;Galán et al 2006;Liguori et al 2007). The usual dNTP concentration between 20µM and 200 µM of each of the four dNTPs gave optimal balance among the yield, specificity and fidelity (Innis and Gelfand 1990).…”

Application of real-time PCR for detection of Lawsonia intracellularis and Brachyspira hyodysenteriae in fecal samples from pigs

“…These findings are in agreement with our experience (data not shown) that bacterial DNA isolated by silica membrane-based methods is better purified and of higher quality. Finally, according to studies performed by Freschi et al (2005) the efficiency of PCR performed on samples subjected to the boiling-centrifugation DNA extraction method was very similar to those achieved by using the commercial kit. However, the speed and low cost of the boiling-centrifugation technique must be emphasized.…”

“…Although average Ct values for both analyzed variants of extractions were similar and statistically no differences were observed, a commercially available Genomic Mini kit, based on silica-membrane, was chosen for the further analysis. The choice was supported by the results obtained by other authors (Song et al 2004, Freschi et al 2005, Nelson et al 2010. Pilot studies performed by Song et al (2004) have confirmed that the use of commercial kit for DNA extraction produces high-quality DNA free of PCR-inhibiting substances.…”

In Vitro Probiotic Potential of Hemophilin-producing Strains of Haemophilus haemolyticus

“…Given the high volume of samples generated from growth experiments, a cheap and high-throughput DNA extraction method was required to reliably distinguish and quantify NTHi and Hh in co-culture. Extraction utilizing thermal lysis has previously been shown to be an efficient and cost-effective method to harvest bacterial DNA for quantitative real-time PCR from suspensions of several bacterial species in a range of sample matrices (36–41). Crude DNA extraction methods are also prone to contamination with PCR inhibitors originating from sample matrices (39, 42).…”

Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification

“…To ensure the cleaner PCR product and lower background, a lower concentration of primer (0.4 µM) than the one used so far (0.8 µM) was tested. Despite the typical PCR primer concentrations range from 0.1 to 1 µM, in general, the optimal primer concentrations were from 0.1 to 0.5 µM (Innis and Gelfand 1990) and this range of primer concentration has been used with a relative frequency (Maneu et al 2000;Andrighetto et al 2000Freschi et al 2005;Galán et al 2006;Liguori et al 2007). The usual dNTP concentration between 20µM and 200 µM of each of the four dNTPs gave optimal balance among the yield, specificity and fidelity (Innis and Gelfand 1990).…”

Application of real-time PCR for detection of Lawsonia intracellularis and Brachyspira hyodysenteriae in fecal samples from pigs

“…These findings are in agreement with our experience (data not shown) that bacterial DNA isolated by silica membrane-based methods is better purified and of higher quality. Finally, according to studies performed by Freschi et al (2005) the efficiency of PCR performed on samples subjected to the boiling-centrifugation DNA extraction method was very similar to those achieved by using the commercial kit. However, the speed and low cost of the boiling-centrifugation technique must be emphasized.…”

“…Although average Ct values for both analyzed variants of extractions were similar and statistically no differences were observed, a commercially available Genomic Mini kit, based on silica-membrane, was chosen for the further analysis. The choice was supported by the results obtained by other authors (Song et al 2004, Freschi et al 2005, Nelson et al 2010. Pilot studies performed by Song et al (2004) have confirmed that the use of commercial kit for DNA extraction produces high-quality DNA free of PCR-inhibiting substances.…”

In Vitro Probiotic Potential of Hemophilin-producing Strains of Haemophilus haemolyticus

“…Given the high volume of samples generated from growth experiments, a cheap and high-throughput DNA extraction method was required to reliably distinguish and quantify NTHi and Hh in co-culture. Extraction utilizing thermal lysis has previously been shown to be an efficient and cost-effective method to harvest bacterial DNA for quantitative real-time PCR from suspensions of several bacterial species in a range of sample matrices (36–41). Crude DNA extraction methods are also prone to contamination with PCR inhibitors originating from sample matrices (39, 42).…”

Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification

“…To ensure the cleaner PCR product and lower background, a lower concentration of primer (0.4 µM) than the one used so far (0.8 µM) was tested. Despite the typical PCR primer concentrations range from 0.1 to 1 µM, in general, the optimal primer concentrations were from 0.1 to 0.5 µM (Innis and Gelfand 1990) and this range of primer concentration has been used with a relative frequency (Maneu et al 2000;Andrighetto et al 2000Freschi et al 2005;Galán et al 2006;Liguori et al 2007). The usual dNTP concentration between 20µM and 200 µM of each of the four dNTPs gave optimal balance among the yield, specificity and fidelity (Innis and Gelfand 1990).…”