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.…”

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).…”

“…The samples were thawed at room temperature, and centrifuged to remove the cell debris. In the boiling process described by Freschi et al (2005), the samples were centrifuged, followed by re-suspension in sterile distilled water, heated to 95ºC for 10 min, cooled on ice and centrifuged at 13,000 x g for 3 min. The supernatant was used for PCR assay.…”

A refrigeração no diagnóstico de Salmonella spp. utilizando o método microbiológico tradicional e reação em cadeia da polimerase em carcaças de frango

“…Tal situação não é exclusiva do Brasil, já que índices elevados de contaminação têm sido também observados em outros países que também implementaram programas semelhantes de redução do patógeno em carcaças de frango (SIMMONS et al, 2003;FLETCHER, 2006). Para a detecção do agente nas carcaças, os programas preconizam o emprego de métodos microbiológicos convencionais (MC), que, além de dispendiosos, podem levar até sete dias para a obtenção do resultado (FRESCHI et al, 2005). Já os métodos rápidos, como a Reação em Cadeia pela Polimerase (PCR), podem produzir respostas em poucas horas, além de serem altamente sensíveis e específicos.…”