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Mini Kit (mini + kit)
Selected AbstractsDetection of Helicobacter pylori DNA by a Simple Stool PCR Method in Adult Dyspeptic PatientsHELICOBACTER, Issue 4 2005Nazime ABSTRACT Introduction.,Helicobacter pylori is the major agent causing peptic ulcer, gastric cancer and mucosa-associated lymphoid tissue (MALT) gastric lymphoma. A simple stool polymerase chain reaction (PCR) method was performed and compared with the gold standards for the diagnosis of H. pylori infection. Material and methods., A total of 54 adult patients (mean age, 46.41 ± 13.12 years) with dyspeptic symptoms from Gastroenterology at Dokuz Eylül University Hospital between May and November 2003 were included. Two antrum and corpus biopsies were taken from each patient. Infection by H. pylori was defined as positivity and negativity of the gold standards. DNA extraction of stool specimens was done using QIAamp DNA Stool Mini Kit (QIAGEN) and PCR conditions included amplification and reamplification steps using the H. pylori ureA gene specific primers (HPU1, HPU2) and were visualized on 1% agarose gel stained with ethidium bromide. Results., Forty-six of 54 patients (85.2%) were diagnosed positive and eight (14.8%) were negative for H. pylori infection by the gold standard methods. Thirty-two patients were positive (59.3%) and 22 of them (40.7%) were detected negative by stool PCR method. The stool PCR method and gold standard methods showed a statistical difference for the detection of H. pylori infection (p < .0001). Sensitivity, specificity, likelihood ratio, and positive and negative predictive values were 65.22%, 75%, 2.61%, 93.75%, and 27.7%, respectively. Discussion., The PCR on the stool specimens resulted as being a very specific test. We suggest that a simple stool PCR method that we developed can be used to detect H. pylori, virulence genes, and in drug resistance studies either first line diagnostic methods in the laboratory or in the clinical management of dyspeptic patients. [source] Improvement in fetal DNA extraction from maternal plasma.PRENATAL DIAGNOSIS, Issue 1 2007Evaluation of the NucliSens Magnetic Extraction system, the QIAamp DSP Virus Kit in comparison with the QIAamp DNA Blood Mini Kit Abstract Objective Prenatal diagnostic assays have been developed using free fetal DNA circulating in the maternal blood of pregnant women. Efficient DNA extraction is crucial for a robust analysis. To improve fetal DNA yield, we tested two manual extraction methods,the NucliSens Magnetic Extraction (NMAG) system and the QIAamp DSP Virus Kit (QDSP),against our current standard method, the widely used QIAamp DNA Blood Mini Kit (QDNA). Methods The fetal DNA yield of the two extraction systems was evaluated using the RHD exon 7 as target in DNA extracts of 75 plasma samples from pregnant RhD-negative women, known to have given birth to RhD-positive infanto. The total DNA yield was evaluated in 23 samples, targeting GAPDH. Results The fetal DNA yield was improved by a mean factor of 1.7 using the NMAG system, and improved by a mean factor of 1.5 using the QDSP. The total DNA yield was improved by a mean factor of 2.3 using the NMAG system, and by a mean factor of 1.3 using the QDSP. Conclusion Both extraction systems tested were superior to our standard with regard to DNA yield. This improvement may have a great impact on the success of genotyping in early pregnancy. Copyright © 2007 John Wiley & Sons, Ltd. [source] ORIGINAL ARTICLE: Are Polymorphisms in the ACE and PAI-1 Genes Associated with Recurrent Spontaneous Miscarriages?AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 6 2009Chelsi Goodman Problem, To determine whether the ACE D/D genotype or the combination of PAI-1 4G/4G and ACE D/D genotypes may serve as a risk factor for recurrent pregnancy loss. Method of study, Buccal swabs were obtained from 120 women experiencing recurrent pregnancy loss and from 84 fertile control women. DNA was extracted from the buccal swab samples using the Qiagen DNA Mini Kit (Qiagen), followed by multiplex polymerase chain reaction (PCR). PCR products were analyzed for the ACE gene polymorphism, which consists of the insertion or deletion (I/D) of a 287-bp fragment in intron 16, and the PAI-1 4G/4G genotype. Results, No significant differences in specific ACE gene mutations were observed when patients experiencing recurrent miscarriage were compared with control women. When the frequencies of homozygous mutations for ACE D/D and PAI-I 4G/4G were compared between recurrent aborters and controls, again no significant differences in the prevalence of the combination of these gene mutations were noted. Conclusion, Homozygosity for the D allele of the ACE gene and the combination of the D/D genotype with two 4G alleles of the PAI-1 promoter gene are not associated with a significant increase in the risk of recurrent miscarriage. [source] DNA Extraction from Olive Oil and PCR Amplification of Microsatellite MarkersJOURNAL OF FOOD SCIENCE, Issue 1 2005Raffaele Testolin And ABSTRACT: DNA was extracted from single-cultivar of cold-pressed (virgin) unfiltered and cotton-filtered olive oils that were stored at 4 °C for up to a year using different DNA extraction kits and protocols. DNA was amplified using original and nested primers designed on 6 microsatellites loci of the UDO series. The most consistent results in terms of successful single sequence repeat amplifications were achieved using the Qiagen QIAamp DNA stool extraction kit, slightly modified and applied to oil sample amounts as small as 200 ,L without any pretreatment. The kit allowed getting polymerase chain reaction (PCR) amplicons visible on gel and scorable peaks at the automatic sequencer for all 6 markers analyzed. Less consistent results were achieved with other kits, such as the Promega Wizard Magnetic DNA Purification System for Food, the LB Link-Biotech ExtMan 50,100 Evolution, the Qiagen Plant Mini kit, and the standard cetyltrimethyl-ammonium bromide-based DNA extraction protocol. The integration in the protocols of further tools, such as the hexane-based phase separation, the addition of water or NaCl solutions to the oil, the precipitation and the use of the pellet, and others, did not result in any substantial use. PCR amplifications that gave low DNA yields were improved by adopting the nested PCR technique, which uses the product of the 1st PCR as a template for a 2nd PCR carried out by means of internal primers. Conclusions are drawn as to the applicability of the method to trace the identity of single-cultivar virgin olive oils. Further work is required to check the sensitivity of the method in determining the varietal composition of blended oils, especially in detecting alleles from cultivars present in only small amounts. [source] Rapid preparation of cyanobacterial DNA for real-time PCR analysisLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2008J.P. Rasmussen Abstract Aims:, To develop a rapid preparation method for real-time PCR analysis of cyanobacteria from cultures or field samples. Methods and Results:, Field samples and cultures containing Anabaena circinalis, Cylindrospermopsis raciborskii or Microcystis aeruginosa were subjected to three cell disruption treatments: (i) heating during thermocycling, (ii) microwave irradiation in the presence of detergent and (iii) probe sonication. Treated samples were directly added to the PCR reaction and analysed on two different real-time devices. A statistically significant difference was evident in the cycle thresholds for each of the treatments in all but one culture and one environmental sample, sonication and microwave treatments performing better than direct addition. The microwave treatment was also compared to the Qiagen DNA Mini kit and performance was equivalent when treated samples were analysed as above. Conclusions:, Whilst microwave treatment was slightly less effective than probe sonication across all samples, it was more amenable to processing multiple samples and significantly better than heat treating the sample during thermocycling. Significance and Impact of the Study:, The microwave method described here is a simple, rapid and effective preparation method for cyanobacterial DNA that can be easily deployed in the field, making the most of the speed and flexibility offered by fixed and portable real-time PCR devices. [source] Development of a real-time PCR-based method for detection of Xylophilus ampelinusPLANT PATHOLOGY, Issue 1 2007T. Dreo A real-time PCR MGB-probe-based detection method specific to Xylophilus ampelinus, the cause of grapevine bacterial blight, was developed. Used in combination with the DNeasy plant mini kit, the sensitivity of X. ampelinus detection was approximately 100 cells from tissue extracts, surpassing the sensitivity of an existing nested PCR method at least tenfold. In field samples a high correlation was observed between real-time PCR cycle threshold (Ct) values obtained and X. ampelinus isolation on artificial media. Isolation was successful from samples with Ct values below 25. Lower concentrations of X. ampelinus, with Ct values up to 36, could also be reliably detected in real-time PCR. The newly developed method offers a reliable and sensitive test for X. ampelinus, suitable as a screening test, complementary to isolation on media or other methods, and could also be used for fast and specific identification of isolated colonies and for relative quantification of X. ampelinus bacteria. [source] | |||||||||||||