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ABSTRACT: The most important restriction for the detection in water samples is the low concentration of Giardia intestinalis cysts, additional difficulty is the presence of PCR inhibitors. We have carried out trials in order to assess the sensitivity of semi-nested PCR and TaqMan real time PCR on the basis of DNA extracted from G. intestinalis cysts coming from spiked environmental and distilled water samples, filtrated with the use of Filta-Max® equipment (1623 Method). Removal of inhibitors was carried out with addition of BSA in different concentrations. During the filtration and concentration of water samples, losses of cysts have been recorded. Moreover, addition of BSA to the PCR and real time PCR mix increases the sensitivity of reaction. The optimal concentration of BSA for semi‑nested PCR was 15 and 20 ng/μl, whereas for real time PCR 5 ng/μl.
Parasite (Paris, France) 11/2011; 18(4):341-3. · 1.00 Impact Factor
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ABSTRACT: The aim of the presented study was to work out an effective method of extraction of DNA from Giardia intestinalis cysts as well as a sensitive and specific method for detection of DNA of this protozoan using a polymerase chain reaction (PCR). Twelve protocols for DNA extraction have been compared. Purification and extraction of DNA were preceded by additional actions in order to destroy the cysts' wall. The highest effectiveness of DNA extraction was obtained in case of alternating application of freezing the samples in liquid nitrogen and their incubation in water bath in the temperature of 100 degrees C, and then the extraction with the QIAamp DNA Tissue Mini Kit (QIAGEN)--T kit--with an all night long incubation with proteinase K in 56 degrees C. Effectiveness of DNA extraction with the use of each kit after extraction with each treatment was measured by nested PCR product of beta-giardin gene fragment and C(T) values of real time PCR of the SSU rRNA gene of G. intestinalis. The detection limit, defined as the lowest number detected in 100% cases, was 100 cysts per 200 microl when effectiveness was evaluated with nested PCR and 50 oocysts with real time PCR after extraction DNA with T kit. Results of our comparative studies have shown that all stages preceding the molecular detection of G. intestinalis DNA are equally important, and materially influence on the final effect and this version of method seems to be very useful for the sensitive detection of DNA of G. intestinalis.
Parasite (Paris, France) 12/2010; 17(4):299-305. · 1.00 Impact Factor
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ABSTRACT: Allergic asthma is a chronic inflammatory disease in which interleukin-18 (IL-18) plays an important role. However, there are controversial reports on IL-18 promoter polymorphism as an independent marker of asthma susceptibility. The aim of the present study was to examine the IL-18 promoter polymorphism in patients with allergic asthma. Two hundred and thirty-one patients with allergic asthma from a Polish population diagnosed according to the National Heart, Lung, and Blood Institute (NHLBI)/WHO guidelines were examined. An allele-specific polymerase chain reaction was used to analyse polymorphisms at positions -137 and -607 in the promoter region of the IL-18 gene. Neither in the -607 C>A nor in the -137 G>C promoter polymorphism were there any differences observed between the total group of asthmatic patients and the controls in the frequencies of genotypes, alleles, diplotypes or haplotypes. In patients with severe asthma, the -607 CC and -137 GG genotypes were observed significantly more frequently (P = 0.03 for both), whereas in patients with mild and moderate asthma, the -137 CC genotype was more prevalent than in the former group. The strongest difference between mild to moderate and severe asthma was observed in -137 allele frequencies (P = 0.006). The results of the present study suggest that the -137 G allele and the C-G/C-G diplotype seem to be involved in the pathogenesis of the severe form of asthma.
Tissue Antigens 11/2007; 70(4):314-8. · 2.59 Impact Factor
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ABSTRACT: Skotarczak B., M. Adamska, M. Supron: Blood DNA Analysis for Ehrlichia (Anaplasma) phagocytophila and Babesia spp. of Dogs from Northern Poland. Acta Vet. Brno 2004, 73:347-351. The most common pathogen transmitted by the tick Ixodes ricinus is the spirochete Borrelia burgdorferi, rarely Ehrlichia bacteria and protozoans from the Babesia genus. The aim of this paper was determine if infested to ticks dogs are a reservoir for E. phagocytophila and Babesia spp. and examine the possibility of coinfection. Canine blood was sampled, part of the material originated from dogs exhibiting symptoms of borreliosis. In an earlier study, the samples were screened for DNA from B. burgdorferi sensu lato. In order to screen for E. phagocytophila and Babesia spp. DNA, a PCR-based method was used with the following primers: EHR521/EHR747 for Ehrlichia and FOR1/REV1 for Babesia. In 192 samples only two contained E. phagocytophila DNA. One of these samples originated from a healthy canine, the other from an individual with symptoms of borreliosis. The examined samples were not positive for Babesia spp DNA. Coinfection was not discovered. The low level of E. phagocytophila infection may indicate that the domestic dog is not a reservoir for Ehrlichia and Babesia in Szczecin and northwestern Poland. Moreover, this area does not have populations of the brown dog tick (Rhipicephalus sanguineus) or Dermacentor reticulatus -both of which are vectors of E. canis and B. canis and commonly induce ehrlichiosis and babesiosis in canines. Ehrlichia (Anaplasma) phagocytophila, Babesia spp. dogs, PCR
Acta Veterinaria Brno 01/2004; 73(3):347. · 0.43 Impact Factor
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Pneumonologia i alergologia polska: organ Polskiego Towarzystwa Ftyzjopneumonologicznego, Polskiego Towarzystwa Alergologicznego, i Instytutu Gruzlicy i Chorob Pluc 02/1992; 60 Suppl 2:96-100.
