EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES

Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

A comparison of four DNA extraction protocols for the analysis of urine from patients with visceral leishmaniasis

Introduction Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplification. Methods A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplification, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. Results With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. Conclusion Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most efficient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost.

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

Extraction of Trypanosoma cruzi DNA from food: a contribution to the elucidation of acute Chagas disease outbreaks

Abstract: INTRODUCTION: Before 2004, the occurrence of acute Chagas disease (ACD) by oral transmission associated with food was scarcely known or investigated. Originally sporadic and circumstantial, ACD occurrences have now become frequent in the Amazon region, with recently related outbreaks spreading to several Brazilian states. These cases are associated with the consumption of açai juice by waste reservoir animals or insect vectors infected with Trypanosoma cruzi in endemic areas. Although guidelines for processing the fruit to minimize contamination through microorganisms and parasites exist, açai-based products must be assessed for quality, for which the demand for appropriate methodologies must be met. METHODS: Dilutions ranging from 5 to 1,000 T. cruzi CL Brener cells were mixed with 2mL of acai juice. Four Extraction of T. cruzi DNA methods were used on the fruit, and the cetyltrimethyl ammonium bromide (CTAB) method was selected according to JRC, 2005. RESULTS: DNA extraction by the CTAB method yielded satisfactory results with regard to purity and concentration for use in PCR. Overall, the methods employed proved that not only extraction efficiency but also high sensitivity in amplification was important. CONCLUSIONS: The method for T. cruzi detection in food is a powerful tool in the epidemiological investigation of outbreaks as it turns epidemiological evidence into supporting data that serve to confirm T. cruzi infection in the foods. It also facilitates food quality control and assessment of good manufacturing practices involving acai-based products.

Chemical and physical attributes and calcium, magnesium, potassium, and phosphorus in the soil of murundu fields in Brazil

Fields of murundus (FM) are wetlands that provide numerous ecosystem services. The objectives of this study were to evaluate the chemical [organic carbon (OC), P, K+, Ca2+, Mg2+, Al3+ and H+Al] and physical [texture and bulk density (Bd)] soil attributes and calculate the organic matter (OM) and nutrient stock (P, Ca, Mg, and K) in soils of FM located in the Guapore River basin in Mato Grosso. Thirty-six sampling points were selected, and soil samples were collected from two environments: the murundu and plain area surrounding (PAS). At each sampling point, mini trenches of 0.5 × 0.5 × 0.4 m were opened and disturbed and undisturbed soil samples were collected at depths of 0-0.1, 0.1-0.2, and 0.2-0.4 m. In the Principal Component Analysis the variables H+Al (49%) and OM (4%) were associated with the F1 component and sand content (47%) with the F2 component. The FM had lower pH values and higher concentrations of K+, P, and H+Al than PAS at all depths (p < 0.05). Additionally, FM stocked up to 433, 360, 205, and 11 kg ha-1 of Ca, Mg, K, and P, respectively, for up to a depth of 0.2 m. The murundu stored two times more K and three times more P than that in the PAS. Our results show that the FM has high sand content and Bd greater than 1.5 Mg m-3, high acidity, low OC content, and low nutrient concentrations. Thus, special care must be taken to preserve FM such that human intervention does not trigger environmental imbalances.

Laser assisted extraction of pacemaker and implantable defibrillator leads

OBJECTIVE: To analyze the results of laser-assisted extraction of permanent pacemaker and defibrillator leads. METHODS: We operated upon 36 patients, whose mean age was 54.2 years, and extracted 56 leads. The reasons for extracting the leads were as follows: infection in 19 patients, elective replacement in 13, and other causes in 4 patients. The mean time of catheter placement was 7.5±5.5 years. Forty-seven leads were from pacemakers, and 9 were from defibrillators. Thirty-eight leads were in use, 14 had been abandoned in the pacemaker pocket, and 4 had been abandoned inside the venous system. RESULTS: We successfully extracted 54 catheters, obtaining a 96.4% rate of success and an 82.1% rate for complete extraction. The 2 unsuccessful cases were due to the presence of calcium in the trajectory of the lead. The mean duration of laser light application was 123.0±104.5 s, using 5,215.2±4,924.0 pulses, in a total of 24.4±24.2 cycles of application. Thirty-four leads were extracted from the myocardium with countertraction after complete progression of the laser sheath, 12 leads came loose during the progression of the laser sheath, and the remaining 10 were extracted with other maneuvers. One patient experienced cardiac tamponade after extraction of the defibrillator lead, requiring open emergency surgery. CONCLUSION: The use of the excimer laser allowed extraction of the leads with a 96% rate of success; it was not effective in 2 patients who had calcification on the lead. One patient (2.8%) had a complication that required cardiac surgery on an emergency basis.

Sulfur fractions and carbon-nitrogen-phosphorus-sulfur relationships in some Brazilian and Iowa soils

O atual conhecimento relativo à distribuição em percentagem das várias frações de enxofre nos solos provem principalmente dos estudos dos solos de regiões temperadas. Em vista disso, este estudo foi conduzido para determinar as frações do S e as relações C-N-P-S em alguns solos da região subtropical dos Estados de São Paulo e do Paraná, Brasil, e comparar estes valores nestes solos com aqueles nos solos do Estado de Iowa, dos Estados Unidos da América do Norte. As análises das frações de enxofre nos solos dos dois países, indicaram que os solos do Brasil contem sulfato inorgânico adsorvido. Expressos como percentagem do S total, os solos do Brasil acusaram de 5 a 23% (média 11%) de S-sulfato inorgânico, de 20 a 65% (média 40%) de S-ester sulfato, de 5 a 12% (média 7%) de S-ligado ao Carbono e de 24 a 59% (média 42%) de S orgânico não identificado. As percentagens correspondentes nos solos de Iowa foram de 2 a 8% (média 5%) de S-sulfato inorgânico, de 43 a 60% (média 50%) de S-ester sufato, de 7 a 18% (média 11%) de enxofre ligado ao carbono e de 30 a 39% (média 34%) de S orgânico, não identificado. Outrossim, não foi encontrado o enxofre inorgânico não-sulfato em nenhum dos solos analisados. Houve grandes variações nas relações C, N, Ρ e S entre solos brasileiros quando comparados com aqueles do Iowa.

Studies on the mineral nutrition and fertilization of the passion fruit plant (Passiflora edulis f. flavicarpa): II - placement of fertilizer phosphorus

A localização do superfosfato (marcado com P32) no maracujá em produção foi estudada em condições de plantação comercial. Verificou-se que as aplicações em sulcos circulares ou faixas superficiais ao redor da planta tem eficiência equivalente sendo esses métodos três vezes superiores à localização do adubo em furos no solo. A pulverização foliar, por sua vez, mostrou-se 20 vezes mais eficiente que a aplicação no solo de acordo com os dois primeiros métodos.

Studies on the mineral nutrition and fertilization of the passion fruit plant (Passiflora edulis F. flavicarpa): II. placement of fertilizer phosphorus

1. Tagged superphosphate was applied to 2.5 year old passion fruit plants from a commercial plantation established in a sandy loam. 2. 100 grams of the fertilizer were distributed in the following ways: in a circular furrow 20 cm around the plant 40 cm from the stems; in a circular strip 10 cm wide, 40 cm from the stems; in six holes around the plants, 40 cm from the stems 20 cm deep, 2.5 cm in diameter. 3. 10 grams of the fertilizer in 11 of water were sprayed to the leaves. 4. Three weeks after the treatments were made, leaf samples were taken for analysis. 5. Determinations of specific activities both in the leaves and in the fertilizer used have shown that R in the plant was derived from the superphosphate in the following relative proportions (by making the first treatment equal to 100): circular furrow = 100; circular strip = 120; holes = 30; foliar spray = 230.

Increasing the efficiency of utilization of soil and fertilizer phosphorus in the subtropical and tropical agricultural systems C

As a rule, soils of the subtropical and tropical regions, in which rainfall is not limiting, are acidic, and low in phosphorus, and, to a less extent, in other macro and micronutrients as well, such a sulfur, boron and zinc. The establishment of a permanent agricultural prac. tice therefore, demands relatively high usage of liming and phosphatic fertilization, to begin with. Several approaches, not mutually exclusive, could be used in order to increase the efficiency of utilization of soil and fertilizer phosphorus so that, goal of diminishing costs of production is reached. The use of liming materials bringing up pH to 6.0-6.5 causes the conversion of iron and aluminum phosphates to more available calcium phosphates; on the other hand, by raising calcium saturation in the exchange complex, it improves the development and operation if the root system which allows c or a higher utilization of all soil nutrients, including phosphorus, and helps of stand water deficits which may occur. The role of mycorrhizal fungi should be considered as a way of increasing soil and fertilizer P utilization, as well as the limitations thereof. Screening of and breeding for varieties with higher efficiency of uptake and utilization of soil and fertilizer phosphorus leads to a reduction in cost of inputs and to higher benefit/cost ratios. Corrective fertilization using ground rock phosphate helps to saturate the fixation power of the soil thereby reducing, as a consequence, the need for phosphorus in the maintenance fertilization. Maintenance fertilization, in which soluble phos-phatic sources are used, could be improved by several means whose performance has been proved: limimg, granula tion, placement, use of magnesium salts. Last, cost of phosphate fertilization could be further reduced, without impairing yields, through impairing yields, through changes in technology designed to obtain products better adapted to local conditions and to the availability or raw materials and energy sources.

Schistosoma mansoni antigenic extracts obtained by different extraction procedures

Solubilization of Schistosoma mansoni antigens was obtained by agitation of adult worms in a 3M KCl solution. The protein contents of the KCl extrats varied from 0.35 to 0.96 mg/ml. Sera from 97 patients with hepatointestinal shistosomiasis and viable eggs in stools from a Brazilian endemic area were studied by immunoelectroomophoresis and Ouchterlony immunodiffusion methods with the KCl extract and with another antigen, obtained by homogenization of adult schistosomes in saline. The rate of positiveness of immunoprecipitation deterctions by immunoelectroomophoresis with the KCl extract was 53.5%. A correlation was verified between methods of detection and extration procedures, resulting in a better association of the extract obtained by agitation in 3M KCl and immunoelectroomophoresis.

Effect of magnesium chloride and guanidinum chloride on the extraction of components of extracellular matrix from chicken cartilage

In order to evaluate the effect of chaotropic agents on proteoglycan and non-collagenous proteins, chicken xiphoid cartilage was treated with guanidine-HCI and MgCl2 in different concentrations (1M to 5M), and different periods of time (12, 24, 48 and 72hr). The maximum yield of uronic acid was obtained with 3M MgCl2 (73.3 per cent). Concentrations of 4M and 5M of MgCl2 showed that much less uronic acid was removed, 55.3 per cent and 38.1 respectively. Extraction with 3M MgCl2 and 3M guanidine-HCl resulted better efficiency when performed for 48 hr. Analysis by SDS-PAGE of the extracts obtained with guanidine-HCl and MgCl, in different concentrations pointed out that most components are equally removed with the two solvents, showing that the extraction with MgCl2 is an alternative assay to remove non-collagenous proteins from extracellular matrix.

Cheap, rapid and efficient DNA extraction method to perform multilocus microsatellite genotyping on all Schistosoma mansoni stages

Schistosomes are endoparasites causing a serious human disease called schistosomiasis. The quantification of parasite genetic diversity is an essential component to understand the schistosomiasis epidemiology and disease transmission patterns. In this paper, we propose a novel assay for a rapid, low costly and efficient DNA extraction method of egg, larval and adult stages of Schistosoma mansoni. One euro makes possible to perform 60,000 DNA extraction reactions at top speed (only 15 min of incubation and 5 handling steps).