Protocol for DNA extraction of Cryptosporidium spp. oocysts in fecal samples

Molecular characterization of Cryptosporidium spp.oocysts in clinical samples is useful for public health since it allows the study of sources of contamination as well as the transmission in different geographical regions. Although widely used in developed countries, in Brazil it is restricted to academic studies, mostly using commercial kits for the extraction of genomic DNA, or in collaboration with external reference centers, rendering the method expensive and limited. The study proposes the application of the modifications recently introduced in the method improving feasibility with lower cost. This method was efficient for clinical samples preserved at -20 °C for up to six years and the low number of oocysts may be overcomed by repetitions of extraction.

Evaluation of three different DNA extraction methods from blood samples collected in dried filter paper in Plasmodium subpatent infections from the Amazon region in Brazil

Asymptomatic Plasmodium infection is a new challenge for public health in the American region. The polymerase chain reaction (PCR) is the best method for diagnosing subpatent parasitemias. In endemic areas, blood collection is hampered by geographical distances and deficient transport and storage conditions of the samples. Because DNA extraction from blood collected on filter paper is an efficient method for molecular studies in high parasitemic individuals, we investigated whether the technique could be an alternative for Plasmodium diagnosis among asymptomatic and pauciparasitemic subjects. In this report we compared three different methods (Chelex®-saponin, methanol and TRIS-EDTA) of DNA extraction from blood collected on filter paper from asymptomatic Plasmodium-infected individuals. Polymerase chain reaction assays for detection of Plasmodium species showed the best results when the Chelex®-saponin method was used. Even though the sensitivity of detection was approximately 66% and 31% for P. falciparum and P. vivax, respectively, this method did not show the effectiveness in DNA extraction required for molecular diagnosis of Plasmodium. The development of better methods for extracting DNA from blood collected on filter paper is important for the diagnosis of subpatent malarial infections in remote areas and would contribute to establishing the epidemiology of this form of infection.

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.

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).

Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction

We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 µg = 6 units) afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25oC) for 35 days, the enzyme lost 10% of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K.

DNA extraction and quantification from touch and scrape preparations obtained from autopsy liver cells

The objective of the present study was to develop a simplified low cost method for the collection and fixation of pediatric autopsy cells and to determine the quantitative and qualitative adequacy of extracted DNA. Touch and scrape preparations of pediatric liver cells were obtained from 15 cadavers at autopsy and fixed in 95% ethanol or 3:1 methanol:acetic acid. Material prepared by each fixation procedure was submitted to DNA extraction with the Wizard® genomic DNA purification kit for DNA quantification and five of the preparations were amplified by multiplex PCR (azoospermia factor genes). The amount of DNA extracted varied from 20 to 8,640 µg, with significant differences between fixation methods. Scrape preparation fixed in 95% ethanol provided larger amount of extracted DNA. However, the mean for all groups was higher than the quantity needed for PCR (50 ng) or Southern blot (500 ng). There were no qualitative differences among the different material and fixatives. The same results were also obtained for glass slides stored at room temperature for 6, 12, 18 and 24 months. We conclude that touch and scrape preparations fixed in 95% ethanol are a good source of DNA and present fewer limitations than cell culture, tissue paraffin embedding or freezing that require sterile material, culture medium, laboratory equipment and trained technicians. In addition, they are more practical and less labor intensive and can be obtained and stored for a long time at low cost.

Use of real-time PCR to evaluate two DNA extraction methods from food

The DNA extraction is a critical step in Genetically Modified Organisms analysis based on real-time PCR. In this study, the CTAB and DNeasy methods provided good quality and quantity of DNA from the texturized soy protein, infant formula, and soy milk samples. Concerning the Certified Reference Material consisting of 5% Roundup Ready® soybean, neither method yielded DNA of good quality. However, the dilution test applied in the CTAB extracts showed no interference of inhibitory substances. The PCR efficiencies of lectin target amplification were not statistically different, and the coefficients of correlation (R²) demonstrated high degree of correlation between the copy numbers and the threshold cycle (Ct) values. ANOVA showed suitable adjustment of the regression and absence of significant linear deviations. The efficiencies of the p35S amplification were not statistically different, and all R² values using DNeasy extracts were above 0.98 with no significant linear deviations. Two out of three R² values using CTAB extracts were lower than 0.98, corresponding to lower degree of correlation, and the lack-of-fit test showed significant linear deviation in one run. The comparative analysis of the Ct values for the p35S and lectin targets demonstrated no statistical significant differences between the analytical curves of each target.

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.

Extraction of genomic DNA from Melipona quadrifasciata (Hymenoptera: Apidae, Meliponinae)

The objective of the present study was to test three different procedures for DNA extraction of Melipona quadrifasciata based on existing methods for DNA extraction of Apis, plants and fungi. These methods differ in the concentrations of specific substances in the extraction buffer. The results demonstrate that the method used for Apis is not adequate for DNA extraction from M. quadrifasciata. On the other hand, with minor modifications this method and the methods for plants and fungi were adequate for DNA extraction of this stingless bee, both for adults and larvae

Extraction of DNA from honey and its amplification by PCR for botanical identification

The physiochemical and biological properties of honey are directly associated to its floral origin. Some current commonly used methods for identification of botanical origin of honey involve palynological analysis, chromatographic methods, or direct observation of the bee behavior. However, these methods can be less sensitive and time consuming. DNA-based methods have become popular due to their simplicity, quickness, and reliability. The main objective of this research is to introduce a protocol for the extraction of DNA from honey and demonstrate that the molecular analysis of the extracted DNA can be used for its botanical identification. The original CTAB-based protocol for the extraction of DNA from plants was modified and used in the DNA extraction from honey. DNA extraction was carried out from different honey samples with similar results in each replication. The extracted DNA was amplified by PCR using plant specific primers, confirming that the DNA extracted using the modified protocol is of plant origin and has good quality for analysis of PCR products and that it can be used for botanical identification of honey.

A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis

Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100°C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.

Inhibition of the polymerase chain reaction by sputum samples from tuberculosis patients after processing using a silica-guanidiniumthiocyanate DNA isolation procedure

With the objective to evaluate PCR-mediated detection of Mycobacterium tuberculosis DNA as a diagnostic procedure for diagnosis of tuberculosis in individuals attending ambulatory services in Primary Health Units of the City Tuberculosis Program in Rio de Janeiro, Brazil, their sputum samples were collected and treated with a DNA extraction procedure using silica-guanidiniumthiocyanate. This procedure has been described to be highly efficient for extraction of different kind of nucleic acids from bacteria and clinical samples. Upon comparing PCR results with the number of acid-fast bacilli, no direct relation was observed between the number of bacilli present in the sample and PCR positivity. Part of the processed samples was therefore spiked with pure DNA of M. tuberculosis and inhibition of the PCR reaction was verified in 22 out of 36 (61%) of the samples, demonstrating that the extraction procedure as originally described should not be used for PCR analysis of sputum samples.