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PubMedDate not supplied

The detection of Plasmodium falciparum and P. vivax in DNA-extracted blood samples using polymerase chain reaction.

Seventeen pairs of published primer sets were compared for their relative sensitivity to detect malaria DNA extracted from blood samples, which were obtained from Pakistani patients suffering from malaria. The primer sets investigated consisted of: (i) 9 pairs of direct primers and 3 sets of nested primers for detecting Plasmodium falciparum, (ii) 2 pairs of direct primers and 2 sets of nested primers for detecting P. vivax, and (iii) 1 set of multiplex primers for detecting both P. falciparum and P. vivax, simultaneously. After a miniscreen of 9 DNA-extracted blood samples using the 17 primer sets stated above, 5 primer sets were short-listed (based on their superior sensitivity) and used for a maxi-screen of DNA extracted from 126 microscopy-positive blood samples from Pakistan, with the following results. (i) For the detection of P. falciparum, the direct primer pair 'PF1 + PF2' gave a sensitivity of 95% and the nested primer set 'RIT405 + RIT406/RIT371 + RIT372' gave a sensitivity of 97%. (ii) For the detection of P. vivax, the direct primer pair 'Forward + Reverse' and the nested primer set 'PLF + UNR/PLF + VIR' both gave a sensitivity of 94%. (iii) The nested multiplex primer set 'rPLU5 + rPLU6/rFAL1 + rFAL2 + rVIV1 + rVIV2' gave a sensitivity of 97% and 96% for P. falciparum and P. vivax, respectively. It was concluded that the nested multiplex primer set was the most optimal primer set to use for the detection of malaria DNA extracted from blood samples. Furthermore, the nested multiplex primer set has the advantage of simultaneously detecting and differentiating between P. vivax and P. falciparum.

Animals↗
PubMed2003

Comparison of methods for DNA extraction from paraffin-embedded tissues and buccal cells.

Both paraffin-embedded tissue specimens and buccal cells are excellent resources for large-scale molecular epidemiological studies. In order to identify the optimal method for DNA extraction, we compared three methods: (1) modified phenol-chloroform protocol; (2) simple boiling method; and (3) DNA Extraction Mini Kit. For paraffin-embedded tissue specimens, amplification of the beta-globin gene sequence was successful in 30 of 34 (88.2%) by the simple boiling method, 29 of 34 (85.3%) samples using DNA extracted by the phenol-chloroform method, and 18 of 34 (52.9%) by the DNA Mini Kit. For buccal cells, amplification of the beta-globin gene sequence was successful in 16 of 17 (94.1%) DNA samples extracted by the phenol-chloroform method, 2 of 16 (12.5%) by the simple boiling method, and 12 of 16 (75%) by the DNA Mini Kit. Both the simple boiling method and the phenol-chloroform method are better methods for DNA isolation from paraffin-embedded tissue specimens, and the phenol-chloroform method is the best method for DNA extraction from buccal cells.

Cheek↗
PubMedDate not supplied

[DNA extraction from hard dental tissues].

Two different standard ways of DNA extraction (salting out and phenol-chloroform methods) were assayed in order to recovery nucleic acids from dental tissues. The DNA extracted was tested for purity by means of transverse alternating field electrophoresis (TAFE) using Saccharomyces cerevisiae chromosomes as markers. Both extraction methods give similar qualitative and quantitative results being a DNA yield from hard dental tissues approximately 30% of those extracted from the whole tooth. Our results indicate salting out as a preferable method due to its rapidity and usefulness.

DNA↗
PubMed2002

Qualitative detection of Legionella species in bronchoalveolar lavages and induced sputa by automated DNA extraction and real-time polymerase chain reaction.

Molecular assays for qualitative detection of Legionella spp. in clinical specimens were evaluated. DNA extraction was done either with a fully automated DNA extraction protocol on the MagNA Pure LC System or with manual DNA extraction. Amplification and detection were done by real-time polymerase chain reaction (PCR) on the LightCycler (LC) instrument. Oligonucleotides were derived from the 16S rRNA gene of Legionella spp. The assays included a specially designed DNA fragment as Legionella-specific internal control. For both molecular assays, the detection limit was determined to be 5 CFU per LC PCR run. Sixty-one clinical specimens were tested with the molecular assays. Results were compared to culture. Five samples were found to be positive with the molecular assays. Three of them were positive in culture. No inhibition was found throughout the whole study. In conclusion, the molecular assays described may lead to safe and early diagnosis of Legionnaires' disease. They proved to be suitable for the routine molecular diagnostics laboratory.

Adult↗
PubMed1994

[Method of DNA extraction and mecA, femA and PBP2' in multi-drug resistant bacterial strains].

Recently, it has become more important to establish rapid and reliable methods for identifying bacterial species and their drug resistances because of the high incidence of nosocomial infection caused by multi-drug resistant bacteria. At first, several enzymes were tested to evaluate the efficacy of DNA extraction. N-acetylmuramidase, lysozyme and achromopeptidase were most effective to extract DNA from E. faecalis, E. faecium and S. aureus, respectively. After achromopeptidase extraction, the mecA gene was amplified by polymerase chain reaction (PCR) in clinically isolated MRSA (95 strains), MSSA (66), MRSE (methicillin-resistant S. epidermidis, 37), MSSE (methicillin-sensitive S. epidermidis, 1), S haemolyticus(5), S.hominis(1) and others (Enterococcus, Pseudomonas, and Streptococcus, total 45). PCR products were analyzed by agarose gel electrophoresis. The positive rates were 95% (MRSA), 1.5% (MSSA), and 97% (MRSE). The mecA gene was also positive in 2/2 of methicillin-resistant S.haemolyticus and 2/3 of methicillin-sensitive S.haemolyticus and 1/1 of methicillin-resistant S.hominis. The mecA gene was not detected in 45 non-Staphylococcal strains. MecA and femA gene by PCR and PBP 2' by IRMA were further detected in newly isolated MRSA (20 strains), MSSA(20), MRSE(14), MSSE(1) and S.simulans(2). Complete correlation between MPIPC susceptibility and mecA were found. The femA gene was positive in 39/40 of S. aureus, and 0/14 of S.epidermidis, 2/3 of S.simulans. PBP2' were positive in 20/20 of MRSA, 0/20 of MSSA, 14/14 of MRSE, 0/1 of MSSE, 1/1 of methicillin-resistant S.simulans, and 0/2 of methicillin-sensitive S.simulans. In conclusion, diagnoses of MRSA by simultaneous detection of mecA and femA gene as well as PBP2' are similarly useful because of their specificity and rapidity.

Bacterial Proteins↗
PubMed2006

Simple salting-out method for DNA extraction from formalin-fixed, paraffin-embedded tissues.

The aim of this study was to standardize a method of DNA extraction from formalin-fixed and paraffin-embedded tissues (PETs) using a salt solution to precipitate protein and isopropanol to precipitate DNA. The samples were submitted to a DNA extraction method in which two different concentrations of ammonium acetate (2 and 4M) were compared with a phenol-chloroform extraction method and with a commercial DNA isolation kit. DNA was qualified and quantified by spectrophotometer analysis, electrophoresis, and amplification by PCR. The 167 and 268bp fragments of APC and beta-globin genes, respectively, were amplified equally from DNA extracted by all tested methods and in all cases. However, the 536bp fragment of beta-globin gene was not amplified in all cases. According to our results, the extraction method using ammonium acetate proved to be simple and suitable for obtaining DNA of good quality, which can be easily amplified by PCR.

Acetates↗
PubMed1992

DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities.

A protocol was devised which permitted the extraction of DNA from deep marine sediments up to 503 m below the sea floor. These sediments have been laid down over the last 3 million years. 16S rRNA gene sequences were amplified from the DNA by the polymerase chain reaction. The details of the successful extraction and polymerase chain reaction methodology varied between samples from different depths. This emphasizes the attention to detail required to allow the diversity of bacteria in these deep sediments to be studied.

Bacteria↗
PubMed2002

Cellophane based mini-prep method for DNA extraction from the filamentous fungus Trichoderma reesei.

BACKGROUND: Methods for the extraction of DNA from filamentous fungi are frequently laborious and time consuming because most of the available protocols include maceration in liquid nitrogen after the mycelium has been grown in a liquid culture. This paper describes a new method to replace those steps, which involves the growth of the mycelium on cellophane disks overlaid on solid medium and the use of glass beads for cell wall disruption. RESULTS: Extractions carried out by this method provided approximately 2 microg of total DNA per cellophane disk for the filamentous fungus Trichoderma reesei. To assess the DNA's quality, we made a PCR (Polymerase Chain Reaction) amplification of a gene introduced by a transformation in this fungus's genome (hph gene), with successful results. We also confirmed the quality of the DNA by the use of Southern blotting to analyze the presence of the same gene, which was easily detected, resulting in a sharply defined and strong band. CONCLUSIONS: The use of this method enabled us to obtain pure DNA from Trichoderma reesei, dispensing with the laborious and time-consuming steps involved in most protocols. The DNA obtained was found to be suitable for PCR and Southern blot analyses. Another advantage of this method is the fact that several samples can be processed simultaneously, growing the fungus on multiple well cell culture plates. In addition, the absence of maceration also reduces sample handling, minimizing the risks of contamination, a particularly important factor in work involving PCR.

Cell Wall↗
PubMed1997

Comparison of three DNA extraction methods on bone and blood stains up to 43 years old and amplification of three different gene sequences.

Extraction of amplifiable DNA-from degraded human material in the forensic context remains a problem, and maximization of yield and elimination of inhibitors of the Polymerase Chain Reaction (PCR) are important issues which rarely feature in comparative studies. The present work used PCR amplification of three DNA sequences (HLA DPB1, amelogenin and mitochondrial) to assess the efficiency of three methods for extracting DNA (sodium acetate, magnetic beads and glass-milk) from 32 skeletal samples and 25 blood stains up to 43 years old. The results, analyzed using multivariate statistics, confirmed that the extraction method was crucial to the subsequent detection of amplification products; the glass-milk protocol performed better than sodium acetate, which was better than magnetic beads. Successful amplification also depended on gene sequence, multiple copy mitochondrial sequences performing best; however, with the singly copy sequences, the longer HLA DPB1 (327 bp) being detected just as often as the shorter amelogenin (106/112 bp). Amplification products were obtained more frequently from blood stains than bone, perhaps reflecting differences inherent in the material, and from younger compared with older specimens, though plateauing seemed to occur after 10 years. PCR inhibitors were more frequent in sodium acetate extracts.

Amelogenin↗
PubMed2004

Palmitoylputrescine, an antibiotic isolated from the heterologous expression of DNA extracted from bromeliad tank water.

Heterologous expression of large fragments of microbial DNA extracted directly from environmental samples (environmental DNA, or eDNA) in easily cultured hosts should provide access to some of the natural products produced by previously uncultured bacteria. The natural product antibiotic palmitoylputrescine (1) was isolated from Escherichia coli transformed with a cosmid (pCSLF16) containing DNA extracted directly from Costa Rican bromeliad tank water. In this report we describe the characterization of this antibiotic and its biosynthetic gene.

Anti-Bacterial Agents↗
PubMed1994

Cloning and sequencing of genomic DNA extracted from archival human temporal bone sections.

Cloning techniques allow the engineering and production of highly purified DNA. Further advances in molecular biology have provided the means to identify DNA sequences in a rapid fashion. Sequencing methods can identify mutations, deletions, polymorphisms, or confirm a known genetic sequence. The use of these techniques in clinical medicine has made it possible to accurately diagnose infectious diseases and determine the molecular etiology of many genetic disorders and malignancies. In this study, DNA extracted from archival, celloidin-embedded temporal bone sections has been cloned and sequenced using these techniques. We amplified, cloned, and sequenced varicella-zoster viral DNA extracted from archival temporal bone sections from patients who had herpes zoster oticus. The application of cloning and sequencing techniques to DNA extracted from archival temporal bones provides the methodology to study temporal bone pathology at the molecular level.

Base Sequence↗
PubMed1996

Increasing DNA extraction yield from saliva stains with a modified Chelex method.

Recovery, preservation and analysis of body fluid stains is an important aspect of forensic science. PCR-based typing of DNA extracted from recovered stains is often a crucial method to identify a perpetrator or exclude an innocent suspect. This paper reports an improved method of extracting genomic DNA from saliva stains deposited on human skin in simulated bite mark situations. Results of organic (phenol-chloroform) extraction and Chelex extraction were compared to a modified Chelex method developed by the authors. Modifications include pre-extraction preparation with proteinase K and incubations at 56 degrees C and 100 degrees C plus microconcentration of the solution. Quantification results using the classical Chelex extraction method showed that 31.9 +/- 4.22% of the deposited DNA was recovered, but using the modified Chelex extraction method DNA recovery was increased to 47.7 +/- 6.90%. The quantity and quality of extracted DNA was shown to be adequate for PCR-based typing at two STR loci.

Bites and Stings↗
PubMedDate not supplied

Investigation of thermic transition and renaturation of high polymerized DNA extracted from thyroid nodule.

The study was carried out on 16 cases of mixed thyroid hypertrophy in which total or partial thyroidectomy was performed. Fragments collected intraoperatively from the thyroid nodule were used for extraction of high polymerized DNA. At the same time with quantitative evaluation of DNA, RNA and proteins were also assayed. The thermic transition mean temperature of the DNA extracted from the thyroid nodule is compared to other standards (DNA-HP-standard, calf thymus DNA, normal leukocytic DNA) and the thermic transition curves are presented. Hyperchromicity after thermic denaturation and renaturation is analysed and expressed in per cent values.

DNA↗
PubMedDate not supplied

Comparison of four rapid DNA extraction techniques for conventional polymerase chain reaction testing of three Mycobacterium spp. that affect birds.

Mycobacteriosis is an avian disease that is most commonly caused by Mycobacterium avium or Mycobacterium genavense. In order to optimize molecular laboratory tests for diagnosing mycobacteriosis in birds, we compared four methods of rapid DNA extraction with isolates of M. avium, M. genavense, and Mycobacterium fortuitum. DNA extraction methods included enzymatic lysis, boiling for 30 min followed by enzymatic lysis, four cycles of freezing and thawing followed by enzymatic lysis, and bead beating followed by enzymatic lysis. The DNA yield and purity for the four methods were evaluated by spectrophotometry and compared. The bead beating with enzymatic lysis technique yielded significantly purer and higher concentrations of extracted DNA compared with other DNA extraction methods. All four methods yielded extraction products for all three organisms that were successfully amplified by polymerase chain reaction (PCR) for a fragment in the 65-kD heat shock protein gene. Subjectively, the PCR amplification products were most abundant for samples extracted by bead beating with enzymatic lysis.

Animals↗
PubMed1999

Optimized DNA extraction to improve reproducibility of short tandem repeat genotyping with highly degraded DNA as target.

The reproducibility of short tandem repeat (STR) genotyping of highly degraded DNA is often reduced due to artifacts generated during polymerase chain reaction (PCR) amplification. The frequency and amount of these artifacts are related to the quality and quantity of the DNA amplified. Consequently, the aim of this investigation was the optimization of DNA extraction to increase the reproducibility of STR genotyping of samples containing highly degraded DNA. Starting from a standard extraction protocol, systematic variation of individual parameters resulted in optimized protocols for three categories of ancient human bone material (different degrees of DNA degradation) and a consensus protocol for the extraction of a broad range of ancient DNA preservation states.

DNA↗
PubMed1999

Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis.

A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient, yet with the advantage of being much less time- and labor-intensive. Five of the 10 tested RAPD primers produced reproducible polymorphisms among five human origin G. duodenalis strains, and grouping of these strains based on RAPD profiles was in agreement among these primers. The consistent classification of two standard laboratory reference strains, Portland-1 and WB, in the same group confirmed previous results using other fingerprinting methods, indicating that the reported simple DNA extraction methods and the selected primers are useful in RAPD for molecular characterization of G. duodenalis strains.

Animals↗
PubMed2003

The sensitivity of PCR detection of Cryptosporidium oocysts in fecal samples using two DNA extraction methods.

BACKGROUND: The implementation of cost-effective intervention strategies for zoonotic protozoa relies on the development of sensitive and accurate diagnostic methods. We carried out a study to evaluate the accuracy of a PCR method for the detection of Cryptosporidium spp. oocysts in fecal samples from cattle. METHODS: Fecal samples were spiked with different numbers of oocysts and the limit of detection of the method was determined. Two methods of DNA extraction were assessed: glass beads and freeze-thawing using liquid nitrogen. A nested PCR approach was developed targeting the Cryptosporidium SSU rRNA and TRAP-C2 genes. Agreement between the diagnosis of Cryptosporidium spp. at the SSU rRNA and TRAP-C2 loci was quantified using the kappa-coefficient. RESULTS: Compared with the freeze-thawing method, the glass beads method was found to be a better way of extracting DNA from Cryptosporidium oocysts (sensitivities were 83 and 100%, respectively). The limits of detection for glass beads and freeze-thaw were low, 1 and 10 oocyst/g fecal samples, respectively. Forty-six percent of the field samples previously classified as negative for Cryptosporidium parvum by the flotation-concentration and enzyme-linked immunosorbent assay methods showed DNA with the PCR protocol. CONCLUSION: Primers for SSU rRNA are more successful in producing an amplification than primers for the TRAP-C2 gene which makes the former PCR protocol the approach of choice for detecting Cryptosporidium parvum oocysts in field samples.

Animals↗
PubMed2006

Evaluation of soil bacterial biomass using environmental DNA extracted by slow-stirring method.

A simple and rapid method (slow-stirring method) for extracting environmental DNA (eDNA) from soils was constructed by physical mild stirring with chemical treatment. eDNA was extracted efficiently with minimal damage from various kinds of soil. The amount of eDNA and soil bacterial biomass showed a linear proportional relation [Y=(1.70x10(8))X, r2=0.96], indicating that bacterial biomass could be evaluated by quantifying levels of eDNA. Consequently, the average bacterial biomass in an agricultural field was calculated as 5.95x10(9) cells/g sample, approximately 10-100 times higher than that in non- and oil-polluted fields.

Bacteria↗