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PubMed2002

A comparison of five methods for extraction of bacterial DNA from human faecal samples.

The purity of DNA extracted from faecal samples is a key issue in the sensitivity and usefulness of biological analyses such as PCR for infectious pathogens and non-pathogens. We have compared the relative efficacy of extraction of bacterial DNA (both Gram negative and positive origin) from faeces using four commercial kits (FastDNA kit, Bio 101; Nucleospin C+T kit, Macherey-Nagal; Quantum Prep Aquapure Genomic DNA isolation kit, Bio-Rad; QIAamp DNA stool mini kit, Qiagen) and a non-commercial guanidium isothiocyanate/silica matrix method. Human faecal samples were spiked with additional known concentrations of Lactobacillus acidophilus or Bacteroides uniformis, the DNA was then extracted by each of the five methods, and tested in genus-specific PCRs. The Nucleospin method was the most sensitive procedure for the extraction of DNA from a pure bacterial culture of Gram-positive L. acidophilus (10(4) bacteria/PCR), and QIAamp and the guanidium method were most sensitive for cultures of Gram-negative B. uniformis (10(3) bacteria/PCR). However, for faecal samples, the QIAamp kit was the most effective extraction method and led to the detection of bacterial DNA over the greatest range of spike concentrations for both B. uniformis and L. acidophilus in primary PCR reactions. A difference in extraction efficacy was observed between faecal samples from different individuals. The use of appropriate DNA extraction kits or methods is critical for successful and valid PCR studies on clinical, experimental or environmental samples and we recommend that DNA extraction techniques are carefully selected with particular regard to the specimen type.

Bacteria↗
PubMed1996

Differentiation of Entamoeba histolytica and E. dispar DNA from cysts present in stool specimens by polymerase chain reaction: its field application in the Philippines.

It has been established that two distinct species exist within what was originally known as Entamoeba histolytica. These are E. dispar and E. histolytica, for the nonpathogenic and pathogenic forms, respectively. Differentiation of these two organisms is of great clinical importance since they are morphologically indistinguishable and both forms can infect the human intestinal cavity to different degrees. A simple and rapid DNA-extraction method that can be used directly on formalin-fixed stool specimens has been developed. The extracted DNA was used for the identification of the species existing in the stools by polymerase chain reaction (PCR). A total of 72 randomly collected stool samples from the Philippines were analyzed. In all, 19 samples reacted with E. dispar primers, resulting in the expected 101-bp PCR products; however, none reacted with E. histolytica primers. Furthermore, sensitivity assay suggests that genomic DNA from as few as five cysts can be used as a template for PCR. These observations imply that the use of genomic DNA directly extracted from formalin-fixed stool specimens for PCR amplification is a useful tool for obtaining a sensitive and accurate diagnosis that can be applied even in epidemiology studies.

Animals↗
PubMed2026

Selective Extraction of Genomic DNA From Animal Tissues Using a Hydrophobic Magnetic Ionic Liquid.

The development of green and efficient methods for genomic DNA extraction from animal tissues is crucial for molecular diagnostics, food traceability, and genetic research. Conventional methods often involve toxic reagents, multiple centrifugation steps, and are time-consuming. In this study, a hydrophobic magnetic ionic liquid (MIL), N-octyl-4-dimethylaminopyridinium hexafluorophosphate MIL ([C8DMAP][PF6]‑Ni MIL), was synthesized and applied for the selective extraction of genomic DNA from various animal tissues. The material exhibited strong paramagnetic behavior, high thermal stability, and excellent hydrophobicity, enabling rapid phase separation under an external magnetic field. A mechanical shaking-assisted extraction method was developed, and key parameters including temperature, time, shaking speed, and [C8DMAP][PF6]-Ni MIL dosage were systematically optimized. The method demonstrated high selectivity for DNA over proteins, RNA, and amino acids, with a maximum recovery rate of 78.06 ± 1.91%. Compared to a commercial DNA extraction kit, the [C8DMAP][PF6]-Ni MIL-based approach provided higher yields from several tissues, including mouse liver, brain, and rabbit lung. Furthermore, the [C8DMAP][PF6]-Ni MIL could be reused for at least six cycles while maintaining extraction efficiency. This work not only provides a high-performance material for DNA extraction, but also demonstrates a sustainable and easily retrievable liquid-phase separation strategy, offering a generalizable platform for complex sample pretreatment.

Animals↗
PubMed1997

Differential replication of a single N-2-acetylaminofluorene lesion in the leading or lagging strand DNA in a human cell extract.

DNA replication in eucaryotic cells is a complex process involving a variety of proteins that synthesize the leading and lagging strand in an asymmetric, coordinated manner. To investigate the effect of this asymmetry on the translesion synthesis of bulky lesions, we have constructed SV40 origin-containing plasmids with site-specific N-2-acetylaminofluorene adduct on either leading or lagging strand templates. These plasmids have been incubated with DNA replication-competent extracts made from human HeLa cells. Two-dimensional agarose gel electrophoresis analyses reveal a strong blockage of fork progression only when the N-2-acetylaminofluorene adduct is located on the leading strand template. Morever, the analysis revealed that replication with HeLa cell extracts of SV40 origin-dependent plasmids functions in both directions from the origin with equal efficiency but, probably due to an important asynchrony at the formation of the two forks, proceeds unidirectionally for a large number of individual molecules. The validity of the in vitro replication approach to study the fidelity of both leading- and lagging strand synthesis is discussed with regard to these new data.

2-Acetylaminofluorene↗
PubMed2006

Comparison of methods of extracting Salmonella enterica serovar Enteritidis DNA from environmental substrates and quantification of organisms by using a general internal procedural control.

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 x 10(3) to 1.8 x 10(3) CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.

Animals↗
PubMed1992

A simple method for the extraction of baculovirus DNA.

We have developed a method using lysozyme for DNA extraction from Baculoviruses using as a model Lymantria dispar nuclear polyhedrosis virus (LdNPV) obtained from infected larvae. This method proved to be quick, inexpensive and the extracted DNA was successfully used in molecular hybridization experiments.

Animals↗
PubMed1991

Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease.

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8-14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligonucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F508 deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F508 primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.

Adult↗
PubMed2002

DNA-based methods for the detection and the identification of phytoplasmas in insect vector extracts.

DNA extraction and storage methods have been evaluated with laboratory-reared leafhoppers and/or field-collected leafhoppers and psyllids. Detection of four different phytopathogenic phytoplasmas, belonging to three taxonomic groups, has been achieved by several direct or nested polymerase chain reaction (PCR) methods with such DNA extracts. Reactions differed in both the 16/23S ribosomal primer pairs used and the specific assay and cycling conditions. Merits and possible hindrances of the various primer pairs, in relation to insect DNA extracts, are discussed. However, identification of the phytoplasma(s) necessarily relied on comparison of the polymorphism in length of the amplified DNA fragments obtained by restriction with appropriate endonucleases. Endonuclease digestion is crucial for determining the identity (subgroup affiliation) of phytoplasmas of the same groups that can be carried by an individual vector.

Animals↗
PubMed2001

DNA polymerase epsilon is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts.

DNA polymerase epsilon (Pol epsilon) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases alpha and delta (Pol alpha and delta), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Pol epsilon in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Pol epsilon is indeed required for chromosomal DNA replication. In Pol epsilon-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Pol alpha are loaded onto the chromatin template in Pol epsilon-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Pol epsilon, along with Pol alpha and Pol delta, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.

Animals↗
PubMed2006

Rapid detection of the fluoroquinolone resistance-associated ParC mutation in Neisseria gonorrhoeae using TaqMan probes.

BACKGROUND: In fluoroquinolone-resistant Neisseria gonorrhoeae, the amino acid mutations in the fluoroquinolone-resistant determining region (QRDR) of the parC gene are an important factor. The aim of the present study was to develop a rapid detection method of a serine 88 to proline substitution in parC which we previously showed as having significantly higher fluoroquinolone minimal inhibitory concentrations (MIC) using the TaqMan discrimination system. METHODS: We extracted DNA from 90 urine or urethral swab samples obtained from male patients with urethritis caused by N. gonorrhoeae. After DNA extraction, they were subjected to real-time polymerase chain reaction (PCR) using a TaqMan discrimination system and compared with the results of conventional DNA sequencing. RESULTS: Of the 90 samples, the TaqMan technique result showed 13 samples that were classified as having a serine 88 to proline mutation in parC, and 77 samples that did not have a serine 88 to proline mutation in parC. The classifications of all samples completely corresponded to those determined by conventional DNA sequencing. We also found that N. gonorrhoeae with a serine 88 to proline mutation in parC have a significantly higher MIC to ciprofloxacin than that without a serine 88 to proline mutant in parC. CONCLUSIONS: The present genotyping method of real-time PCR using a TaqMan discrimination system could be applied to the rapid detection of a serine 88 to proline amino acid mutation in parC of N. gonorrhoeae. This point mutation is significant for the determination of fluoruquinolone resistance. This rapid detection system may lead to the prevention of use of noneffective antimicrobial agents and a decrease of resistant strains.

DNA Topoisomerase IV↗
PubMed2004

Detection of transgenes in soybean via a polymerase chain reaction and a simple bioluminometric assay based on a universal aequorin-labeled oligonucleotide probe.

The recombinant photoprotein aequorin was used as a reporter in highly sensitive and automatable hybridization assays for the analysis of transgenic sequences in genetically modified organisms (GMO). The terminator of the nopaline synthase gene (NOS) from Agrobacterium tumefaciens and the 35S promoter sequence were detected in genetically modified soybean. The endogenous, soybean-specific, lectin gene was also detected for confirmation of the integrity of extracted DNA. A universal detection reagent was produced through conjugation of aequorin to the oligonucleotide (dA)(30). Biotinylated (through PCR) products for the three target sequences were captured onto streptavidin-coated wells, and one strand was removed by NaOH treatment. The immobilized single-stranded DNAs were then hybridized with oligonucleotide probes consisting of a target-specific segment and a poly(dT) tail. This allowed the subsequent determination of all hybrids through the use of the (dA)(30)-aequorin conjugate as a universal reagent. The bound aequorin was measured by adding Ca(2+) and integrating the light emission for 3 s. As low as 2 pM (100 amol per well) of amplified DNA was detectable for all three targets, with a signal-to-background ratio of about 2. The analytical range extended up to 2000 pM. As low as 0.05% GMO content in soybean can be detected with a signal-to-background ratio of 8.2. The overall repeatability of the proposed assay, including DNA extraction, PCR, and hybridization assay, ranged from 7.5-19.8%. The use of a (dA)(30)-aequorin conjugate renders the assay configuration general for any target DNA, provided that the specific probe carries a poly(dT) tail.

Aequorin↗
PubMed2003

Molecular detection methods developed for a systemic rickettsia-like bacterium (RLB) in Penaeus monodon (Decapoda: Crustacea).

Molecular detection methods were developed to aid in the diagnosis of a rickettsia-like bacterium (RLB) which caused severe mortalities of farm-raised Penaeus monodon in Madagascar. Using primers derived from the 16S rRNA gene of bacteria, a PCR assay was optimized to amplify this region of the genome of the RLB, using extracted DNA from infected P. monodon tissue as the template. The resulting amplified PCR product was sequenced and 2 novel primers were selected from the variable region of the gene. These primers amplified a 532 bp fragment of DNA originating from the rickettsia-infected samples. The PCR assay was optimized and tested on DNA extracted from specific pathogen-free (SPF) P. vannamei tissue and several other strains of bacteria. The PCR assay with the rickettsia-specific primers was specific for this RLB and did not amplify the other DNA samples tested. The 532 bp PCR-amplified fragment was labeled with digoxigenin (DIG) for in situ hybridization assays. This probe was tested on SPF, RLB and bacteria-infected shrimp specimens preserved in Davidson's fixative. The probe was specific for both natural and experimental rickettsial infections. Hybridization with this probe required a stringent temperature of 65 degrees C, otherwise cross-reactivity was observed with other types of bacteria.

Animals↗
PubMed2000

Molecular beacon polymerase chain reaction detection of Escherichia coli O157:H7 in milk.

A fluorescently labeled oligonucleotide probe (molecular beacon) was applied to detect Escherichia coli O157:H7 in artificially contaminated skim milk during polymerase chain reaction (PCR) amplification of extracted DNA. The probe was designed to hybridize with a region of the slt-II gene coding for the A subunit and to fluoresce when the hairpin-stem conformation was linearized upon hybridization to the target sequence. The molecular beacon was incorporated into PCR reactions containing DNA extracted from artificially contaminated skim milk. The degree of fluorescence was monitored in PCR reactions containing 10(3), 10(5), and 10(7) CFU of E. coli O157:H7 per ml and was found to correlate with the amount of template in each reaction. Fluorescence significantly increased above background levels by cycle 8, 14, or 14 in reactions containing DNA from the 10(7)-, 10(5)-, or 10(3)-CFU/ml template, respectively (P < 0.05). Molecular beacon PCR demonstrated positive results more rapidly than traditional agarose gel electrophoresis analysis of PCR products. Use of molecular beacons allows real-time monitoring of PCR reactions, and the closed-tube format allows simultaneous detection and confirmation of target amplicons without the need for agarose gel electrophoresis and/or Southern blotting. This is the first report of a stem-and-loop molecular beacon being applied for direct detection of a pathogen in food.

Animals↗
PubMed1999

[Detecting microorganisms in soil by PCR and molecular hybridization].

PCR and molecular hybridization provide new methods to detect microorganisms in environment. A simple, sensitive protocol was developed to detect Bacillus thuringiensis HD-1 in soil by PCR and Southern Blotting in this study. Methods of extracting DNA from soil and purification were compared. By a modified total DNA extraction from soil and purification, followed by PCR and Southern Blotting, strain HD-1 released into soil can be detected with a sensitivity of 2.2 x 10(2) cells/g soil. By using other specific primers, this detection protocol can be widely used in the research of ecology and risk assessment of genetically engineered microorganisms released into environment.

Bacillus thuringiensis↗
PubMed2003

Analysis of human fecal material for autosomal and Y chromosome STRs.

Human stool samples from eight volunteers were stored under various conditions and extracted by three different procedures. Fecal material and tissue paper soiled with fecal material obtained from a crime scene were also extracted. Extracted DNA was amplified using the AmpFlSTR Profiler Plus, AmpFlSTR COfiler, and the AmpFlSTR Identifiler PCR amplification kits for the detection of the autosomal STR allelic patterns. DNA extracted from the male volunteers and from the soiled tissue paper evidence sample was also amplified using the Y-PLEX 6 and Y-PLEX 5 amplification kits. Analysis of the amplified products was carried out by capillary electrophoresis on the ABI PRISM 310 Genetic Analyzer. Autosomal and Y-STR profiles obtained from the fecal material were concordant with the results from the donors' buccal swabs.

Alleles↗
PubMed2000

[Studies on identification of circumsporozoite protein genotyping of Plasmodium vivax].

OBJECTIVE: To develop a new method of genotyping circumsporozoite protein (CSP) gene for identification of field isolates of Plasmodium vivax. METHODS: Improved Chelex-100 ion-exchange method was used to extract DNA from blood filter paper samples, nested PCR and allele-specific PCR techniques, agarose gel electrophoresis analysis and dot/southern blotting-probe hybridization were employed for amplification, resolution and identification of the diagnostic fragments. RESULTS: Using the nest-allele-specific PCR assay reported here, small amounts of DNA extracted from a piece of blood filter paper sample were amplified which produced three different size ranges of diagnostic bands: 650-770 bp PV-species-specific band, 170-230 bp diagnostic band for temperate zone family and 588 bp band for PV type-2. The sizes and patterns of the bands produced by the reference strains were consistent with those of designed target sequences. Of 59 examined isolates from 6 provinces of China, 42 temperate zone family strains, 15 tropical zone family strains and two PV type-2 strains were identified. CONCLUSION: 1, Three genotype strains of P. vivax mentioned above could be identified by this method with only two rounds of PCR and without probe hybridization. 2, The preliminary results showed that PV type-1 including temperate zone family strains and tropical zone family strains as well as PV type-2 strains are present in China. In addition, another CSP genotype with both sequence characteristics of temperate zone and tropical zone family might also be present in China.

Animals↗
PubMed2006

Accuracy of fetal gender determination in maternal plasma at 5 and 6 weeks of pregnancy.

OBJECTIVE: To assess the viability of the early diagnosis of fetal gender in maternal plasma before 7 weeks of pregnancy by real-time polymerase chain reaction (real-time PCR), starting at 5 weeks of pregnancy. METHOD: Peripheral blood was collected from pregnant women, starting at 5 weeks of gestation. After centrifugation, plasma was separated for fetal DNA extraction. DNA was analyzed by quantitative real-time PCR for two genomic regions, one on the Y chromosome (DYS-14) and the other shared by both sexes (ss-globin), by the TaqMan Minor Groove Binder (MGB) probe assay. The results of the examinations were compared to fetal gender determined after delivery. RESULTS: A total of 79 examinations of fetal DNA in maternal plasma were performed for 52 pregnant women. Accuracy according to gestational age was 92.6% (25 of 27 cases) at 5 weeks, and 95.6% (22 of 23 cases) at 6 weeks. These results also demonstrate that fetal DNA is present at low concentrations in maternal plasma at 5 weeks (8.5 genome equivalents (GE)/mL) and 6 weeks (34.1 GE/mL) of pregnancy. CONCLUSION: Quantitative real-time PCR and TaqMan MGB probes specific for the detection of fetal gender in maternal plasma starting at 5 weeks of gestation have good sensitivity and excellent specificity.

Chromosomes, Human, Y↗
PubMed1997

Transfection of the inner cell mass and lack of a unique DNA sequence affecting the uptake of exogenous DNA by sperm as shown by dideoxy sequencing analogues.

PURPOSE: The purpose of this study was to determine whether exogenous DNA internalized into blastocysts after transference from DNA-carrier sperm are localized at the inner cell mass or trophoblast cells and to identify differences in uptake of exogenous DNA fragments by sperm due to unique DNA sequences. METHODS: Mouse blastocysts at the hatching stage were exposed to migrating human sperm cells carrying exogenous DNA fragments synthesized from the E6-E7 conserved gene regions of human papillomavirus (HPV) types 16 and 18. After an interaction period of 2 hr, the transfected blastocysts were washed several times to remove extraneous sperm and the blastocysts were dissected into groups of cells derived from the inner cell mass and trophoblasts. The cells were analyzed by polymerase chain reaction (PCR) for the presence of HPV DNA fragments. In the second part of the experiment, thawed donor (N = 10) sperm cells were pooled, washed, and divided into two fractions. The first (control) fraction was added with formalin and further divided and added with a 35S-radiolabeled G, A, T, or C sequencing mixture. The second fraction was similarly treated but the formalin step was omitted from the treatment. After an hour of incubation at 37 degrees C, the sperm specimens were washed several times by centrifugation and DNA extracted by the GeneReleaser method. The extracted DNA were processed on sequence gels, and the autoradiographs analyzed. RESULTS: Mouse blastocysts transfected by carrier sperm with DNA from HPV types 16 and 18 showed localization of the HPV DNA to both the inner cell mass and trophoblast cells. Negative controls consisting of untreated human sperm and untreated mouse blastocysts did not reveal any evidence of HPV DNA. The positive sperm control generated expected DNA fragments from HPV types 16 and 18. In the second experiment, the intensities of the DNA fragments in the G, A, T, and C columns from low to high molecular weights were not different from the positive control bands. Band intensities of the four sequencing columns were similar. Formalin pretreatment of the sperm inhibited uptake of the DNA fragments from the smallest to the largest DNA molecules. CONCLUSIONS: Exogenous DNA taken into blastocysts are localized to both the inner cell mass and trophoblast cells. Only live sperm exhibited the capacity to carry various sizes of exogenous DNA, suggesting the involvement of active cell membrane mechanism in the transference process. The results showed that DNA fragments terminating in any of the four nucleotides were equally taken up by the sperm cell. Fragments of DNA produced by the sequencing reaction failed to identify a unique DNA sequence that would facilitate or inhibit the sperm from taking up exogenous DNA.

Animals↗