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PubMed1977

DNA contained by two densonucleosis viruses.

The DNA contained by particles of densonucleosis viruses 1 and 2 were analyzed within the particle, and properties of DNA extracted from these particles were determined. The DNA appears to exist as a single-stranded molecule with limited secondary structure within particles, as assessed by spectral changes induced by formaldehyde, melting profiles, and circular dichroism studies. The single-stranded DNA had an apparent molecular weight of 1.9 X 10(6) to 2.2 X 10(6) as assessed by differences in the molecular weight of virus particles and top component and percentage of nucleic acid. DNA extracted from virus particles in low-salt buffers possessed properties typical of a single-stranded molecule. Double-stranded DNA could be extracted from virus particles under appropriate high salt and elevated temperature. The linear double-stranded DNA extracted from both viruses had a molecular weight of about 3.9 X 10(6) to 4.1 ZX 10(6) determined by neutral sedimentation and electron microscopy and an equivalent genome size determined by reassociation kinetics. About 87% of the DNA was homologous between the two viruses.

Animals↗
PubMed2003

Identification of Cryptosporidium spp. oocysts in United Kingdom noncarbonated natural mineral waters and drinking waters by using a modified nested PCR-restriction fragment length polymorphism assay.

We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities of Cryptosporidium spp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65 degrees C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted from Cryptosporidium parvum oocysts, C. muris (RN 66), C. baileyi (Belgium strain, LB 19), human-derived C. meleagridis, C. felis (DNA from oocysts isolated from a cat), and C. andersoni was used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymes DraI and VspI. Discrimination between C. andersoni and C. muris isolates was confirmed by a separate, subsequent digestion with DdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. The Cryptosporidium species detected in these finished water samples was C. parvum genotype 1. This method consistently and routinely detected >5 oocysts per sample.

Animals↗
PubMed1998

Helicobacter pylori acquistion of metronidazole resistance by natural transformation in vitro.

AIM:To study whether Helicobacter pylori is naturally transformable.METHODS:Transformation was performed in BHI broth supplemented with horse serum and yeast extract. Genomic DNA extracted from a metronidazole resistant H.pylori strain was added to H. pylori broth culture. The mixture was incubated at microaerophilic atmosphere. The DNA-treated cells were plated on blood agar containing 8mg/L metronidazole to select for transformants. Sterile distilled water was used as a negative DNA control.The DNA profiles of transformants were compared with that of their parent strains by randomly amplified polymorphic DNA (RAPD) fingerprinting.RESULTS:Transformation ofH. pylori with DNA from a metronidazole resistant strain as a marker was demonstrated. Out of the 12 strains of H. pylori tested, 9 (75%) strains were found to be transformable. The transformation frequencies ranged from 3.4?10(-6) to 2.4 10(-4). By RAPD, DNA fingerprints of the transformants and their parent strains showed no change in DNA profiles though transformants were all resistant to metronidazole as compared with their metronidazole-sensitive parent strains.CONCLUSION:Helicobacter pylori is naturally transformable which might be one of the ways that H. pylori develops resistance to metronidazole.

Journal Article↗
PubMed2005

Extensive human DNA contamination in extracts from ancient dog bones and teeth.

Ancient DNA (aDNA) sequences, especially those of human origin, are notoriously difficult to analyze due to molecular damage and exogenous DNA contamination. Relatively few systematic studies have focused on this problem. Here we investigate the extent and origin of human DNA contamination in the most frequently used sources for aDNA studies, that is, bones and teeth from museum collections. To distinguish contaminant DNA from authentic DNA we extracted DNA from dog (Canis familiaris) specimens. We monitored the presence of a 148-bp human-specific and a 152-bp dog-specific mitochondrial DNA (mtDNA) fragment in DNA extracts as well as in negative controls. The total number of human and dog template molecules were quantified using real-time polymerase chain reaction (PCR), and the sequences were characterized by amplicon cloning and sequencing. Although standard precautions to avoid contamination were taken, we found that all samples from the 29 dog specimens contained human DNA, often at levels exceeding the amount of authentic ancient dog DNA. The level of contaminating human DNA was also significantly higher in the dog extracts than in the negative controls, and an experimental setup indicated that this was not caused by the carrier effect. This suggests that the contaminating human DNA mainly originated from the dog bones rather than from laboratory procedures. When cloned, fragments within a contaminated PCR product generally displayed several different sequences, although one haplotype was often found in majority. This leads us to believe that recognized criteria for authenticating aDNA cannot separate contamination from ancient human DNA the way they are presently used.

Animals↗
PubMed2002

Reliability of nested PCR for detection of Chlamydia pneumoniae DNA in atheromas: results from a multicenter study applying standardized protocols.

The present multicenter study was designed to find explanations for the discrepancies in the reported rates of detection of Chlamydia pneumoniae DNA in endarterectomy specimens. Coded identical sets of (i) a C. pneumoniae DNA dilution series (panel 1; n = 10), (ii) spiked control tissue specimens (panel 2; n = 10 specimens, including 5 negative controls), and (iii) endarterectomy specimens (panel 3; 15 atheromas, 5 negative controls) were analyzed at four laboratories by three standardized DNA extraction methods in each laboratory and a nested touchdown PCR protocol targeting the ompA gene of C. pneumoniae. Panel 1 samples were correctly identified as positive to levels of 0.3 inclusion-forming units (IFU)/PCR mixture (100%) and 0.03 IFU/PCR mixture (50%). All negative controls were correctly reported as negative. Panel 2 samples were identified as C. pneumoniae positive to levels of 0.01 IFU/PCR mixture (100%) and 0.005 IFU/PCR mixture (91%), independent of the DNA extraction method used, and only one false-positive result was reported. For panel 3 samples, 5 of 240 (2%) analyses (in which DNA extractions and PCR were performed at the same laboratory) were positive; the positive specimens were from three endarterectomy specimens and two negative controls. After exchange of DNA extracts between laboratories, 13 of 15 atheroma samples were C. pneumoniae DNA positive in at least 1 of a series of 48 analyses per atheroma sample; however, the overall positivity rate did not exceed 5% (33 of 720 analyses) and therefore was lower than that for the negative controls (8%; 19 of 240 analyses). Not a single positive result could be achieved when all panel 3 extracts (n = 240 analyses) were reamplified by a 16S rRNA PCR followed by hybridization with a C. pneumoniae-specific probe. Statistical analyses demonstrated that positive results did not occur in an independent and random fashion and could most likely be explained by amplicon carryover at the nested PCR level as well as amplicon introduction during DNA extraction, but not by the patterns of distribution of very low target levels or a certain DNA extraction protocol. The results of studies by nested PCR for detection of the prevalence of C. pneumoniae will always be questionable.

Arteriosclerosis↗
PubMed2001

Microarray sampling-platform fabrication using bubble-jet technology for a biochip system.

The fabrication of microarrays containing PCR-amplified genomic DNA extracts from mice tumors on a Zetaprobe membrane using a modified thermal ink-jet printer is described. A simple and cost-effective procedure for the fabrication of microarrays containing biological samples using a modified bubble-jet printing system is presented. Because of their mass-produced design, ink-jet printers are a much cheaper alternative to conventional spotting techniques. The usefulness of the biochip microarray platform is illustrated by the detection of human fragile histidine triad (FHIT), a tumor suppressor gene. Subcutaneous carcinomas were induced with MKN/FHIT and MKN/E4 cell lines in immunodeficient mice. Several weeks into their development, the tumors from both groups of mice were removed and subjected to DNA extraction by lysis of tissue samples. The extracted DNA samples were amplified by PCR (30 cycles) using the primers corresponding to nucleotides 2 to 18 of the FHIT sequence. The resulting solution was transferred to the individual reservoirs of a three-color cartridge from a conventional thermal ink-jet printer (HP 694C), and arrays were printed on to a Zetaprobe membrane. After spotting, these membranes were used in a hybridization assay, using fluorescent probes, and detected with a biochip.

Acid Anhydride Hydrolases↗
PubMed1993

Rapid extraction of high purity chromosomal DNA from Serratia marcescens.

Rapid non-specific degradation of Serratia marcescens DNA extracted with guanidium thiocyanate, occurred within 10 min of incubation with restriction endonuclease enzymes. The described modified method based on chemical and enzymatic deproteinization produced preparations of Ser. marcescens DNA of high yield and quality which did not autodegrade when incubated with restriction endonucleases.

Chromosomes, Bacterial↗
PubMed2000

An evaluation of the relevance of routine DNA typing of fingernail clippings for forensic casework.

DNA extracted from fingernail clippings of victims in forensic cases is a possible source of DNA from the perpetrator in cases where victims struggled or defended themselves. The source of this DNA on a victim's fingernails could possibly originate from contact with the suspect's blood, saliva, semen or scratched skin. In this technical note we evaluate the relevance of routine DNA typing of fingernail clippings in the forensic biology laboratory when, in real casework, normally only small quantities of nail material is sent. This was carried out by extracting DNA from fingernail clippings from a number of volunteers, before and after aggressively scratching other volunteers. No blood was drawn from the scratching, but skin flakes were observed under the nails before cutting and subsequent DNA typing. The DNA extracted was then typed using the STR systems: HUMTHO1, HUMTPOX and HUMCSF1PO (CTT triplex) and the system of D1S80. These profiles were compared with profiles achieved by similar typing of buccal swabs as a reference from each volunteer. In this study, the profile detected from each volunteer's clippings was the same before and after scratching, and matched the profile of the corresponding volunteer as defined by typing each volunteer's reference buccal swab. Fingernail clippings that are sent to our lab in actual casework are usually so small that additional treatment by swabbing or removing debris from below the clipping is not possible. For this reason, in this simulation the entire clippings were used for DNA extraction, to maximize the possibility of finding an additional profile. In conclusion, the findings from this study show that although the profiles obtained when typing fingernail clippings are those of the donors themselves, we suggest that typing of fingernail clippings should be carried out in forensic cases only when relevant. We would suggest that fingernail clippings not be routinely sent to the biology laboratory as items of evidence to be tested.

Forensic Medicine↗
PubMed2005

Pneumocystis jiroveci in HIV/AIDS patients: detection by FTA filter paper together with PCR in noninvasive induced sputum specimens.

OBJECTIVES: To detect P. jiroveci (previously named P. carinii) by PCR using FTA filter paper to extract the DNA, from noninvasive induced sputum samples of HIV/AIDS patients. MATERIAL AND METHOD: Fifty two HIV/AIDS patients suspected of Pneumocystis jiroveci pneumonia (PJP) in King Chulalongkorn Memorial Hospital were recruited. Both cytological method and PCR with FTA filter paper technique were performed to detect P jiroveci from each specimen. RESULTS: The detectability rate of P. jiroveci infection was 21%. The PCR with FTA filter paper method was 4 folds much more sensitive than Giemsa staining technique. P. jiroveci was detected in 18% of the HIV/AIDS patients in spite of receiving standard PJP prophylaxis. CONCLUSION: Detection of P. jiroveci by using FTA filter paper together with PCR in induced sputum samples could detect more cases of P. jiroveci infection than by using cytological method. DNA extraction using the FTA filter paper was more rapid and convenient than other extraction methods. The causes of failure of PJP prophylaxis should be evaluated.

AIDS-Related Opportunistic Infections↗
PubMed2000

A PCR-colorimetric microwell plate hybridization assay for detection of Mycobacterium tuberculosis and M. avium from culture samples and Ziehl-Neelsen-positive smears.

Differentiation between Mycobacterium tuberculosis and M. avium is essential for the treatment of mycobacterial infections. We have developed an easy and rapid detection assay for the diagnosis of mycobacterial diseases. This is a PCR-hybridization assay based on selective amplification of a 16S rRNA gene sequence using pan-Mycobacterium primers followed by hybridization of the amplification products to biotinylated M. tuberculosis and M. avium-specific probes. A total of 55 mycobacterial isolates were tested. For all isolates, results concordant with those of conventional identification methods were obtained. Moreover, we developed a method for extraction of DNA from Ziehl-Neelsen-positive smears which allows the recovery of intact target DNA in our PCR-hybridization assay. Our method was able to confirm all culture results for 59 Ziehl-Neelsen-positive smears from clinical specimens (35 sputum, 11 lymph node biopsy, 6 stool, 4 pus, 2 urine, and 1 pericardial fluid specimens). These data suggest that our PCR-hybridization assay, which is simple to perform and less expensive than commercial probe methods, may be suitable for the identification of M. tuberculosis and M. avium. It could become a valuable alternative approach for the diagnosis of mycobacterial infections when applied directly to DNA extracted from Ziehl-Neelsen-positive smears as well.

Colorimetry↗
PubMed2005

Real-time quantitative PCR detection of Mycobacterium avium subsp. paratuberculosis and differentiation from other mycobacteria using SYBR Green and TaqMan assays.

Sensitive real-time sequence detection methods based on two different chemistries were developed for Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of Johne's disease in cattle. One is based on the detection of SYBR Green bound to PCR products and the second method is more specific, detecting the cleavage of a fluorogenic (TaqMan) probe bound to a target sequence during primer extension phase. Novel primers and probes that amplify small fragments (<80 bp) of the Map specific insertion sequence, IS900 were designed. Both the SYBR Green and TaqMan assays are sensitive, able to detect 4 fg of DNA extracted from Map strain ATCC19698. This amount of DNA corresponds to the detection of 0.8 cells. Map cells were quantified directly from 7H9 broth using the SYBR Green assay and compared to dilutions of DNA extracted from an equivalent number of cells. The SYBR Green assay of 7H9 broth resulted in a minimum detectable limit of 0.07 cells (equivalent to 0.34 fg of DNA). Media ingredients were not observed to interfere with the assay. Since no extraction step was necessary in the direct cell measurements, direct detection was ten-fold more sensitive than detection of extracted DNA. Both SYBR Green and TaqMan assays are highly specific for the detection of Map. They did not detect any closely related members of the avium complex, other species of mycobacteria, or related genera that are likely to be present in environmental samples. No reporter signal was detected during TaqMan assays performed with 100 pg of template DNA from the non-Map organisms.

Animals↗
PubMed2006

Use of hybridization probes in a real-time PCR assay on the LightCycler for the detection of methicillin-resistant Staphylococcus aureus.

The rapid and accurate identification of methicillin-resistant Staphylococcus aureus (MRSA) is of great importance for the affected patient, the involved ward, and the microbiological laboratory. Resistance to methicillin is encoded by the mecA gene in S. aureus. Because routine laboratory diagnostics may be time consuming and because species differentiation encounters a variety of difficulties, molecular techniques detecting both the mecA and a S. aureus-specific gene are used for rapid and accurate detection and identification of MRSA. Various protocols, including the manual extraction of DNA have been established. In this chapter, the identification of MRSA based on simultaneous detection of the mecA gene and the S. aureus-specific Sa442 DNA fragment using automated DNA extraction and real-time polymerase chain reaction is described. This method is an attractive alternative to labor-intensive manual protocols and can easily be incorporated into the diagnostic microbiology laboratory workflow, with the ability to obtain results within 4 h.

Bacterial Proteins↗
PubMed1996

Survival of luxAB-marked Alcaligenes eutrophus H850 in PCB-contaminated soil and sediment.

A rifampicin-resistant PCB-degrading Alcaligenes eutrophus H850 strain was marked with luxAB reporter genes and designated H850Lr. This strain was enumerated in soil by viable plating and counting of light-emitting colonies. The marked strain was also inoculated into soil and sediment microcosms contaminated with PCBs and treated with rhamnolipid biosurfactants produced by Pseudomonas aeruginosa UG2Lr or inoculated with the P. aeruginosa UG2Lr strain. A. eutrophus H850Lr exhibited similar survival in sandy loam soil in the absence or presence of PCBs over 56 days. Survival of A. eutrophus H850Lr in PCB-contaminated sediment was less than in sandy soil under the same incubation conditions. Addition of P. aeruginosa UG2 rhamnolipids to soil increased the culturable indigenous heterotrophic population, and numbers of A. eutrophus H850Lr cells. P. aeruginosa UG2Lr cells did not affect survival of A. eutrophus H850, as cell enumerations after 2 months were the same as in microcosms containing only A. eutrophus H850 inoculum. P. aeruginosa UG2Lr survived in soils as demonstrated by the slight decrease in CFU from 1 x 10(8) to 2 x 10(6) CFU cm-3 after 2 months. Direct extraction of DNA from soil and purification for use in PCR amplification using primers specific for the bphC gene detected 8 x 10(2) A. eutrophus H850Lr CFU g-1 soil in PCB-contaminated soils. Colony lifts of bacteria isolated from microcosms containing PCB-contaminated soil did not hybridize with LB400 bphC probe. However, enrichment of PCB-contaminated soil with biphenyl, followed by DNA extraction and probing with bphC gene probe detected indigenous PCB-degrading bacteria containing a similar gene sequence in PCB-contaminated sediment. This study demonstrates the usefulness of using the lux reporter system in monitoring bacterial survival in PCB-contaminated soils and sediments.

Alcaligenes↗
PubMed2003

Molecular detection of anthrax spores on animal fibres.

AIMS: To develop a rapid, specific and sensitive diagnostic test for the detection of the spores of Bacillus anthracis on commercial samples of animal fibres (e.g. wool and cashmere). METHODS AND RESULTS: Extraction of DNA from spores using a mechanical disruption method based on bead beating was evaluated but subsequently abandoned as it compromised the sensitivity of the overall protocol. A multiplex PCR and two nested amplification reactions designed for B. anthracis were developed during this study. CONCLUSIONS: A simple selective incubation step in combination with multiplex PCR was found to be more effective than generic DNA extraction coupled to a sensitive nested amplification reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid diagnostic test could be applied to the analysis of commercial fibre samples for the detection of anthrax as required by health and safety legislation resulting in considerable savings in time and expense.

Animals↗
PubMed1995

Extraction and purification of microbial DNA from petroleum-contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis.

We investigated the use of multiplex polymerase chain reaction (PCR) techniques coupled with Southern analysis to detect xenobiotic-degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt-2 (pWW0), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWW0, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide-stained gels of the initial PCR reaction indicated detectable amplification of between 10(0) to 10(6) cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 microL) of the first reaction increased the ethidium bromide-based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradative cells, at a lower detection limit of approximately 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells.

Alcaligenes↗
PubMed2005

Eight-hour PCR-based procedure for the detection of Salmonella in raw oysters.

The aim of this study was to evaluate the ability of a nested PCR system to detect Salmonella senftenberg in raw oysters. The specific primers of the PCR were derived from the invA gene sequence, essential for Salmonella invasiveness into epithelial cells. First, for the extraction of DNA, four methods (guanidine isothiocyanate, E.Z.N.A. Mollusc Kit, Chelex-100, and lysis with detergents) were compared. A nested PCR method combined with 3.5 h pre-enrichment in buffered peptone water (BPW) and DNA extraction by the resin Chelex-100 is proposed for the detection of S. senftenberg in oyster samples. The detection limit of the method is less than 0.1 CFU/ml (<1 CFU/g of oyster). This procedure is shown to be an excellent tool for the sensitive detection of S. senftenberg from naturally contaminated oysters, with results being obtained within 8 h.

Animals↗
PubMed2006

Development of a real-time Staphylococcus aureus and MRSA (SAM-) PCR for routine blood culture.

The notification of "Gram-positive cocci, possibly staphylococcus" in a blood culture drawn from a seriously ill patient is responsible for a large amount of vancomycin prescribing in institutions where methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of bacteraemia. A duplex real-time TaqMan polymerase chain reaction targeting the species-specific nuc gene, and the mecA gene encoding methicillin-resistance, was developed as a tool for rapid identification and detection of S. aureus and methicillin-resistance, and optimised for immediate as-needs testing. Three different DNA extraction methods achieved varying DNA quality, with PCR inhibition the main problem. Serial blood cultures (n=120) identified as possible staphylococci on Gram stain from our clinical laboratory were examined. There was one false negative result for a methicillin-resistant Staphylococcus epidermidis, which was positive on repeat testing, and one false negative result due to DNA extraction failure for MRSA from peritoneal dialysate inoculated into blood culture medium. Sensitivity and specificity of 97% and 100%, respectively, were obtained for mecA; and sensitivity and specificity of 98% and 100%, respectively, for nuc. Detection of slow-growing coagulase-negative staphylococci as co-infecting strains may be reduced. The assay quickly and reliably identified S. aureus in mixed infection, and identified methicillin resistance in both S. epidermidis and S. aureus strains.

Bacterial Proteins↗