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PubMed2002

Biodiversity (Communications arising): suspect evidence of transgenic contamination.

Quist and Chapela claim that transgenic DNA constructs have been introgressed into a traditional maize variety in Mexico, and furthermore suggest that these constructs have been reassorted and introduced into different genomic backgrounds. However, we show here that their evidence for such introgression is based on the artefactual results of a flawed assay; in addition, the authors misinterpret a key reference to explain their results, concluding that reassortment of integrated transgenic DNA occurs during transformation or recombination.

Artifacts↗
PubMed2005

Isolation of nontuberculous mycobacteria in Zambia: eight case reports.

The isolation of nontuberculous mycobacteria (NTM) raises the question of their clinical significance, especially in an African setting. We found a high percentage of NTM isolated from various specimens, including ones that are normally sterile, among 213 patients in Zambia. Because tuberculosis can affect all parts of the body, we decided to include patients who had signs and symptoms in any part of the body for more than 2 weeks. Most patients had tractus respiratorius (80%) and tractus digestivus (10%) symptoms. During three consecutive days, sputum was collected and two separate sputum specimens were cultured for mycobacteria. Depending on the clinical picture, pleural effusion, ascites, abscess material, or enlarged lymph nodes were also cultured for mycobacteria. A specimen from one sterile body site was collected from 25 patients (60% human immunodeficiency virus [HIV] positive). NTM were isolated from 8 of these 25 specimens. Mycobacterium lentiflavum was isolated from four patients, and Mycobacterium goodii was isolated from one patient. In order to exclude the possibility of laboratory cross-contamination, a novel amplified fragment length polymorphism DNA typing method for M. lentiflavum was developed. Genetic variation was detected, rendering the likelihood of laboratory contamination unlikely. Clinically relevant infection due to NTM occurs in both HIV-positive and HIV-negative patients in Zambia, and their clinical impact seems to be underestimated. This is the first report of M. lentiflavum and M. goodii infections in Africa.

AIDS-Related Opportunistic Infections↗
PubMed2003

Mitochondrial cytochrome b of the Lyakhov mammoth (Proboscidea, Mammalia): new data and phylogenetic analyses of Elephantidae.

The phylogenetic relationships between recent Elephantidae (Proboscidea, Mammalia), that is to say extant elephants (Asian and African) and extinct woolly mammoth, have remained unclear to date. The prevailing morphological scheme (mammoth grouped with Asian elephant) is either supported or questioned by the molecular results. Recently, the monophyly of woolly mammoths on mitochondrial grounds has been demonstrated (Thomas, et al., 2000), but it conflicts with previous studies (Barriel et al., 1999; Derenko et al., 1997). Here, we report the partial sequencing of two mitochondrial genes: 128 bp of 12S rDNA and 561 bp of cytochrome b for the Lyakhov mammoth, a 49,000-year-old Siberian individual. We use the most comprehensive sample of mammoth (11 sequences) to determine whether the sequences achieved by former studies were congruent or not. The monophyly of a major subset of mammoths sequences (including ours) is recovered. Such a result is assumed to be a good criterion for ascertaining the origin of ancient DNA. Our sequence is incongruent with that of Yang et al. (1996), though obtained for the same individual. As far as the latter sequence is concerned, a contamination by non-identified exogenous DNA is suspected. The robustness and reliability of the sister group relation between Mammuthus primigenius and Loxodonta africana are examined: down-weighting saturated substitutions has no impact on the topology; analyzing data partitions proves that the support of this clade can be assigned to the most conservative phylogenetic signal; insufficient taxonomic and/or characters sampling contributed to former discordant conclusions. We therefore assume the monophyly of "real mammoth sequences" and the (Mammuthus, Loxodonta) clade.

Animals↗
PubMed1999

Quantitative PCR for Listeria monocytogenes with colorimetric detection.

An enzyme-linked immunosorbent assay (ELISA)-mediated polymerase chain reaction (PCR) technique was developed to detect and quantify Listeria monocytogenes in food products. The bacterial DNA was extracted from artificially contaminated food and co-amplified with a synthetic internal standard (IS) using primers specific for the target gene coding for the invasive-associated protein (i.a.p.), a virulence factor of L. monocytogenes (i.a.p.) or IS in the presence of fluorescein-dUTP PCR products were hybridized with biotinylated probes designed for the i.a.p. or IS, and then the hybrids were bound to a streptavidin-coated ELISA plate. An alkaline phosphatase-conjugated antibody to fluorescein was added to the plate and in the presence of substrate, PCR products were quantitated based on an optical density reading. The detection limit for L. monocytogenes experimentally inoculated into milk samples and channel catfish fillets was 20 CFU/ml and 1-2 CFU/g, respectively. Little or no cross reaction was detected in the presence of other spoilage and pathogenic organisms such as Pseudomonas aeruginosa, Aeromonas hydrophila, Escherichia coli, and Staphylococcus aureus. The ELISA-mediated PCR technique, when compared to traditional methods, is more rapid (2 working days) for detecting and quantifying L. monocytogenes.

Animals↗
PubMed2006

Survival of thermophilic spore-forming bacteria in a 90+ year old milk powder from Ernest Shackelton's Cape Royds Hut in Antarctica.

Milk powder taken to Antarctica on Shackelton's British Antarctic Expedition in 1907 was produced in New Zealand by a roller drying process in the first factory in the world dedicated to this process. Thermophilic bacilli are the dominant contaminants of modern spray-dried milk powders and the 1907 milk powder allows a comparison to be made of contaminating strains in roller-dried and spray-dried powders. Samples of milk powder obtained from Shackelton's Hut at Cape Royds had low levels of thermophilic contamination (< 500 cfu ml-1) but the two dominant strains (Bacillus licheniformis strain F and Bacillus subtilis) were typical of those found in spray-dried powders. Soil samples from the floor of the hut also contained these strains, whereas soils distant from the hut did not. Differences in the RAPD profiles of isolates from the milk powder and the soils suggest that contamination of the milk from the soil was unlikely. It is significant that the most commonly encountered contaminant strain in modern spray-dried milk (Anoxybacillus flavithermus strain C) was not detected in the 1907 sample.

Animals↗
PubMed1995

CD8+ T lymphocytes in the lung of acquired immunodeficiency syndrome patients harbor human immunodeficiency virus type 1.

Human immunodeficiency virus-1 (HIV-1) infection of CD8+ lymphocytes has been described in several in vitro culture systems, but whether CD8+ cells are a target and also serve as a reservoir for infection in vivo as yet is unknown. We addressed this issue in patients with acquired immunodeficiency syndrome (AIDS)-related lower respiratory tract chronic inflammation, which is characterized by a massive influx of CD8+ HIV-1-specific cytotoxic T lymphocytes (CTL). Proviral load in lung T lymphocytes and their subpopulations was evaluated by using the DNA-polymerase chain reaction (PCR) technique on cells retrieved by bronchoalveolar lavage. To avoid the possibility that the presence of HIV-1 DNA could be caused by contaminating CD4+ cells, serial dilutions of highly purified CD8+ cells were also analyzed by PCR. Our findings showed that lung CD8+ cells harbor and express HIV-1. To explore the possible mechanisms leading to pulmonary CD8+ lymphocyte infection, we evaluated CD4 gene expression on highly purified CD8+ cells by means of reverse transcriptase PCR. Despite the lack of membrane CD4 reactivity, we could show that CD8+ cells may express CD4 RNA. Coinfection of lung CD8+ cells harboring proviral HIV-1 sequences by viral agents capable of inducing CD4 expression (ie, HHV-6) was not detected. Our data indicate that not only CD4+ T lymphocytes and macrophages, but also CD8+ cells, may represent a target and/or a reservoir for HIV-1 in vivo, and suggest that lung CD8+ lymphocytes could derive from precursors equipped with enough CD4 molecules to become HIV-1 permissive. Aside from the cell-to-cell contact between activated HIV-1 specific CTL and relevant targets, the infection of precursors could represent an additional mechanism accounting for the infection of pulmonary CD8+ cells and their functional impairment.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Detection and characterization of Salmonella associated with tropical seafood.

The prevalence of Salmonella in seafood samples collected from the southwest coast of India was studied by conventional culture and by a DNA based molecular technique, polymerase chain reaction (PCR). While conventional culture techniques detected Salmonella in only 20 out of the 100 samples analyzed, direct enrichment lysate PCR detected 52 as positive for Salmonella. A set of three different PCR primers viz., hns, invA and invE were used. It was observed that hns primer detected Salmonella in a significantly higher number of samples. Fourteen out of nineteen isolates belonged to serovar S. enterica Weltevreden. S. Weltevreden isolates were genotyped yielding 4 different patterns both by RAPD and ERIC-PCR but when combined, the overall results discriminated the isolates of S. Weltevreden into 6 different types. This suggests that genetically diverse Salmonella Weltevreden are prevalent in seafood.

Animals↗
PubMed2001

Mycobacterium tuberculosis complex DNA from an extinct bison dated 17,000 years before the present.

In order to assess the presence of tuberculosis in Pleistocene bison and the origin of tuberculosis in North America, 2 separate DNA extractions were performed by 2 separate laboratories on samples from the metacarpal of an extinct long-horned bison that was radiocarbon dated at 17,870+/-230 years before present and that had pathological changes suggestive of tuberculosis. Polymerase chain reaction amplification isolated fragments of tuberculosis DNA, which were sequenced, and on which spoligotyping was also performed to help determine its relationship to the various members of the Mycobacterium tuberculosis complex. Extensive precautions against contamination with modern M. tuberculosis complex DNA were employed, including analysis of paleontologic and modern specimens in 2 geographically separate laboratories.

Animals↗
PubMed1994

Polymerase chain reaction for monitoring human papillomavirus contamination of medical personnel during treatment of genital warts with CO2 laser and electrocoagulation.

Genital warts and intraepithelial neoplasia caused by infection with human papillomavirus are usually treated with CO2 laser or electrocoagulation. In this study, contamination of personnel and operating theatres with human papillomavirus DNA during treatment sessions was investigated. Samples were taken from the nostrils, nasolabial folds and conjunctiva of the operating physician before and after operating sessions and from Petri dishes left open in the operating theatres. Human papillomavirus DNA was demonstrated by the polymerase chain reaction technique. The results show that there is a risk of contamination of the operator by human papillomavirus DNA, detectable with the polymerase chain reaction technique, during both CO2 laser and electrocoagulation treatment.

Aerosols↗
PubMed2001

[Study on allele-specific diagnostic PCR of the traditional Chinese medicines of the deers].

AIM: To develop a convenient and accurate method of DNA molecular marker for the identification of traditional Chinese medicines made of deers, consisting of pilose antler, penis and testis, tendon and foetus. METHODS: Based on the analysis of DNA sequence of mitochondrial Cyt b gene from original animals of both genuine crude drugs, Cervus nippon and Cervus elaphus, and adulterants, a pair of allele-specific primers named as ILu01-L and ILu01-H were designed for distinguishing geniune crude drugs of deers from their adulterants. RESULTS: The results of diagnostic PCR annealing at 64 degrees C for original animals showed that a 365 bp fragment was only amplified from DNA templates of Cervus nippon and Cervus elaphus. For the identification of medicinal materials total of 43 samples from 6 packages were tested under the same reaction conditions except for DNA templates extracted from these crude drugs. Only 9 samples mentioned above was shown to generate positive amplificon. The result indicate that of 8 samples from 1 package of pilose antler and only 1 sample of deer tendon was genuine crude drug. After that, 3 amplified fragments selected randomly were performed with sequencing analysis with the purpose of verifying the results from diagnostic PCR. Data from sequencing confirmed the reliability of diagnostic PCR identification. CONCLUSION: The diagnostic primers designed in the present study were highly specific for Cervus nippon and Cervus elaphus, and they could be used for the authentication of traditional Chinese medicines made from the deer. The quality of the crude drugs of the deer in the current market is a problem and more effective quality control for these traditional Chinese medicines is urgently needed.

Alleles↗
PubMed2003

Using a portable real-time PCR assay to detect Salmonella in raw milk.

The purpose of this study was to determine the efficacy of a portable real-time PCR system in detecting Salmonella spp. in raw milk. The 200 bulk milk samples chosen for this study constituted a subset of the samples for a larger study; this subset contained 24 samples that were culture positive for Salmonella and 176 that were culture negative. Milk was both plated directly on selective agar and plated after enrichment in selective media. Presumptive Salmonella colonies were isolated by direct culturing of five samples, while Salmonella was isolated from the remaining 19 positive samples only after enrichment. Presumptive Salmonella isolates were serotyped, and isolates from 22 samples were confirmed to be Salmonella isolates. PCR assays of culture-positive milk prior to enrichment yielded no evidence of Salmonella. DNA extracts of bacterial pellets from the enriched samples were analyzed for Salmonella by real-time PCR with the Ruggedized Advanced Pathogen Identification Device (RAPID). Fifty-four samples from the enrichment pellets tested positive for Salmonella by real-time PCR. Two samples that tested positive for Salmonella by culture and serotyping tested Salmonella negative by real-time PCR. Serotyping identified isolates from these samples as Salmonella Montevideo. All DNA extracts of Salmonella Montevideo isolates tested positive for Salmonella by real-time PCR. Thirty-three samples tested negative by culture and positive by real-time PCR. These results indicate that the portable real-time PCR system appears to be a useful tool for detecting Salmonella in raw milk. Additionally, the combination of enrichment and real-time PCR techniques used in this study can yield results in 24 h, compared with the 48 to 72 h required for traditional culture.

Animals↗
PubMed1991

Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells.

We have used the nested polymerase chain reaction (PCR) combined with fluorescence-activated cell sorting to define sites of latency of human cytomegalovirus (HCMV) in the peripheral blood of healthy subjects. Peripheral blood mononuclear (PBM) cells were separated into T cell or non-T cell populations and monocytes, and were then analysed by PCR for the presence of HCMV DNA. In five of six seropositive subjects, HCMV was found predominantly in the non-T cell population. Further analysis suggested that the virus was present in adherent cells and CD14+ cells. In three of nine seronegative subjects we could demonstrate HCMV DNA, which we do not believe was due to contamination, reproducibly by PCR. In one of these seronegative subjects, HCMV DNA was present predominantly in the non-T cell fraction of PBM cells. No HCMV DNA was detectable in the remaining six seronegative subjects. We conclude that, within the PBM cells of normal asymptomatic seropositive and some seronegative subjects, HCMV is present predominantly in the monocyte fraction. In addition, the detection of HCMV sequences in seronegative subjects may indicate that infection with HCMV is more widespread than conventional seroepidemiology suggests.

Antibodies, Viral↗
PubMed1997

DNA adducts as exposure biomarkers and indicators of cancer risk.

Quantitation of DNA adducts in human tissues has been achieved with highly sensitive techniques based on adduct radiolabeling, antisera specific for DNA adducts or modified DNA, and/or adduct structural characterization using chemical instrumentation. Combinations of these approaches now promise to elucidate specific adduct structures and provide detection limits in the range of 1 adduct/10(9) nucleotides. Documentation of human exposure and biologically effective dose (i.e., chemical bound to DNA) has been achieved for a wide variety of chemical carcinogens, including polycyclic aromatic hydrocarbons (PAHs), aromatic amines, heterocyclic amines, aflatoxins, nitrosamines, cancer chemotherapeutic agents, styrene, and malondialdehyde. Due to difficulties in exposure documentation, dosimetry has not been precise with most environmental and occupational exposures, even though increases in human blood cell DNA adduct levels may correlate approximately with dose. Perhaps more significant are observations that lowering exposure results in decreasing DNA adduct levels. DNA adduct dosimetry for environmental agents has been achieved with dietary contaminants. For example, blood cell polycyclic aromatic hydrocarbon-DNA adduct levels were shown to correlate with frequency of charbroiled meat consumption in California firefighters. In addition, in China urinary excretion of the aflatoxin B1-N7-guanine (AFB1-N7-G) adduct was shown to increase linearly with the aflatoxin content of ingested food. Assessment of DNA adduct formation as an indicator of human cancer risk requires a prospective nested case-control study design. This has been achieved in one investigation of hepatocellular carcinoma and urinary aflatoxin adducts using subjects followed by a Shanghai liver cancer registry. Individuals who excreted the AFB1-N7-G adduct had a 9.1-fold adjusted increased relative risk of hepatocellular carcinoma compared to individuals with no adducts. Future advances in this field will be dependent on chemical characterization of specific DNA adducts formed in human tissues, more-precise molecular dosimetry, efforts to correlate DNA adducts with cancer risk, and elucidation of opportunities to reduce human DNA adduct levels.

Biomarkers↗
PubMed1994

[Methods for the detection of viral contamination in food of animal origin].

Contamination of foods of animal origin with pathogenic human viruses may occur during handling or through polluted water. Most of these viruses are pathogens originating from the human gastrointestinal tract. They can be transmitted by the consumption of contaminated food and often cause disease. A survey is given of DNA- and RNA-viruses that may occur as contaminants of foods. In addition, the classical methods for detecting viral contaminations in foods are summarized. They are based on the effects after virus inoculation of cell cultures. Besides the fact that these methods are not economic and time consuming, they do not permit detection of some of the most important foodborne gastroenteritis viruses. The possibility of replacing these methods by detecting the viral genomes using hybridization and polymerase chain reaction (PCR) is discussed.

Animals↗
PubMed1985

Epidermal Langerhans cells--a cycling cell population.

The limited number of Langerhans cells (LC) in human epidermis and the resultant technical difficulties have left open the question of LC kinetics. In the present study using flow cytometry (FCM) we have applied 3 methods to estimate LC-DNA distribution: (1) FCM-DNA measurement on highly enriched LC suspensions, (2) FCM-correlated analysis of DNA and OKT-6(+) cells in total epidermal cell suspensions, (3) LC-enriched suspensions (70-90%) were FACS (fluorescence-activated cell sorter) sorted on microscopic slides, and stained with the Feulgen technique, and DNA was measured densitometrically. In the latter method, contaminating keratinocytes were counterlabeled with antikeratin serum to eliminate them from LC-DNA estimation. All 3 in vitro analyses clearly showed that human LC are a cycling cell population in the epidermis. The number of LC in S (1.3-3.3%) and G2/M (1.0-2.5%) phase compares with those found for keratinocytes. Assuming that this percentage of keratinocytes in S and G2/M phases is sufficient to maintain the structural integrity of the epidermis, it was suggested that LC may represent a stable, self-reproducing cell population in normal epidermis.

Cell Count↗
PubMed2001

A technical note on microsatellite DNA instability studied in archival paraffin-embedded tissues.

The authors present brief overview of problems associated with analysis of microsatellite sequences in DNA from archival paraffin-embedded tissue. This methodology can serve several diagnostic and research purposes provided appropriate precautions are taken to extract DNA of acceptable quality with small admixture of contaminants. Also PCR amplification step requires some modifications compared to routine reactions involving DNA from fresh or freshly-frozen tissues.

DNA↗
PubMed1999

Unique strains of SV40 in commercial poliovaccines from 1955 not readily identifiable with current testing for SV40 infection.

SV40 was first identified as a contaminant of poliovaccines used from 1955 until 1963. Recently, SV40 has been detected in several human tumors. The virus detected in human tumors often contained only one 72-bp enhancer in the regulatory region, in contrast to the SV40 originally isolated from poliovaccines, which contained two 72-bp enhancers. The origin of viruses with one 72-bp enhancer in humans was unknown, because it was thought that these viruses were not present in poliovaccines. It was also thought that all poliovaccine vials produced from 1955 until 1963 had been discarded, thus the possibility that one 72-bp virions contaminated those vials could not be tested. We unexpectedly obtained what appear to be the last available vials of poliovaccine produced in 1955. In these vials, we detected and sequenced SV40 containing only one 72-bp enhancer in the regulatory region. The tissue culture cytopathic test currently used in the United States to screen oral poliovaccines was designed to detect rapidly proliferating SV40 virions containing two 72-bp enhancers. We found that this test is not sensitive enough to detect low amounts of the slow-replicating SV40 virions containing one 72-bp enhancer. This virus was easily detected in the same cells by immunostaining and PCR. Twelve current vials of poliovaccines tested uniformly negative for SV40, suggesting that the precaution of preparing poliovaccines from kidneys obtained from monkeys bred in isolated colonies prevented SV40 contamination. Our data demonstrate that humans were exposed to SV40 viruses with both one 72-bp enhancer and two 72-bp enhancers SV40 through contaminated vaccines. Our data also suggest that instead of cytopathic tests, immunohistochemical and/or molecular studies should be used to screen poliovaccines for SV40 to completely eliminate the risk of occasional contamination.

Animals↗