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PubMed1997

Validation studies of the CTT STR multiplex system.

Studies were performed to define the typing conditions and evaluate the forensic applicability of multiplex amplification of three STR loci, CSF1PO, TPOX, and THO1. Results were obtained using the GenePrint STR System (Promega Corporation, Madison, WI) Kit. To determine the utility of the GenePrint STR System for forensic casework analyses, the following experiments were conducted: 1) analysis of mixed body fluid; 2) determination of the sensitivity of detection; and 3) evaluation of results from biological samples from casework. In addition, the following simulated forensic conditions were assayed to detect whether or not there may be adverse effects on the ability to type these loci: 1) chemical contaminant effects on the DNA in body fluid samples; 2) the effects on DNA from samples deposited on various substrates; 3) the consequences of micro-organism contamination; and 4) the effect of sunlight and storage conditions on the integrity of the STR profiles/DNA. The data demonstrate that STR typing of biological samples exposed to a variety of environmental insults yields reliable results and that the analysis of the STR loci CSF1PO, TPOX, and THO1 can be applied in a forensic setting.

Alleles↗
PubMed1998

Formation of DNA adducts in tissues of mouse progeny through transplacental contamination and/or lactation after administration of a single dose of ochratoxin A to the pregnant mother.

Ochratoxin A (OTA) is a mycotoxin which has been detected in foods of plant origin, in edible animal tissues, and in human sera, urine, and milk in many countries. OTA is nephrotoxic and carcinogenic in mice and rats and is suspected to play a key role in the etiology of Balkan endemic nephropathy and/or associated urinary tract tumors. In the present study, some early signs of genetic impairment, including the presence of DNA adducts in target tissues from the progeny of mice after administration of a single OTA dose during late pregnancy, have been investigated. By the 32P-postlabeling method, several characteristic DNA adducts with the same Rf values were detected in kidney and liver of both the OTA-treated mice and their progeny the fetus and the offspring. No adduct was found in tissues from control animals. Different adducts were most important in kidney and liver DNA and some were organ-specific. High levels of DNA adducts were detected in the kidneys of male progeny, whereas in the female progeny and the mothers they were detected almost exclusively in the liver. This result correlates well with the carcinogenicity in mice: the target organ for males is the kidney, while for females it is the liver. High levels of DNA adducts were also found in fetuses. These results provide evidence for a direct genotoxic action of OTA in the progeny through transplacental contamination, which constitutes a new serious health hazard of exposure to this toxin.

Administration, Oral↗
PubMed2006

Chromosome aberrations and DNA strand breaks in glaucous gull (Larus hyperboreus) chicks fed environmentally contaminated gull eggs.

In this present laboratory study, our results suggest that a complex mixture of pollutants found in the marine environment exerts genotoxic effects on glaucous gull (Larus hyperboreus) chicks fed environmentally contaminated gull eggs. Chromosome aberrations, quantified by cytogenetic analysis of blood cells, and DNA strand breaks, quantified by agarose gel electrophoresis and image data analysis, were determined in glaucous gull chicks fed environmentally contaminated gull eggs (exposed group) and in chicks fed hen eggs (control group). For both female and male gulls, the fraction of damaged metaphases was quantitatively higher in exposed than in control groups. On the other hand, the differences between the control and the exposed groups were more relevant when the chromosomal aberration data were treated as group totals rather than at the individual level. Consistent results were obtained in the DNA strand break analyses. The control group appeared to display a greater median molecular length (MML) than the exposed group.

Animals↗
PubMed2026

ViralQC: a tool for assessing completeness and contamination of predicted viral contigs.

MOTIVATION: Viruses represent the most abundant biological entities on Earth, playing vital roles in diverse ecosystems. Cataloging viruses across various environments is essential for understanding their properties and functions. Metagenomic sequencing has emerged as the most comprehensive method for virus discovery. However, distinguishing viral sequences from the vast background of microbial organisms in metagenomic data remains a significant challenge. Existing tools experience varying degrees of false positive rates due to noise in sequencing and assembly, and the integration of proviruses into microbial genomes. This highlights the urgent need for an accurate and efficient method to evaluate the quality of viral contigs. RESULTS: To address these challenges, we introduce ViralQC, a tool designed to assess the quality of viral contigs or bins. ViralQC identifies microbial contamination within putative viral sequences using an ensemble framework powered by DNA and protein foundation models and estimates completeness by analyzing protein organization. We evaluated ViralQC on multiple datasets and compared its performance against the state-of-the-art tool, CheckV. Leveraging both DNA and protein foundation models, ViralQC achieves higher sensitivity on contamination detection for contigs longer than 10 kbp while maintaining comparable accuracy. Additionally, ViralQC delivers more accurate estimation on contigs with completeness > 50%. AVAILABILITY: The source code of ViralQC is available via: https://github.com/ChengPENG-wolf/ViralQC.

Software↗
PubMed1985

Purification of circular DNA using benzoylated naphthoylated DEAE-cellulose.

Un-nicked circular DNA can be separated from protein, RNA, and other DNA in a simple three-step protocol consisting of exonuclease III digestion, extraction with benzoylated naphthoylated DEAE-cellulose (BND cellulose) in 1 M NaCl, and alcohol precipitation of the remaining supercoiled DNA. Exonuclease III treatment introduces single-stranded regions into contaminating linear and nicked circular DNA. This DNA, together with most RNA and protein, is adsorbed onto BND cellulose leaving form I DNA in solution. The protocol can be used to purify analytical as well as preparative amounts of supercoiled DNA. This procedure is a substitute for cesium chloride-ethidium bromide gradient ultracentrifugation and gives a comparable yield of pure form I DNA. Other classes of DNA can be isolated by changing the pretreatment step. Selective digestion of linear DNA with lambda exonuclease permits the isolation of both nicked circular and supercoiled DNA while brief heat-induced or alkali-induced denaturation leads to the recovery of rapidly reannealing DNA. In large-scale purifications, the basic protocol is usually preceded by one or more BND cellulose extractions in 1 M NaCl to remove contaminants absorbing UV or inhibiting exonuclease III.

Cellulose↗
PubMed2005

Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR.

Although PCR and RT-PCR provided a valuable approach for detection of pathogens, the high level of sensitivity of these assays also makes them prone to false positive results. In addition to cross-contamination with true positive samples, false positive results are also possible due to "carry-over" contamination of samples with amplicon DNA generated by previous reactions. To reduce this source of false positives, amplicon generated by reactions in which dUTP was substituted for dTTP can be degraded by uracil DNA glycosylase (UNG). UNG does not degrade RNA but will cleave contaminating uracil-containing DNA while leaving thymine-containing DNA intact. The availability of heat-labile UNG makes use of this approach feasible for RT-PCR. In this study, real-time RT-PCR was used to quantify UNG degradation of amplicon DNA and the effect of UNG on RNA detection. Using the manufacturers' recommended conditions, complete degradation of DNA was not observed for samples containing 250 copies of amplicon DNA. Doubling the UNG concentration resulted in degradation of the two lowest concentrations of DNA tested, but also resulted in an increase of 1.94 cycles in the CT for RNA detection. To improve DNA degradation while minimizing the effect on RNA detection, a series of time, temperature and enzyme concentrations were evaluated. Optimal conditions were found to be 0.25 U UNG per 25 microl reaction with a 20 min, 30 degrees C incubation prior to RT-PCR. Under these conditions, high concentrations of amplicon DNA could be degraded while the CT for RNA detection was increased by 1.2 cycles.

Arterivirus↗
PubMedDate not supplied

Advantage of a rapid extraction method of HIV1 DNA suitable for polymerase chain reaction.

We describe a new protocol for extraction of DNA suitable for HIV1 gene amplification from clinical samples using "Chelex-100" chelating resin. Comparison was made with the classic proteinase K extraction method; 154 specimens were extracted with both methods and subjected to PCR (polymerase chain reaction). The Chelex-100 procedure optimized the yield of DNA recovery and minimized contamination due to sample manipulation. It decreased false negative results due to PCR inhibitors. A DNA sample suitable for use in PCR was obtained in 30 minutes. Chelex-100 treatment is a simple, rapid and low-cost method for DNA extraction in clinical laboratories.

Cells, Cultured↗
PubMed2005

[HOXD13 polyalanine tract expansion in synpolydactyly: mutation detection and prenatal diagnosis in a large Chinese family].

OBJECTIVE: Synpolydactyly (SPD, MIM 186000), also known as syndactyly type II, is a dominantly inherited limb malformation with incomplete penetrance and variable expressivity. Polyalanine tract expansion in HOXD13 has been shown to be the disease-causing mutation in SPD. The present study was designed to identify mutation in HOXD13 and to provide prenatal diagnosis, in a large Chinese SPD family consisting of 54 individuals. METHODS: The proband and 4 other affected individuals in the family were evaluated physically and radiologically to ascertain the SPD phenotype. Genomic DNA was extracted from peripheral blood samples obtained from 18 family members (9 affected and 9 unaffected), and from amniotic fluid and chorionic villus samples obtained from the proband during her two consecutive pregnancies. With the use of a pair of specific primers, a fragment of 161bp was amplified by polymerase chain reaction (PCR) to cover the imperfect GCN triplet repeat sequence in exon 1 of HOXD13 encoding the 15-residue polyalanine tract. The PCR products were detected by agarose gel electrophoresis, and sequenced after cloning into pMD18T vector. To confirm prenatal diagnosis, haplotype analysis was also performed by allele-typing three microsatellite markers, including the intronic CA repeats in HOXD13. RESULTS: Digital and radiographic findings indicated a typical SPD phenotype in the family. These included 3/4 finger syndactyly and 4/5 toe syndactyly with an extra digit in the syndactylous web. Unilateral finger syndactyly in the proband, unilateral toe syndactyly in 2 individuals, bilateral brachydactyly of the fifth toes in 1 individual, and clinodactyly of the fifth fingers in 4 individuals were also observed, indicating variable expressivity. Gel electrophoresis of the PCR products showed an additional longer fragment in all 9 affected individuals but not in the unaffected ones. Sequence analysis of the longer fragment revealed a 9-alanine expansion. The expansion was detectable in DNA from the amniotic fluid and chorionic villus samples. Furthermore, haplotype analysis ruled out potential contamination of the maternal DNA. These suggested that the two fetuses carried the same polyalanine expansion. CONCLUSION: HOXD13 polyalanine expansion was detected in a large Chinese family with SPD and prenatal diagnosis of two affected fetuses was achieved. This is the first report on prenatal diagnosis of SPD by detecting the HOXD13 polyalanine expansion in the Han population of the Chinese mainland.

Base Sequence↗
PubMed1999

Contaminations occurring in fungal PCR assays.

Successful in vitro amplification of fungal DNA in clinical specimens has been reported recently. In a collaboration among five European centers, the frequency and risk of contamination due to airborne spore inoculation or carryover contamination in fungal PCR were analyzed. The identities of all contaminants were specified by cycle sequencing and GenBank analysis. Twelve of 150 PCR assays that together included over 2,800 samples were found to be contaminated (3.3% of the negative controls were contaminated during the DNA extraction, and 4.7% of the PCR mixtures were contaminated during the amplification process). Contaminants were specified as Aspergillus fumigatus, Saccharomyces cerevisiae, and Acremonium spp. Further analysis showed that commercially available products like zymolyase powder or 10x PCR buffer may contain fungal DNA. In conclusion, the risk of contamination is not higher in fungal PCR assays than in other diagnostic PCR-based assays if general precautions are taken.

Acremonium↗
PubMed1999

Detection of Salmonella contamination in food samples by dot-ELISA, DNA amplification and bacterial culture.

A dot-blot enzyme-linked immunosorbent assay (dot-ELISA) employing a genus Salmonella specific monoclonal antibody (MAb) was used for detection of the bacteria in food samples in comparison with the conventional culture method and the DNA amplification. Among the 200 chicken and pork samples (100 each) tested, 9% and 33%, 7% and 20% and 7 and 23% were positive for salmonellae by the dot-ELISA, the culture method and the DNA amplification, respectively. Statistical analyses revealed that the sensitivity, specificity, efficacy, and positive and negative predictive values of the detection of Salmonella in the food samples by dot-ELISA compared with the culture method were 93.33%, 91.76%, 92%, 66.66% and 98.73%, respectively. Comparison of the DNA amplification and the culture method revealed the sensitivity, specificity, efficacy, and positive and negative predictive values of 100%, 91.58%, 92%, 65.21% and 100%, respectively. The dot-ELISA and the DNA amplification results were in a better agreement when the two assays were compared. The sensitivity, specificity, efficacy, positive and negative predictive values of the dot-ELISA compared to the DNA amplification were 91.3%, 100%, 98%, 100% and 97.5%, respectively. From this study, the dot-ELISA is rapid, simple, sensitive, specific at low cost with limited amount of infectious waste to be disposed and offers another advantage in that it detects only the smooth LPS of Salmonella which implies the possible presence of the virulent organisms.

Animals↗
PubMed1997

[A preliminary study on the identification of crude snake drugs by molecular genetic markers].

DNA was amplified from 11 samples of crude snake drugs by 2 random primers. "Wushaoshe" (Zaocys dhumnades) and its substitutes, Elaphe taeniurus, E. rufodorsata, E. carinata and Sinonatrix annularis, and "Jinqian Baihyashe" (Bungarus multicinctus) and its adulterants, Dinodon rufozonatum and Sinonatrix annularis, can be distinguished by electrophoresis pattern of the amplified products. The results showed that the molecular genetic marker technique can be used as a new method for the identification of crude snake drugs.

Animals↗
PubMed1993

Photochemical inactivation of cell-associated human immunodeficiency virus in platelet concentrates.

Photochemical decontamination (PCD) of platelet concentrates, with adequate preservation of platelet function, has been shown using 8-methoxypsoralen (8-MOP) and long wavelength UV light (UVA). To further evaluate this technique, models for the inactivation of pathogenic human cell-associated viruses and integrated proviral sequences are required. We have assessed the ability of the PCD technique to inactivate cell-associated human immunodeficiency virus 1 (HIV-1) in platelet concentrates. We correlated PCD inhibition of HIV-1 infectivity with 8-MOP-DNA adduct formation in contaminating nucleated cells, and measured the inhibition of polymerase chain reaction (PCR)-mediated amplification of cellular DNA sequences as a surrogate for inactivation of integrated proviral nucleic acid sequences. After PCD treatment (8-MOP 300 micrograms/mL, UVA 17 mW/cm2) for 60 minutes, 0.5 x 10(6) plaque-forming units (PFU)/mL of cell-associated HIV-1 were inactivated and no virus was detectable by infectivity assay. After 60 minutes of PCD, 15 8-MOP-DNA adducts per 1,000 bp were formed, while in the absence of UVA, no adducts were formed. PCR-mediated amplification of a 242-bp cellular DNA sequence (HLA-DQ-alpha) was inhibited when greater than eight psoralen-DNA adducts per 1,000 bp were present. These studies indicate that high titers of cell-associated HIV-1 in platelet concentrates were inactivated by PCD, and the numbers of 8-MOP-DNA adducts in nucleated cells were sufficient to inhibit amplification of DNA segments that encode for as few as 80 amino acids. Based on the frequency of 8-MOP-DNA adducts, for the 10-kb HIV-1 genome, the probability of an integrated genome without at least one 8-MOP adduct after 60 minutes of PCD was 10(-33).

Blood Component Transfusion↗
PubMed2001

Random amplified polymorphic DNA typing applied to the study of cross-contamination by Listeria monocytogenes in processed food products.

The presence of Listeria spp. was investigated in 369 samples of cooked meat products and 52 of smoked salmon. Incidences of 17.6% for cooked meat and 38.5% for smoked salmon samples were found. All Listeria monocytogenes isolates (34 from meat products and 16 from smoked salmon) were typed serologically and by random amplified polymorphic DNA (RAPD) typing using primers HLWL74 (5'-ACGTATCTGC-3'), HLWL85 (5'-ACAACTGCTC-3'), and OMP-01 (5'-GTI'GGTGGCT-3'). Strains from cooked meat products were characterized and compared in relation to their origin. The detection of identical strains in products of different type and brand packed on the same date suggested cross-contamination, probably during the slicing process. All L monocytogenes isolates from smoked salmon were indistinguishable by serotyping and RAPD, suggesting that this strain was highly disseminated and adapted to the treatment used for the preservation of this food. RAPD subtypes were analyzed using GelCompar version 4.1 software and the unweighted pair method using arithmetic averages, and six groups with at least 78% similarity were established. Serotyping and RAPD results were in concordance, although RAPD showed a higher discriminatory power with L. monocytogenes isolates from meat products. RAPD is an easy method that could be useful to detect cross-contamination occurring during postprocessing manipulations.

Animals↗
PubMed2004

Development of a simulation model to assess the impact of contamination in casework using STRs.

Because contamination is usually tube-specific, negative controls cannot give assurance that an associated batch of extracted casework material is contaminant-free. However, it is possible to use them to predict the level of overall (undetected) contamination that is processed by an operational DNA unit. A MATLAB-based program was used to combine results of negative controls with actual casework DNA profiles to assess the probability that laboratory contaminants will give rise to reportable profiles (along with their likelihood ratios). Using data from an operational DNA unit as an example, it was demonstrated that the risk is inextricably linked to guidelines used to interpret DNA profiles. We have demonstrated how computer-based models can predict the levels of contamination expected in the process and, in addition, how the process can be made more robust by changing reporting guidelines. There is a need to compare DNA profiles against staff and plasticware elimination databases in order to determine sources of contamination. The likeliest outcome of a contamination event is false exclusion.

Computer Simulation↗
PubMedDate not supplied

Molecular, forensic and haplotypic inconsistencies regarding the identity of the Ekaterinburg remains.

BACKGROUND: A set of human remains unearthed near Ekaterinburg, Russia has been attributed to the Romanov Imperial Family of Russia and their physician and servants. That conclusion was officially accepted by the Russian government following publication of DNA tests that were widely publicized. The published study included no discussion of major forensic discrepancies and the information regarding the burial site and remains included irregularities. Furthermore, its conclusion of Romanov identity was based on molecular behaviour that indicates contamination rather than endogenous DNA. The published claim to have amplified by PCR a 1223 bp region of degraded DNA in a single segment for nine individuals and then to have obtained sequence of PCR products derived from that segment without cloning indicates that the Ekaterinburg samples were contaminated with non-degraded, high molecular weight, 'fresh' DNA. AIM: Noting major violations of standard forensic practices, factual inconsistencies, and molecular behaviours that invalidate the claimed identity, we attempted to replicate the findings of the original DNA study. SUBJECT: We analysed mtDNA extracted from a sample of the relic of Grand Duchess Elisabeth, sister of Empress Alexandra. RESULTS: Among clones of multiple PCR targets and products, we observed no complete mtDNA haplotype matching that reported for Alexandra. The consensus haplotype of Elisabeth differs from that reported for Alexandra at four sites. CONCLUSION: Considering molecular and forensic inconsistencies, the identity of the Ekaterinburg remains has not been established. Our mtDNA haplotype results for Elisabeth provide yet another line of conflicting evidence regarding the identity of the Ekaterinburg remains.

Bone and Bones↗
PubMed2004

New developments in the diagnosis of bloodstream infections.

New techniques have emerged for the detection of bacteria in blood, because the blood culture as gold standard is slow and insufficiently sensitive when the patient has previously received antibiotics or in the presence of fastidious organisms. DNA-based techniques, hybridisation probes, and PCR-based detection or protein-based detection by mass spectroscopy are aimed at rapid identification of bacteria and provide results within 2 h after the first signal of growth in conventional blood cultures. Also, detection of microorganisms directly in blood by pathogen-specific or broad-range PCR assays (eubacterial or panfungal) shows promising results. Interpretation is complex, however, because of detection of DNA rather than living pathogens, the risk of interfering contamination, the presence of background DNA in blood, and the lack of a gold standard. As these techniques are emerging, clinical value and cost-effectiveness have to be assessed. Nevertheless, molecular assays are expected eventually to replace the current conventional microbiological techniques for detection of bloodstream infections.

Bacteremia↗