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PubMed2005

Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR.

The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P<0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The Master Pure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA, including DNA from Aspergillus and Candida species. In conclusion, the six extraction methods produce markedly differing yields of fungal DNA and thus can significantly affect the results of fungal PCR assays. No single extraction method was optimal for all organisms.

Aspergillosis↗
PubMed1993

Purification of forensic specimens for the polymerase chain reaction (PCR) analysis.

Purification methods of deoxyribonucleic acid (DNA) from degraded and contaminated forensic samples were investigated for polymerase chain reaction (PCR) analysis. DNA extracted from putrefied tissue or bloodstains sometimes contained the copurified contaminant, that was identified as the porphyrin compound (hematin). When contaminated but less degraded DNA was analyzed by PCR, it was necessary to eliminate the impurity by anion exchange column chromatography or chelating resin preparation, and ultrafiltration using Centricon microconcentrators. When highly degraded DNA was analyzed, trace amounts of high molecular weight DNA was recovered by electroelution method, and then further purified by both column chromatography and ultrafiltration. From thus purified samples, the amelogenin gene for sex determination could be amplified by dual PCR technique.

Adipose Tissue↗
PubMed1996

[Personal identification using DNA polymorphism--the identification of forensic biological materials].

We performed personal identification by DNA polymorphism using forensic specimens. DNA was purified by the potassium iodine method instead of the phenol extraction method. When DNA is amplified by PCR, there are inhibitors of PCR such as melanin in hairs or blood and inorganic salts in bone. This problem was solved by the potassium iodine method. The genotype of the ABO blood groups could be determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in all 29 old blood-stain specimens (obtained 11 months-15 years before) and 14 fingerprint specimens on a glass plate. In sexual assaults against women, one key to identifying the suspect is ABO phenotyping or the typing of other polymorphic markers of the seminal fluid in the victim's vagina. However, ABO phenotyping is frequently unsuccessful, since mixtures of fluids cannot be separated to be subjected to conventional methods for the detection of antibody or antigen material. We therefore studied ABO blood group genotyping of sperm DNA isolated from contaminating vaginal fluid by the PCR-RFLP method. Seminal samples of genotypes OO, AO, BO and AB were experimentally mixed with vaginal fluid (OO, AO, BO and AB), and were successfully separated and genotyped by this method. In practice, we also separated and genotyped the seminal DNA of suspects from contaminated postcoital vaginal fluid obtained in 4 sexual assaults. These forensic samples were easily separated and completely genotyped. This reliable ABO genotyping method by PCR-RFLP, using separated sperm DNA, should be of value in forensic identification in sexual assaults. We attempted sex and individual identifications of several forensic specimens by detecting various sex chromosome-specific sequences by the polymerase chain reaction (PCR) method. The specimens were 30 blood stains that were attached to gauze and stored 11 months-15 years (15 males, 14 females and 1 of unknown gender) and 10 bleached white bones (8-about 10000 years after death). Of the known 29 blood stains, the sex determination rate was the highest by DYZ-1 (amplified DNA fragment = 149bp, about 3000 copies on Y chromosome) recombinated with DXZ-1 (28 of the 29 samples) although 27 samples could be determined by DYZ-1 (amplified DNA fragment = 1000bp, about 3000 copies on Y chromosome) recombinated with DXZ-1. The sex determination rate was relatively low by DYZ-3 (amplified DNA fragment = 170bp, about 100 copies on Y chromosome) recombinated with DXZ-1 (23 of the 29 samples). The sex determination rate by two single locus markers, PAB or Amelogenin was markedly low (7 and 9 of 29 samples, respectively). The sex determination rate by the 27H39 locus was 27 of the 29 samples; 14 of the 15 male were determined (A = 186bp, 1; B = 190bp, 7; C = 194bp, 5; D = 198bp, 1; E = 202bp, 0). One unknown gender could be identified as male by multiple locus markers, but could not be identified by any single locus markers except for the 27H39 locus. Of the 10 bleached white bone specimens, 9 could be determined by the combination of DYZ-1 and DXZ-1, but the results of determination were inconsistent with the anthropological characteristics in 3 cases. The 27H39 locus showed polymorphism in 4 of the 6 anthropologically male beached bones. Studies on a new microsatellite on the Y chromosome are needed for evaluation of polymorphism on the Y chromosome because it is not necessary to consider the alleles from the female in the paternity test in male children or female-derived elements in sexual delict, and there is a distance between the alleles using 27H39 alone. In this study, We evaluated the allele distribution of DYS384, DYS388, DYS389, DYS390, DYS391, DYS392, and DYS393, in Japanese subjects and found the usefulness of DYS384 and DYS390. Future studies on personal identification by DNA polymorephism will mainly evaluate short tandem repeat (STR).

ABO Blood-Group System↗
PubMed1991

Amplification and analysis of Miocene plant fossil DNA.

Ancient DNA has been extracted and sequenced from several animal and plant specimens. Previous considerations of the damage to ancient DNA have suggested that both the age and size of DNA fragments that can be retrieved and sequenced may be limited, the former to between several thousand and at most tens of thousands of years old, and the latter to at most a few hundred bases. A recent report of a 770 base pair (b.p.) sequence from the chloroplast gene rbcL from a Miocene Magnolia latahensis leaf indicates that both estimated limitations may be too conservative. Further work has indicated that analysis of Miocene fossil DNA can be replicated, and can, therefore, open up the prospects for future development of the field of molecular palaeontology. Successful amplification of fossil DNA is sometimes confounded by factors inherent to fossil DNA or to samples with minimal amounts of target DNA. Techniques that alter denaturation, reduce inhibitors and the problem of contaminants, and repair DNA prior to polymerase chain reaction amplification can increase the probability of success.

Base Sequence↗
PubMed1996

Evidence against a role for human T-cell lymphotrophic virus type I (HTLV-I) in the pathogenesis of American cutaneous T-cell lymphoma.

We used a standard polymerase chain reaction (PCR)/Southern blot assay (sensitivity > 10(-5)) to detect human T-cell lymphotrophic virus type I (HTLV-I) proviral pX, pol, and env genes in the lesional skin of 42 American patients with cutaneous T-cell lymphoma (CTCL). As in some prior reports using similar methods, a variable proportion of PCR tests were positive (seven of 42 for pX, three of 42 for pol, and two of 37 for env), resulting in an overall positive test rate of 12 of 121 (10%). To determine the significance of these positive test results, we performed several additional studies. D1S80 polymorphism analysis of CTCL cases and HTLV-I PCR analysis of non-CTCL dermatosis controls showed no evidence that positive PCR tests resulted from sample mislabeling, gross HTLV-I contamination, or human endogenous retroviruses. We then modified the standard PCR assay to incorporate ultraviolet (UV) light to destroy low-level PCR contamination. With this modified assay (sensitivity > 10(-5)), only three of 12 previously positive cases were still positive, suggesting that the earlier positives were due to trace contamination of PCR reagents or trace contamination of sample DNA. This interpretation was also supported by: (i) a match between pX and pol sequences cloned from one PCR-positive specimen and the MT4-positive control, (ii) our inability to confirm HTLV-I in any PCR-positive case using genomic dot blotting (sensitivity > 10(-2)), and (iii) negative PCR results when new samples from two of the remaining positive cases were analyzed. Finally, we used our modified UV/ PCR/Southern blot assay to test an additional 28 cases of American CTCL for pX. All of them were negative. Although these studies of 70 cases of American CTCL do not exclude the possibility that another virus is involved in the pathogenesis of this disease, they provide strong evidence against a role for HTLV-I. Furthermore, they emphasize the need for special strategies to control for false-positive PCR tests that can result from even trace levels of contamination with viral DNA. As a consequence, associations between diseases and viruses should be viewed skeptically if they are based primarily on conventional PCR data.

Base Sequence↗
PubMed1996

ABO genotyping of suspects from sperm DNA isolated from postcoital samples in sex crimes.

In sexual assaults against women, one key to identifying the suspect is ABO phenotyping or the typing of other polymorphic markers of the seminal fluid in the victim's vagina. However, ABO phenotyping is frequently unsuccessful, since mixtures of fluids cannot be separated to be subjected to conventional methods for the detection of antibody or antigen material. We therefore studied ABO blood group genotyping of sperm DNA isolated from contaminating vaginal fluid by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Seminal samples of genotypes OO, AO, BO and AB were experimentally mixed with vaginal fluid (OO, AO, BO and AB), and were successfully separated and genotyped by this method. In practice, we also separated and genotyped the seminal DNA of suspects from contaminated postcoital vaginal fluid obtained in 4 sexual assaults. These forensic samples were easily separated and completely genotyped. This reliable ABO genotyping method by PCR-RFLP, using separated sperm DNA, should be of value in forensic identification in sexual assaults.

ABO Blood-Group System↗
PubMed1985

Method for the preparation of plasmid DNA suitable for physicochemical measurements.

A method has been developed for the isolation of plasmid DNA suitable for physical and physicochemical measurements. The procedure is based on the deproteinization of the cleared lysate of bacterial cells (after amplification of plasmids by chloramphenicol) by phenol at pH 8.0 and subsequent removal of chromosomal DNA by means of phenol at pH 4.0 and separation of RNA on a hydroxyapatite column at higher temperature. ColE1 DNA sample was compared with samples of the same DNA prepared by three thus far used methods. Samples obtained by means of the latter methods were contaminated with chromosomal DNA, RNA, or ethidium bromide. The presence of ethidium bromide in the DNA sample was a factor interfering in the electrochemical analysis, chromosomal DNA and RNA were disturbing in the use of other methods. DNA separated by the method devised by us was free of any detectable contaminants and fulfilled the high requirements for sample purity of differential pulse polarography. Measurements performed by means of differential pulse polarography showed that the content of single-stranded segments in superhelical ColE1 DNA is less than 0.15% (i.e. less than 20 bases per molecule). This is in keeping with the notion that a cruciform is formed in this DNA (as a result of tension due to supercoiling) in the region of inverted repeat sequence, containing only 5 bases in the single-stranded loop region.

Animals↗
PubMed1976

Detection of anti-DNA antibody using synthetic antigens. Characterization and clinical significance of binding of poly (deoxyadenylate-deoxythymidylate) by serum.

Virtually all preparations of DNA used to detect antibody to native DNA (nDNA) by binding assays have been found to be subtly contaminated by single stranded DNA. Because recent DNA binding data have directly challenged the unique role previously attributed to these antibodies in systemic lupus erythematosus (SLE), resolution of the consequent ambiguity is of theoretical and practical importance. It is proposed that a synthetic nDNA molecule (dAT) might circumvent this difficulty by being antigenically equivalent to nDNA while, on theoretical grounds, lacking significant contamination with single stranded DNA or other cellular antigens. These expectations were generally confirmed by biochemical and immunological analyses. In clinical studies, sera from 124 pateints with SLE and from controls were examined for their ability to bind dAT. In contrast to results with KB binding, patients with non-SLE rheumatologic disorders were indistinguishable from normals by dAT binding. dAT binding was elevated in 85% of sera from SLE patients with clinically-judged active nephritis but in only 9% of those with inactive renal disease. Active non-renal disease, including cerebritis, was not associated with increased dAT binding. Individual non-lupus sera which bound increased amounts of KB DNA, failed to bind dAT. It is suggested that such binding resulted from contaminating non-nDNA antigens. When elevated, dAT binding, like KB binding, varied with disease activity and might thus be useful as a parameter thereof. In several patients elevated dAT binding led to the finding, on biopsy, of clinically silent, active, diffuse proliferative nephritis. It is concluded that use of synthetic nDNA antigens such as dAT may offer theoretical and practical advantages over naturally-derived preparations in detecting anti-nDNA, both clinically and for investigational purposes. Also, caution is urged in interpreting DNA binding data derived from incompletely characterized systems, particularly with regard to the occurrence of anti-nDNA antibodies in serum.

Adult↗
PubMed2004

Contamination of coagulation factor concentrates with human parvovirus B19 genotype 1 and 2.

Human parvovirus B19 (B19) DNA has frequently been detected in plasma-derived coagulation factor concentrates. Furthermore, transmission of B19 infection was observed, indicating presence of the infectious virus despite routine viral inactivation/removal procedures during the manufacturing process. Recently, human parvovirus DNA isolates, variant from B19, have been identified resulting in classification of B19 virus into three distinct genotypes, with all viruses previously classified as B19 belonging to genotype 1. So far, there is no information available on contamination of clotting factor concentrates with genotype 2. Therefore, we analysed 202 different factor concentrate lots for genotype 1 and 2 DNA by PCR. Analysis of one hundred eighty-one lots representing 13 different products, administered over the last three years, was compared to 21 lots (8 products) used until the early 1980s which had not been treated by viral inactivation procedures. Genotype 1 DNA was detected in 77/181 (42.5%) currently administered lots, and 17/21 (81%) previously used lots. The level of genotype 1 DNA contamination was similar in currently and previously administered concentrates. Genotype 2 DNA was found in 5/202 (2.5%) lots, all of which were co-contaminated with genotype 1 DNA. DNA sequence analysis showed that the PCR-double positive concentrates contained typical genotype 1 and genotype 2 DNA. Because genotype 2 appears to cause a similar spectrum of diseases as genotype 1, simultaneous detection of genotype 2 by nucleic acid amplification testing (NAT), now widely applied to plasma pools for genotype 1, would give an added level of safety to blood products.

Blood Coagulation Factors↗
PubMed2006

Isolation of sperm vesicles from adult male mayflies and other insects to prepare high molecular weight genomic DNA samples.

We describe here a simple and efficient protocol for genomic DNA isolation from adult males of insects: e.g., Ephemeroptera, Odonata, Orthoptera and Dictyoptera. To minimize contamination of external DNA source, the sperm vesicles were isolated from male individuals from which high molecular weight genomic DNA was extracted. According to this protocol, the genomic DNA samples obtained were high quality (intact), and abundant enough for genotyping analyses and molecular cloning. The protocol reported here enables us to process a huge number of individuals at a time with escaping from cross-contamination, and thus it is quite useful for conducting genetic studies at least in some species of insects. The large yield of high molecular weight DNA from single individual may be advantageous for non PCR-based experiments. As a case study of the protocol, partial coding sequences of histone H3 and EF-1alpha genes are determined for some insects with PCR-amplified DNA fragments.

Aging↗
PubMed1991

Reduction of vector contamination in detection of human papillomavirus DNA using full-length genomic DNA probes.

Nucleic acid hybridization techniques are currently the most specific and sensitive procedures available for diagnosing and typing human papillomavirus (HPV) infection. HPV genomic DNA cloned into vector pBR322 or related vectors are commonly used as probes for detection of HPV. When isolating HPV insert DNA however, it is difficult to remove all pBR322 DNA. This can result in false positive readings when clinical specimens harbouring sequences homologous to pBR322 are screened. It was found that up to 200 ng of vector-like sequences in a clinical sample could be blocked by the addition of non-labelled, digested pBR322 sequences to the hybridization reaction.

Animals↗
PubMed1994

Design and use of signature primers to detect carry-over of amplified material.

Signature primer pairs designed for use with the polymerase chain reaction have been developed which can determine if a positive result originated from the intended target nucleic acid or from so-called "carry-over" contamination of previously amplified DNA. The 3' ends of each signature primer, SK339/341, SSK110/111, and SSK58/59 contain a viral specific sequence complementary to regions of either HIV-1, HTLV-I and II respectively. The 5' ends of each primer contain a non-human, non-viral (NHNV) signature sequence including restriction endonuclease sites for subsequent cloning. A fourth set of primers, SK338/340, consist solely of these NHNV sequences and are designed to anneal to any product previously amplified by the viral-specific signature primers. These primers were tested against their corresponding positive and negative DNA targets, to determine their specificity and sensitivity. As expected, the viral-specific signature primers detected the retroviral infected samples while no detectable amplification occurred in negative DNA controls. Primers SK338/340 did not amplify any viral positive or negative template DNA's. Samples spiked with amplified material generated from the viral-specific signature primers could be specifically amplified by the NHNV primers SK338/340. Primers SK338/340 were determined to be more sensitive than the viral-specific signature primers, ensuring the detection of extremely low amounts of carryover. This strategy may be useful in developing other retroviral or non-retroviral primers with a built-in signature sequence that can differentiate false positives from true positives in a subsequent confirmatory test.

Artifacts↗
PubMed1998

Discordant detection of human cytomegalovirus DNA from peripheral blood mononuclear cells, granulocytes and plasma: correlation to viremia and HCMV infection.

BACKGROUND: There exist only few data about the HCMV infection of single positive leukocyte subtypes in immunosuppressed patients. Most reports describe HCMV coinfection of cells of the myelomonocytic line or even T- and B-cell populations. Correlation of positive PCR findings from two major leukocyte fractions and plasma to viremia and HCMV infection in general should contribute to select suitable sources of HCMV DNA for diagnostic purposes. OBJECTIVE: The diagnostic value of qualitative leukoDNAemia of simultaneously isolated peripheral blood mononuclear cells (PBMC), granulocytes as well as plasmaDNAemia was evaluated by comparing the positive results of nested PCR from blood with virus isolation either from leukocytes or from any other sources, with serology and the clinical status of immunosuppressed patients. STUDY DESIGN: PBMC, granulocytes and plasma were prepared of a total of 220 blood samples of 75 immunosuppressed patients with clinically suspected primary or recurrent HCMV infection. In a collective of 35 patients consisting mainly of recipients of marrow or solid organ transplants positive results of leuko- or plasmaDNAemia were correlated with data from HCMV screening and the clinical status. For standardization, HCMV IE Exon 4 DNA was amplified from 100 ng cellular DNA of each leukocyte population. Cross contamination can be excluded. DNA from plasma was extracted by phenol/chloroform. Using this experimental design, HCMV DNA was not detectable in PBMC, granulocytes and plasma of 23 healthy HCMV seropositive blood donors. RESULTS: Leukocyte separation in a collective of 30 patients with positive leukoDNAemia revealed in only 12 cases (40%) double infection of PBMC and granulocytes. In the majority of cases (18 patients, 60%) however, HCMV DNA was detectable in only one leukocyte fraction, either in PBMC or granulocytes. LeukoDNAemia did not correlate to viremia. HCMV DNA amplified from plasma was shown to be cell free. Infectious virus from plasma was not isolated. The predictive value of qualitative nested PCR from blood to detect HCMV infection was high for plasma and decreased in the following sequence: plasma (0.92) > PBMC (0.83) > granulocytes (0.65). CONCLUSIONS: Qualitative nPCR from plasma and PBMC seems to be sufficient to detect (an ongoing) HCMV infection of immunosuppressed patients. However, the rate of single positive leukocyte fractions is approximately 60%. Therefore, viral leukoDNAemia in 40% of cases seems to be restricted to either PBMC or granulocytes. For diagnostic purposes the whole leukocyte population should be used for PCR analysis.

Bone Marrow Transplantation↗