Positive Control DNA Research Articles

Malignant Transformation of Human Cells by Constitutive Expression of Platelet-derived Growth Factor-BB

Platelet-derived growth factors (PDGFs) comprise a family of growth factors strongly implicated in human oncogenesis. A number of human tumors overexpress PDGF family members or have translocations activating PDGF receptors. Whereas the epidemiologic evidence implicating PDGF in human tumors is strong, malignant transformation of human cells by overexpression of PDGF has not been demonstrated. We have previously developed a human cell line by the sequential introduction of large T cells and telomerase, and we have demonstrated that these cells express functionally active PDGF receptor (PDGFR) beta. In order to determine whether growth factor-mediated transformation of human cells could occur, these cells were transduced with a retrovirus encoding PDGF-BB. Constitutive expression of PDGF-BB led to malignant transformation in nude mice. This is the first demonstration of constitutive signaling causing malignant transformation of human cells. Some of the changes that occur because of constitutive growth factor expression can be reversed by the clinically approved tyrosine kinase inhibitor Glivec, whereas other changes are not reversible by tyrosine kinase inhibitors. Our model allows the assessment of epigenetic changes that occur during human carcinogenesis. In addition, these studies provide insight into the clinical failure of tyrosine kinase inhibitors as monotherapy for advanced malignancy.

Development of a novel approach for the production of dried genomic DNA for use as standards for qualitative PCR testing of food-borne pathogens

As part of a multi-centre European project, FOOD-PCR, the feasibility of a novel approach for production of dried bacterial DNA that could be used as certified reference materials (CRM) was assessed. Selected strains of Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157, Campylobacter jejuni and Yersinia enterocolitica were used to produce genomic DNA (gDNA). These preparations gave support to method development for qualitative polymerase chain reaction (PCR) detection methods for food-borne pathogens. Purified gDNA was transformed into stable and dry gDNA by using polypropylene vials as carrier and applying a vacuum-drying technique. The gDNA preparations were shown to be sufficiently stable under ambient transport conditions without cooling and proved to have long-term stability at 5°C of at least 22 months. The dried DNA was easily reconstituted by addition of distilled water then gentle shaking. These studies have shown that production of stable and dry bacterial gDNA material is feasible and could help satisfy the increasing need for certified reference DNA positive control samples in the field of PCR testing for detection and verification of food-borne microbial pathogens.

In vivo transgenic mutation assays

Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of ∼3×10 −5 mutants/locus and 125,000–300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose–response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when scientifically justified. Normally male animals alone are sufficient and normally at least one rapidly proliferating and one slowly proliferating tissue should be sampled. Although, as agreed previously, sequencing data are not normally required, they might provide useful additional information in specific circumstances, mainly to identify and correct for clonal expansion and in some cases to determine a mechanism associated with a positive response.

Precision studies using the ABI Prism�3100 Genetic Analyzer for forensic DNA analysis

Precision studies using the ABI Prism 3100 Genetic Analyzer have been conducted by performing multiple runs of the AmpFlSTR Identifiler allelic ladder, 9947A kit positive control DNA and the 250-base-pair fragment from the internal size standard (GeneScan LIZ-500). Intra-run and inter-run precision data demonstrates the sizing reproducibility of the 3100 instrument for forensic applications. This precision data is utilized to assist in making allele assignments for outlier and variant alleles (i.e., 'off-ladder' alleles). It also provides a means of addressing systematic variations due to temperature fluctuations, polymer (POP-4) or capillary effects, and associated troubleshooting efforts.

Report of Bacterial Leaf Spot on Collards and Turnip Leaves in Ohio.

In 2000, circular water-soaked lesions typical of bacterial leaf spot were observed on leaves of collards (Brassica oleracea L. var. viridis) throughout commercial fields in northwest Ohio. Light brown, rectangular, water-soaked lesions were observed on turnip leaves (Brassica rapa L.). Bacterial streaming from lesions on both crops was observed microscopically. Cream colored, fluorescent colonies were isolated from diseased tissues on Pseudomonas F medium, and eight representative colonies (four from collards and four from turnip) were selected and purified. Fatty acid methyl ester analysis was performed on all of the isolates. Two from collards and two from turnip were identified as Pseudomonas syringae pv. maculicola (mean similarity index = 0.82 [MIDI Inc., Newark, DE]). DNA extracts from pure cultures of the P. syringae pv. maculicola strains were used as template in a polymerase chain reaction (PCR) assay with primers derived from the region of the coronatine gene cluster controlling synthesis of the coronafacic acid moiety found in P. syringae pv. tomato and P. syringae pv. maculicola (CorR and CorF2) (D. Cuppels, personal communication). DNA from P. syringae pv. tomato strain DC3000 and P. syringae pv. maculicola strain 88-10 (2) served as positive controls, while water and DNA from Xanthomonas campestris pv. vesicatoria strain Xcv 767 were used as negative controls. The expected 0.65-kb PCR product was amplified from three of four strains (two from turnip and one from collards) and the positive control DNA, but not from the negative controls. Pathogenicity tests were performed twice on 6-week-old turnip ('Forage Star', 'Turnip Topper', 'Turnip Alamo', 'Turnip 7'), collard ('Champion') and mustard (Brassica juncea L. 'Southern Giant Curl') seedlings using the three PCR-positive strains. Premisted seedlings were spray-inoculated separately with each of the three strains (2 × 108 CFU/ml, 5 ml per plant) and a water control. Greenhouse temperatures were maintained at 20 ± 1°C. For both tests, all strains caused characteristic lesions on all of the crucifer cultivars within 5 days after inoculation; the control plants did not develop symptoms. To satisfy Koch's postulates, one of the turnip strains was reisolated from 'Turnip Topper' plants, and the collard strain was reisolated from 'Champion' plants. The three original and two reisolated strains induced a hypersensitive response in Mirabilis jalapa L. and Nicotiana tabacum L. var. xanthia plants 24 h after inoculation with a bacterial suspension (1 × 108 CFU/ml). The original and reisolated strains were compared using rep-PCR with the primer BOXA1R (1). The DNA fingerprints of the reisolated strains were identical to those of the original strains. To our knowledge, this is the first report of bacterial leaf spot on commercially grown collards and turnip greens in Ohio.

Semiautomated quantification of hepatitis B virus DNA in a routine diagnostic laboratory.

The Cobas Amplicor HBV Monitor test for quantitative determination of hepatitis B virus (HBV) DNA in serum has recently been introduced. To evaluate the performance of this assay in a routine diagnostic laboratory, reproducibility of results was determined with the First European Union Concerted Action HBV Proficiency Panel and the Accurun 325 HBV DNA Positive Control, Series 300. Results for 270 routine serum samples were additionally evaluated. To avoid the retesting of a large number of samples due to titers exceeding the upper limit for the linear range of the assay, sera of patients with chronic hepatitis B (CHB) were diluted prior to the assay to 10(-4) in normal human plasma, which is included in the assay. The mean coefficient of variation was 22.9% for all input HBV DNAs. Of 270 routine serum samples, 182 (150 sera from transplant donors and 32 sera from patients who had recovered from CHB) tested negative. Eighty-six sera were found to be HBV DNA positive; in six sera, HBV DNA levels were found to exceed the upper limit for the linear range of the assay and had to be retested. In the remaining two sera, inhibition occurred. The semiautomated Cobas Amplicor HBV Monitor test showed sufficient reproducibility and helped in avoiding human error. The relatively narrow linear range of detection is a limitation of the new assay.

First Report of Bacterial Canker of Pepper in Ohio.

Light brown, raised lesions were observed on the leaves of bell pepper (Capsicum annuum L.) plants throughout a commercial field in northwest Ohio in 1999. Bacterial streaming from the lesions was observed microscopically. Five representative, pale yellow colonies isolated on yeast dextrose carbonate medium were selected and purified. All isolates induced a hypersensitive response in Mirabilis jalapa L. plants 24 h after inoculation with a 1 × 108 CFU/ml bacterial suspension. All five were identified as Clavibacter michiganensis subsp. michiganensis by fatty acid methyl ester analysis (mean similarity index [S.I.] = 0.76; MIDI, Newark, DE). Identity was confirmed at the University of Hawaii (W. Kaneshiro and A. Alvarez) by carbon substrate utilization pattern (mean S.I. = 0.75; Biolog, Hayward, CA) and positive reactions with C. michiganensis subsp. michiganensis-specific monoclonal antibodies (clones 103-142-1-1 and 103-148-2-1) in ELISA. DNA extracted from lesions and pure cultures was used as template in a polymerase chain reaction (PCR) assay with primers specific for C. michiganensis subsp. michiganensis (CMM-5 and CMM-6) (1). DNA from a known strain of C. michiganensis subsp. michiganensis served as a positive control, while water and DNA from healthy tomato plants were used as negative controls. A 0.6-kb PCR product was amplified from lesions, pure cultures of all five strains, and positive control DNA, but not from the negative controls. Pathogenicity tests were performed twice on 5- to 6-week-old bell pepper (cvs. Collossal, Lafayette, King Arthur, Brigadier, and Commandant) and tomato (cv. Peto 696) plants. Pepper plants were inoculated with each strain by clipping the lowest petioles with scissors that had been dipped into a bacterial suspension (1 × 108 CFU/ml) or by spray inoculation (approximately 5 ml/plant). Tomato plants were inoculated by clipping. Both inoculation methods included a water control. All five strains caused water-soaked lesions on leaves of all pepper varieties within 7 days after spray inoculation. Pepper plants inoculated by clipping did not develop symptoms. DNA extracts from lesions of challenged pepper plants were positive in PCR. All inoculated tomato seedlings exhibited wilting, streaks, and cankers in the stems and necrosis of leaf margins within 15 days after inoculation. None of the control plants developed symptoms. All five strains were re-isolated from inoculated tomato and pepper plants. The original pepper strains and the strains re-isolated from tomatoes were compared using rep-PCR with ERIC primers (4). DNA fingerprints of the re-isolated strains were identical to those of the original strains and were characteristic of C. michiganensis subsp. michiganensis type C. Bacterial canker is a common disease of tomatoes worldwide and has occurred in Ohio for at least 70 years. However, this is the first report of C. michiganensis subsp. michiganensis infecting peppers in Ohio. While the pathogen does not appear to cause systemic disease in peppers, it may serve as a source of inoculum for tomatoes, which are highly susceptible to the disease and often produced in the same greenhouse as peppers or planted in adjacent fields. Bacterial canker has been reported previously from commercial pepper fields in California (2) and Indiana (3).

Lack of evidence for tamoxifen- and toremifene-DNA adducts in lymphocytes of treated patients

Tamoxifen (TAM) is used for the adjuvant treatment of women with breast cancer and has also been recommended as a chemopreventive agent. Among unwanted side effects, TAM was shown to increase endometrial cancer in treated women by mechanisms that are not yet clearly understood. We studied DNA adducts in lymphocytes of female breast cancer patients treated with TAM or toremifene (TOR), a TAM analogue and compared them with adducts formed by TAM in rat liver, where the drug induces tumours. DNA adducts were measured by TLC-(32)P-post-labelling assays. After TLC, all DNA samples including DNA from untreated healthy women showed a faint radioactive zone, where the positive control DNA adducts isolated from the liver of rats treated with TAM migrated. The relative adduct levels were calculated from the radioactivity present in this zone. Means +/- SD of adduct levels per 10(8) nucleotides (associated with this area) were for untreated volunteers (control) 1.83 +/- 1.41 (n = 13), for TAM treatment 2.17 +/- 3.04 (n = 25) and for TOR treatment 1.18 +/- 1.05 (n = 8). Most of the human samples were further analysed by HPLC after labelling with (32)P in order to compare adducts in human DNA with those in liver DNA isolated from TAM-treated rats. None of the human samples showed any peaks at retention times where putative TAM-DNA adducts were eluted. In conclusion, lymphocyte DNA from female patients treated at therapeutic levels did not show evidence of the formation of TAM- or TOR-DNA adducts.

In vivo transgenic mutation assays.

Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage-based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3 x 10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two-thirds the MTD, and one-third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control.

PCR amplification and cycle DNA sequencing analysis of the Chlamydia trachomatis elongation factor Tu gene

Based on the elongation factor Tu (EF-Tu) gene of Chlamydia trachomatis, a pair of oligonucleotide primers CTUFU and CTUFD, were designed to amplify a specific target fragment of 931bp. The PCR assay could detect C. trachomatis in cervical smear specimens obtained from sex workers undergoing routine examination in an STD clinic. Distinct target bands were also amplified from at least 10ng of positive control DNA samples from cultured cells infected with C. trachomatis. PCR with these primers could differentiate C. trachomatis from eight non-chlamydial bacterial species. Further verification could be obtained from the non-digestion of C. trachomatis PCR products by MspA1I restriction endonuclease, in contrast to the digestion of the non-specific PCR products of Klebsiella and Bacillus. Direct cycle DNA sequencing of ∼450bp of the PCR products of four C. trachomatis isolates revealed complete identity of one isolate with the known sequence of serovar F, while the other three isolates harboured three phenotypically silent point mutations at codons 96, 305 and 312 of the EF-Tu gene. The sequence analyses confirm the authenticity of the target bands, reiterate the conservation and role of the EF-Tu gene in protein biosynthesis, and indicate the utility of the primers for the rapid detection of C. trachomatis.

Evaluation of Amplicor MTB test as adjunct to smears and culture for direct detection of Mycobacterium tuberculosis in the French Caribbean

A total of 784 specimens collected from 370 individuals between January and August 1995 were analyzed by using the Amplicor Mycobacterium tuberculosis test (Roche Diagnostic System, Basel, Switzerland), a PCR-based test for the direct detection of organisms of the M. tuberculosis complex. The PCR results were compared with standard bacteriological data, including those obtained by acid-fast microscopy, culture, and biochemical identification as well as final clinical diagnosis for each patient. Several parallel controls were used: the kit DNA positive control, 10(3) CFU of M. tuberculosis, and three negative controls for each independent assay. No false-positive PCR results were obtained, and overall, M. tuberculosis was detected in 20 of 370 individuals screened. Five additional patients during the same time were found to be infected with mycobacteria other than tubercle bacilli; their specimens gave positive smear and/or culture test results, but Amplicor tests were always negative. The sensitivity, specificity, positive predictive value, and negative predictive value for the Amplicor MTB test compared with culture per specimen were 76.7, 97.7, 66.0, and 98.6%, respectively. For resolved cases, these values were, respectively, 69.4, 100, 100, and 96.8%; however, the sensitivity and negative predictive value increased to 90.9 and 99.2%, respectively, if PCR-negative nonrespiratory specimens (gastric washings) were not considered. When only specimens from proven tuberculosis patients were considered (n = 114) and the sum of PCR-positive and/or culture-positive samples from proven tuberculosis patients was considered the total number of positive samples, PCR had a sensitivity of 83.3% compared with 71.6% for culture. Results per patient (about three samples each) yielded 100% sensitivity and 100% specificity. We conclude that the Amplicor MTB test is highly specific and rapid for routine use in a clinical laboratory. However, in order to obtain a higher degree of sensitivity, it should be run as an adjunct to smears and culture with at least three samples for each patient, and a single-sample PCR-negative results must be considered carefully because of potential false-negatives.