Genus-specific Assay Research Articles

Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC)

In the present study, we developed a new real-time PCR system based on the cycling probe technology (CPT), which is composed of two single tube real-time PCR assays: the Fusarium genus-specific assay and the Fusarium solani species complex (FSSC)-specific assay with primers targeting the 28s ribosomal RNA gene. The Fusarium genus-specific assay was shown to be highly specific, detecting all reference Fusarium strains with no cross-reaction with other reference fungal strains, such as Aspergillus spp. and human DNA. The FSSC-specific assay also reacted very specifically with FSSC, except for a cross-reaction with Fusarium lunatum. To validate the real-time PCR system, we tested 87 clinical isolates of Fusarium spp. Identification results from the real-time PCR system were found to be 100% concordant with those from DNA sequencing of EF-1α gene. The sensitivity testing also demonstrated high sensitivity, enabling detection of one copy of standard DNA with good reproducibility. Furthermore, both assays were shown to be extremely sensitive even when fungal cells were mixed with human cells, detecting 3 germinated conidia spiked in 3mL of human blood. To apply our new real-time PCR system to the molecular diagnosis of fusariosis, we evaluated its efficacy using a mouse model of invasive F. solani infection. Plasma and whole blood samples of infected mice were tested using the real-time PCR system. The sensitivity of the real-time PCR system was found to be 100% (n=4) in plasma samples. In contrast, no amplification signal was detected in whole blood samples. This system could provide a rapid and precise diagnostic tool for early diagnosis, which is necessary for appropriate treatment and improvement of prognosis of disseminated fusariosis.

Characterization of the protozoan parasite Marteilia refringens infecting the dwarf oyster Ostrea stentina in Tunisia

Marteilia refringens is a protozoan parasite recognized as a significant pathogen of the European flat oyster Ostrea edulis. The life cycle of this species is still poorly known, although there is evidence of the need for intermediate host(s). In the present study, we have used molecular approaches to identify this parasite in samples of the dwarf oyster Ostrea stentina after reports of massive mortality along the Tunisian coasts. In 2009 we evaluated the status of O. stentina from Monastir and checked if there was an infection with M. refringens, using polymerase chain reaction assays. Of the 103 tested O. stentina, 85 were PCR-positive using a Marteilia genus-specific assay. Additional assays were subsequently carried out on some samples collected in 2010 in Monastir and processed for histology, transmission electron microscopy and complementary molecular analyses. PCR was carried out to amplify the IGS and ITS regions. Histological and transmission electron microscopy analyses allowed us to confirm the presence of this parasite in the digestive gland tissue of O. stentina and to characterize it at the ultrastructural level. This is the first record of the occurrence of M. refringens in the oyster O. stentina along the Tunisian coasts.

Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

The detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium, although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water.

Integrated analysis of microRNA expression and mRNA transcriptome in lungs of avian influenza virus infected broilers

BackgroundAvian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. Our previous study suggested microRNAs (miRNAs) play significant roles in the regulation of host response to AIV infection in layer chickens. The objective of this study was to test the hypothesis if genetic background play essential role in the miRNA regulation of AIV infection in chickens and if miRNAs that were differentially expressed in layer with AIV infection would be modulated the same way in broiler chickens. Furthermore, by integrating with parallel mRNA expression profiling, potential molecular mechanisms of host response to AIV infection can be further exploited.ResultsTotal RNA isolated from the lungs of non-infected and low pathogenic H5N3 infected broilers at four days post-infection were used for both miRNA deep sequencing and mRNA microarray analyses. A total of 2.6 M and 3.3 M filtered high quality reads were obtained from infected and non-infected chickens by Solexa GA-I Sequencer, respectively. A total of 271 miRNAs in miRBase 16.0 were identified and one potential novel miRNA was discovered. There were 121 miRNAs differentially expressed at the 5% false discovery rate by Fisher’s exact test. More miRNAs were highly expressed in infected lungs (108) than in non-infected lungs (13), which was opposite to the findings in layer chickens. This result suggested that a different regulatory mechanism of host response to AIV infection mediated by miRNAs might exist in broiler chickens. Analysis using the chicken 44 K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection.ConclusionsA comprehensive analysis combining both miRNA and targeted mRNA gene expression suggests that gga-miR-34a, 122–1, 122–2, 146a, 155, 206, 1719, 1594, 1599 and 451, and MX1, IL-8, IRF-7, TNFRS19 are strong candidate miRNAs or genes involved in regulating the host response to AIV infection in the lungs of broiler chickens. Further miRNA or gene specific knock-down assay is warranted to elucidate underlying mechanism of AIV infection regulation in the chicken.

Abstract 3940: Determining the mitochondrial DNA repair capacity in peripheral blood mononuclear cells using QPCR

Abstract Mutations in mitochondrial DNA (mtDNA) have been described in many cancer types. The functional significance of these mutations is not totally understood, but it has been suggested that mtDNA mutations may lead to increased ROS production, aerobic glycolysis and growth promotion in cancer cells. Increased mutagenesis in mtDNA may be due to decreased mtDNA repair capacity. However, there are a limited number of methods to specifically determine mtDNA repair capacity. The goal of this work is to apply a gene-specific PCR-based assay that can detect the presence of a variety of lesions in the mtDNA as a tool to determine the mtDNA repair capacity of peripheral blood mononuclear cells (PBMCs). The rationale of the QPCR assay is that lesions that stall the thermostable DNA polymerase on the DNA template during the PCR reaction will lead to a decrease in amplification of the fragment of interest. Because the DNA polymerase amplifies only undamaged templates, amplification is inversely proportional to the presence of damage. We performed dose-response and time-course experiments using the oxidizing agent H2O2 to define the optimal conditions of the QPCR assay to be used to determine mtDNA repair capacity in PBMCs from healthy human subjects. Our dose-response experiments show a dose-dependent reduction in the amplification of the mtDNA amplicon. In these experiments, exposure of PBMCs to 0.1 mM H2O2 for 1-hour resulted in a 50% decrease in the amplification of the mtDNA fragment. Time course experiments showed that 24 hours after treatment there was still a 50% reduction in the amplification of the mtDNA amplicon, suggesting that mtDNA lesions were still present. However, we observed that 48 hours after treatment, the amplification of the mtDNA amplicon reached levels similar to those from untreated control cells. These results indicate that human PBMCs take 48 hours to repair the mtDNA damage induced by 1-hour exposure to 0.1 mM H2O2. These results define the conditions to compare mtDNA repair capacity in PBMCs from cancer patients and healthy subjects. Sponsored by 5U54CA096297-07. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3940.

Apex1 haploinsufficiency leads to age‐dependent increases in mtDNA lesions in liver

The base excision repair pathway repairs DNA lesions induced by reactive oxygen species (ROS). A central component of this pathway is the apurinic/apyrimidinic endonuclease 1 (APEX1). Knockout of both copies of Apex1 results in murine embryo lethality, whereas heterozygous mice (Apex1+/−) are viable and can live to old age. Apex1 haploinsufficiency does lead to a phenotype characterized by increased mutation frequencies in germ cells. Because of the prominent role of the liver in detoxification reactions, we hypothesize that APEX1 plays a prominent role in protecting this organ from the deleterious effects of ROS. Our hypothesis predicts that Apex1 haploinsufficiency will lead to increased DNA lesions, particularly in the mitochondrial genome (mtDNA). For this purpose we determined the amount of mtDNA lesions in liver tissue obtained from 6, 16, and 28‐month old wild type (WT) and Apex1+/− mice using a gene specific assay based on PCR. This assay can detect a variety of DNA lesions such as abasic sites, strand breaks and oxidized bases, among others. The results of these experiments show a significant increase in mtDNA lesions in both WT and Apex1+/− 28‐month‐old mice as compared to 6 month‐old animals. Moreover, the 28‐month‐old Apex1+/− mice show a 2.5 fold increase in mtDNA lesions over aged‐matched WT animals suggesting that Apex1 haploinsufficiency leads to an age‐dependent increase in mtDNA lesions. These results underscore the role of BER in maintaining mtDNA stability in liver. Supported by 5SC3GM084759, 5R25GM061838 and G12RR03051.

Improved Detection of Lassa Virus by Reverse Transcription-PCR Targeting the 5′ Region of S RNA

The method of choice for the detection of Lassa virus is reverse transcription (RT)-PCR. However, the high degree of genetic variability of the virus poses a problem with the design of RT-PCR assays that will reliably detect all strains. Recently, we encountered difficulties in detecting some strains from Liberia and Nigeria in a commonly used glycoprotein precursor (GPC) gene-specific RT-PCR assay (A. H. Demby, J. Chamberlain, D. W. Brown, and C. S. Clegg, J. Clin. Microbiol. 32:2898-2903, 1994), which prompted us to revise the protocol. The design of the new assay, the GPC RT-PCR/2007 assay, took into account 62 S RNA sequences from all countries where Lassa fever is endemic, including 40 sequences generated from the strains in our collection. The analytical sensitivity of the new assay was determined with 11 strains from Sierra Leone, Liberia, Ivory Coast, and Nigeria by probit analysis; the viral loads detectable with a probability of 95% ranged from 342 to 2,560 S RNA copies/ml serum, which corresponds to 4 to 30 S RNA copies/assay. The GPC RT-PCR/2007 assay was validated with 77 serum samples and 1 cerebrospinal fluid sample from patients with laboratory-confirmed Lassa fever. The samples mainly originated from Liberia and Nigeria and included strains difficult to detect in the assay of 1994. The GPC RT-PCR/2007 assay detected virus in all clinical specimens (100% sensitivity). In conclusion, a new RT-PCR assay, based in part on the protocol developed by Demby et al. in 1994, for the detection of Lassa virus is described. Compared to the assay developed in 1994, the GPC RT-PCR/2007 assay offers improved sensitivity for the detection of Liberian and Nigerian Lassa virus strains.

Observation of a Bonamia sp. infecting the oyster Ostrea stentina in Tunisia, and a consideration of its phylogenetic affinities

The small non-commercial oyster Ostrea stentina co-occurs with commercially important Ostrea edulis in the Mediterranean Sea, yet its disposition with respect to the destructive pathogens Bonamia ostreae and Marteilia refringens is unknown. We began an evaluation of the Bonamia spp. infection status of O. stentina from Hammamet, Tunisia, in June 2007 using polymerase chain reaction diagnostics followed by histology and in situ hybridization. Of 85 O. stentina sampled, nine were PCR-positive for a Bonamia sp. using a Bonamia genus-specific assay; of these nine, one displayed the uninucleate microcells associated with oyster hemocytes characteristic of Bonamia spp. There was no associated pathology. DNA sequencing of the parasite from this one infected individual revealed it to be of a member of the Bonamia exitiosa/ Bonamia roughleyi clade, an identification supported by positive in situ hybridization results with probes specific for members of this clade, and by the morphology of the parasite cells: nuclei were central, as in B. exitiosa, not eccentric, as in B. ostreae. There is no basis for identifying the Tunisian parasite as either B. exitiosa or B. roughleyi, however, as these species are genetically indistinguishable. Likewise, there is no basis for identifying any of the other Bonamia spp. with affinities to the B. exitiosa/ B. roughleyi clade, from Argentina, Australia, Spain, and the eastern USA, as one or the other of these named species. Though they are clearly distinct from Bonamia perspora and B. ostreae, justification for drawing species boundaries among the primarily austral microcells with affinities to B. exitiosa and B. roughleyi remains elusive.

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of yellow head virus in shrimp

A real-time reverse transcription loop-mediated isothermal amplification (real-time RT-LAMP) method was applied for detecting the replicase polyprotein-encoding gene of yellow head virus (YHV) in shrimp, Penaeus monodon. It is a novel, gene-specific assay that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions using a set of six specially designed primers that recognize eight distinct sequences of the target gene. This method works with even low copies of DNA and is based on magnesium pyrophosphate turbidity detection by an inexpensive photometer for quantitative analysis. A user-friendly protocol was developed with optimal conditions standardized at 63 °C for 60 min. With this protocol, the assay sensitivity was 10 times higher than the widely used YHV nested RT-PCR system. Cross-reactivity analysis using other shrimp virus DNA/cDNA and YHV-negative shrimp demonstrated high specificity of the assay. The real-time RT-LAMP method was performed also for an internal control gene, EF-1α, to compare with the expressions of the YHV gene in different organs of infected shrimp, and the resulting standard curves showed high correlation coefficient values. These results suggest that this assay is applicable widely as a new quantitative detection method in the pursuit of YHV-free shrimp culture.

Identification of Novel Direct Stat3 Target Genes for Control of Growth and Differentiation

Signal transducer and activator of transcription 3 (Stat3) is a key regulator of gene expression in response to signaling of the glycoprotein 130 (gp130) family cytokines, including interleukin 6, oncostatin M, and leukemia inhibitory factor. Many efforts have been made to identify Stat3 target genes and to understand the mechanism of how Stat3 regulates gene expression. Using the microarray technique, hundreds of genes have been documented to be potential Stat3 target genes in different cell types. However, only a small fraction of these genes have been proven to be true direct Stat3 target genes. Here we report the identification of novel direct Stat3 target genes using a genome-wide screening procedure based on the chromatin immunoprecipitation method. These novel Stat3 target genes are involved in a diverse array of biological processes such as oncogenesis, cell growth, and differentiation. We show that Stat3 can act as both a repressor and activator on its direct target genes. We further show that most of the novel Stat3 direct target genes are dependent on Stat3 for their transcriptional regulation. In addition, using a physiological cell system, we demonstrate that Stat3 is required for the transcriptional regulation of two of the newly identified direct Stat3 target genes important for muscle differentiation.

Development of quantitative gene-specific real-time RT-PCR assays for the detection of measles virus in clinical specimens

Real-time RT-PCR assays targeting sequences in the measles virus (MV) nucleoprotein (N), fusion (F), and hemagglutinin (H) genes were developed for the detection of MV RNA in clinical specimens. Four primer and probe sets each for the N, F, and H genes were evaluated and reaction conditions optimized. Using dilution series of synthetic RNAs, the limits of detection were determined to be approximately 10 copies for each target RNA/reaction. The relationship between C t values and RNA concentration was linear within a range of 10–10 6 RNA copies/reaction, and intra- and inter-assay variability was low. The N gene-specific real-time assay detected MV RNA in 100% of clinical samples from confirmed measles cases compared to 41% by standard RT-PCR. The MV H and F gene-specific real-time assays detected MV RNA in 93% and 82% of these specimens, respectively. Real-time assays could detect RNA from strains representing each active genotype of MV and were also highly specific, as no false positives were identified when samples known to contain other respiratory viruses were tested. Real-time RT-PCR assays will be available to support routine measles laboratory surveillance, to facilitate research projects on pathogenesis that require sensitive and quantitative detection of MV RNA, and to aid in the investigation of serious disease sequelae resulting from natural measles infection or vaccination with measles-containing vaccines.

Response of Pisum Sativum Cytokinin Oxidase/Dehydrogenase Expression and Specific Activity to Drought Stress and Herbicide Treatments

The expression of cytokinin oxidase/dehydrogenase (CKX EC: 1.5.99.12) is subject to fine regulation and it provides a rapid turnover of cytokinins, which serves as a signal for triggering developmental events during plant growth. The activity of this enzyme is believed to be responsible for the changes in cytokinin pool under adverse environmental conditions. CKX gene-specific assay to measure the expression in response to different stress treatments in the tissues of Pisum sativum plants was developed. Pea CKX genes were amplified and sequenced using primers designed from the sequences of Medicago truncatula CKX genes. Expression of two P. sativum CKX genes was assessed using relative-quantification in real time two-step RT-PCR, in leaves and roots of drought-, glufosinate- and atrazine-treated cv. Manuela pea plants. Varied CKX responses support the existence of complicated regulating mechanism of cytokinin oxidase/ dehydrogenase gene expression.

Expression analysis of androgen-responsive genes in the prostate of veal calves treated with anabolic hormones

In order to identify indirect molecular biomarkers of anabolic treatments in veal calves, an animal experiment was performed using two combinations of growth promoters (consisting of boldenone undecylenate and estradiol benzoate, and of testosterone enantate and estradiol benzoate). We selected a set of 12 genes that are known to be androgen responsive in other mammalian species. The expression profile of this set of genes was analysed on prostate samples of veal calves using a real-time RT-PCR approach. For each selected gene the corresponding bovine sequence was obtained and a gene specific real-time assay was optimised and validated. The amplification was shown to be highly specific, linear and efficient. High reproducibility (<1%) and low-test variability (<2.5%) were also been achieved. Messenger RNA levels were quantified in prostate samples, non-parametric analysis of variance showed significant up-regulation of three genes (MAF, ESR1 and AR) and significant down-regulation of four genes (HMGCS1, HPGD, DBI, and LIM) in treated samples when compared with untreated controls. To assess the possibility of identifying hormone-treated animals by molecular means we performed a discriminant analysis that was effective in classifying treated and non-treated samples with an accuracy of 93%. Our results indicate that identification of treatment with steroid hormones in veal calves by means of gene expression analysis is a feasible approach and could be improved increasing both the number of genes and the number of controls analysed.

PCR as a diagnostic tool for brucellosis

Numerous PCR-based assays have been developed for the identification of Brucella to improve diagnostic capabilities. Collectively, the repertoire of assays addresses several aspects of the diagnostic process. For some purposes, the simple identification of Brucella is adequate (e.g. diagnosis of human brucellosis or contamination of food products). In these cases, a genus-specific PCR assay is sufficient. Genus-specific assays tend to be simple, robust, and somewhat permissive of environmental influences. The main genetic targets utilized for these applications are the Brucella BCSP31 gene and the 16S–23S rRNA operon. Other instances require identification of the Brucella species involved. For example, most government-sponsored brucellosis eradication programs include regulations that stipulate a species-specific response. For epidemiological trace back, strain-specific identification is helpful. Typically, differential PCR-based assays tend to be more complex and consequently more difficult to perform. Several strategies have been explored to differentiate among Brucella species and strains, including locus specific multiplexing (e.g. AMOS-PCR based on IS 711), PCR-RFLP (e.g. the omp2 locus), arbitrary-primed PCR, and ERIC-PCR to name a few. This paper reviews some of the major advancements in molecular diagnostics for Brucella including the development of procedures designed for the direct analysis of a variety of clinical samples. While the progress to date is impressive, there is still room for improvement.

Application of the fluorogenic probe technique (TaqMan PCR) to the detection of Enterococcus spp. and Escherichia coli in water samples

A recent PCR detection technique (TaqMan) based on the 5′–3′-exonuclease activity of the Taq DNA polymerase was applied to the detection of indicator organisms in water samples. In this technique, an increasing fluorescence signal is measured online which enables direct assessment of results after PCR without additional detection steps. The test is completed within about 5 h. Two sets of primers and probes were designed and tested: a genus-specific assay for the detection of Enterococcus spp. based on 23S rRNA sequence and an Escherichia coli-specific assay based on the uidA gene sequence. Specificity of the assays was confirmed by testing strains of target bacteria and potential interfering microorganisms. Application of the tests to 55 natural water samples showed the need of an overnight enrichment step to achieve compliance with detection limits of existing regulations. Compared with a parallel microbiological examination of the samples, agreement was 96% with the Enterococcus assay and 98% with the E. coli assay. The rapidity and feasibility of the method point to benefits in drinking water analysis, particularly in emergency situations and, thus, to improved public health management.

Global genome repair of 8-oxoG in hamster cells requires a functional CSB gene product.

Cockayne syndrome (CS) is an autosomal recessive human disease characterized by UV-sensitivity as well as neurological and developmental abnormalities. Two complementation groups have been established, designated CS-A and CS-B. Traditionally, CSA and CSB have been ascribed a function in the transcription-coupled repair (TCR) pathway of nucleotide excision repair (NER) that efficiently removes bulky lesions from the transcribed strand of RNA polymerase II transcribed genes. To assess the role of the CSB protein in the repair of the highly mutagenic base lesion 7,8-dihydro-8-oxoguanine (8-oxoG), we have investigated the removal of this lesion using an in vitro incision approach with cell extracts as well as an in vivo approach with a modified protocol of the gene-specific repair assay, which allows the measurement of base lesion repair in intragenomic sequences. Our results demonstrate that the integrity of the CSB protein is pivotal for processes leading to incision at the site of 8-oxoG and that the global genome repair (GGR) of this lesion requires a functional CSB gene product in vivo.

Homogeneous repair of nuclear genes after experimental stroke.

The repair of oxidative DNA lesions (ODLs) in the nucleus of ischemic cortical brain cells was examined following experimentally induced stroke by occluding the right middle cerebral artery and both common carotid arteries for 60-90 min followed by reperfusion in male long-Evans hooded rats. The control group consisted of sham-operated animals undergoing the same surgery without vessel occlusion. Using a gene-specific assay based upon the presence of Escherichia coli Fpg protein-sensitive sites, we noted that animals with stroke exhibited six and four ODLs per gene in the actin and DNA polymerase-beta genes, respectively. This was increased from one per four copies of each gene in the sham-operated control (p < 0.01). One half of the initial ODLs was repaired within 30 min, and 83% of them were repaired as early as 45 min of reperfusion. There was no further increase when gene repair was measured again at 2 h of reperfusion. The rates of active repair within 45 min of reperfusion were the same in these two genes (p = 0.103, ANOVA). BrdU (10 mg/kg) was administered via intraperitoneal injection at least one day before surgery. We observed that there was no significant incorporation of BrdU triphosphates into genomic DNA during active repair, but there were significant amounts of BrdU triphosphate in nuclear DNA after active repair. The result indicates that genomic repair of ODLs in the brain did not significantly incorporate BrdU, and the initiation of neurogenesis probably starts after the completion of repair in the brain.

Measuring gene-specific nucleotide excision repair in human cells using quantitative amplification of long targets from nanogram quantities of DNA

We have been developing a rapid and convenient assay for the measurement of DNA damage and repair in specific genes using quantitative polymerase chain reaction (QPCR) methodology. Since the sensitivity of this assay is limited to the size of the DNA amplification fragment, conditions have been found for the quantitative generation of PCR fragments from human genomic DNA in the range of 6–24 kb in length. These fragments include: (1) a 16.2 kb product from the mitochondrial genome; (2) 6.2, 10.4 kb, and 15.4 kb products from the hprt gene, and (3) 13.5, 17.7, 24.2 kb products from the human β-globin gene cluster. Exposure of SV40 transformed human fibroblasts to increasing fluences of ultraviolet light (UV) resulted in the linear production of photoproducts with 10 J/m2 of UVC producing 0.085 and 0.079 lesions/kb in the hprt gene and the β-globin gene cluster, respectively. Kinetic analysis of repair following 10 J/m2 of UVC exposure indicated that the time necessary for the removal of 50% of the photoproducts, in the hprt gene and β-globin gene cluster was 7.8 and 24.2 h, respectively. Studies using lymphoblastoid cell lines show very little repair in XPA cells in both the hprt gene and β-globin locus. Preferential repair in the hprt gene was detected in XPC cells. Cisplatin lesions were also detected using this method and showed slower rates of repair than UV-induced photoproducts. These data indicate that the use of long targets in the gene-specific QPCR assay allows the measurement of biologically relevant lesion frequencies in 5–30 ng of genomic DNA. This assay will be useful for the measurement of human exposure to genotoxic agents and the determination of human repair capacity.

Low correlation of serology with detection of Chlamydia trachomatis by ligase chain reaction and antigen EIA.

The aim of the present study was to evaluate the diagnostic accuracy of serology by using new assays for the detection of genus and species-specific IgG, IgM, IgA and secretory IgA antibody in female sex workers. Cervical swabs and first void urine (FVU) from 314 female sex workers were submitted to nucleic acid amplification by ligase chain reaction (LCx, Abbott). Concomittantly, blood samples were tested for the presence of IgG, IgM and IgA antibodies using a genus-specific assay (rELISA, Medac) and species-specific test (SeroCT, Orgenics). Chlamydia trachomatis infection was detected in a total of 30 (9.6%) female sex workers by LCR. With rELISA, seroprevalences for IgG, IgM, and IgA antibody to Chlamydia were 88.9%, 19.1% and 62.7%, respectively. IgG and IgA antibody prevalences against C. trachomatis (SeroCT) were 65.0% and 23.9%, respectively. In comparison to the positive LCR results obtained from cervical swab and/or FVU, the sensitivity of rELISA for Chlamydia IgG, IgA and IgM detection was 93.3%, 83.3% and 16.7%, respectively. With SeroCT, the sensitivity for C. trachomatis-specific IgG and IgA detection was 86.7% and 33.3%, respectively. The specificities of both serologic tests in comparison to LCR were very low. In conclusion, the correlation of serology with active C. trachomatis infection of the lower genital tract is very low. According to our results, serologic testing for Chlamydia can exclude active infection of the lower genital tract with a high reliability (> or = 95%). However, detection of C. trachomatis can only be reliably achieved by nucleic acid amplification assays.

Lack of correlation between repair of DNA interstrand cross-links and hypersensitivity of hamster cells towards mitomycin C and cisplatin

The ability to repair DNA interstrand cross-links may be an important factor contributing to mitomycin C (MMC) and cisplatin cytotoxicities. We have assessed the repair of interstrand cross-links induced by MMC in two MMC-hypersensitive hamster cell mutants and their resistant parental cell line. Using a gene-specific repair assay, we found no evidence for repair of MMC cross-links in either parental or mutant cells, suggesting that persistence of DNA interstrand cross-links is not responsible for the differential toxicity of MMC towards hypersensitive cells. Repair of cisplatin-induced interstrand cross-links was efficient in resistant as well as in mutant cells. Therefore we concluded that a defect in excision repair of interstrand cross-links was not responsible for the cytotoxic effects of MMC and cisplatin in these hypersensitive mutants.