DNA PCR Assay Research Articles

Comparison of commercial and in-house Real-time PCR assays for quantification of Epstein-Barr virus (EBV) DNA in plasma

BackgroundEpstein-Barr virus (EBV) DNA load monitoring is known to be useful for the diagnosis and monitoring of EBV-associated diseases. The aim of this study is to compare the performance of two real-time PCR assays for EBV DNA: a commercial kit as the Q-EBV Real-Time System (Q-EBV PCR, Amplimedical, Turin, Italy) and an in-house assay (EBV RQ-PCR).ResultsThe range of linearity and the degree of precision of the two assays were similar. The clinical sensitivity of Q-EBV PCR was higher for reference samples containing less than 1,000 EBV DNA copies/ml. The absolute quantitative results of the two methods were statistically correlated (R2 = 0.7789; p < 0.0001), with the systematic overestimation by EBV RQ-PCR possibly linked to different amplification efficiency in calibration standards.ConclusionBoth the commercial and the in-house assay may be appropriate for clinical use, but common standards are advisable for comparable absolute values, as these would improve the clinical utility of EBV DNA load measurement.

Evaluation of Dried Whole Blood Spots Obtained by Heel or Finger Stick as an Alternative to Venous Blood for Diagnosis of Human Immunodeficiency Virus Type 1 Infection in Vertically Exposed Infants in the Routine Diagnostic Laboratory

The diagnostic accuracy of the Roche Amplicor human immunodeficiency virus type 1 DNA PCR assay (version 1.5) on DNA extracted from pediatric heel prick dried blood spots using Roche MagNA Pure nucleic acid purification technology was evaluated. The methodologies transfer successfully from the labor-intensive research laboratory to the high-throughput automated routine laboratory.

Prospective Evaluation of the Hybrid Capture 2 and AMPLICOR Human Papillomavirus (HPV) Tests for Detection of 13 High-Risk HPV Genotypes in Atypical Squamous Cells of Uncertain Significance

The use of high-risk human papillomavirus (hrHPV) testing as an adjunct to cervical cytology in population-based screening programs is currently based on DNA hybridization and PCR assays. The aim of this study was to prospectively assess the diagnostic performance of the Hybrid Capture 2 test (HC2; Digene Corporation) in comparison with that of the recently developed PCR-based AMPLICOR HPV test (Roche Molecular Systems) for the detection of 13 hrHPV types. A reverse line blot hybridization assay (Innogenetics) was used as an internal reference standard in discordant cases. Two hundred seventy-one patients with atypical squamous cells of uncertain significance (ASCUS) in cervical samples underwent hrHPV testing. The chi-square test was performed to compare respective proportions. Totals of 160/271 (59%) and 156/271 (58%) were found to be positive for hrHPV with HC2 and AMPLICOR, respectively. Concordant results were obtained for 235 (86.7%) of the 271 samples (kappa statistic, 0.73 +/- 0.04). Considering types 26, 53, and 66 as oncogenic types, negative predictive values (NPVs) of HC2 and AMPLICOR were 92.8% and 87.8%, respectively (difference was not significant), and their respective accuracies were 94.8% and 91.9% (difference was not significant). Considering types 26, 53, and 66 as not oncogenic, the respective HC2 and AMPLICOR NPVs were 92.8% and 97.4% (difference was not significant), and accuracy was significantly higher for the AMPLICOR assay (95.9% versus 90.8% for HC2) (P<0.05). For ASCUS samples, the NPV was 92.8% for HC2 testing and might be compromised if the copy number of HPV DNA was low. The NPV was 97.4% for the AMPLICOR assay and might be compromised if HPV types 26, 53, and 66 were considered oncogenic. The accuracy of these two assays is good and is compatible with routine clinical use in the triage of ASCUS cases.

Prophylaxis Against Cytomegalovirus Infections with Oral Maribavir in Allogeneic Stem Cell Transplant Recipients: Results of a Randomized, Double-Blind, Placebo-Controlled Trial.

Cytomegalovirus (CMV) remains a major threat to recipients of allogeneic stem cell transplants (SCT). Potential toxicities of currently available anti-CMV agents complicate their use in this population. Maribavir (MBV) is an oral antiviral drug with a unique mechanism of action against CMV and a promising safety profile in early clinical studies. This randomized, double-blind, multicenter, dose-ranging study enrolled adult CMV-seropositive recipients of allogeneic SCT. After engraftment, patients were randomized to receive CMV prophylaxis with placebo or MBV at one of three doses for up to 12 weeks. CMV surveillance was performed weekly with both CMV pp65 antigenemia and plasma CMV DNA PCR assays. If CMV was detected, study drug was stopped and preemptive anti-CMV therapy started as per standard practice at each center. A total of 111 patients were enrolled (median age 47 years; 70% myeloablative SCT; 53% related donor). Incidence of CMV infection or disease in the first 100 days post-SCT are shown in the table. The safety profile of maribavir was favorable in this patient population; most adverse events occurred at similar rates in placebo and MBV groups. There was no adverse impact of MBV on neutrophil counts or other laboratory or ECG parameters. Consistent with prior clinical studies, the most notable events that appeared to be associated with MBV were mild-moderate taste disturbance, nausea, and diarrhea. In conclusion, maribavir prophylaxis reduced the rate of CMV infection compared to placebo, with a favorable safety profile in this complex patient population. Phase 3 studies of maribavir in stem cell and solid organ transplant recipients are being planned.PlaceboMBV 100 mg BIDMBV 400 mg QDMBV 400 mg BIDN evaluable28272726CMV infection or disease based on:pp65 antigenemia11 (39%)4 (15%)5 (19%)4 (15%)p=0.046p=0.116p=0.053plasma CMV DNA PCR13 (46%)2 (7%)3 (11%)5 (19%)p=0.001p=0.007p=0.038Use of preemptive anti-CMV Rx16 (57%)4 (15%)8 (30%)4 (15%)p=0.001p=0.051p=0.002CMV disease only3 (11%)000p=0.089p=0.084p=0.091p values represent comparison to placebo (stratified by myeloablative or nonmyeloablative SCT)

Enterotoxigenic Escherichia coli with STh and STp Genotypes Is Associated with Diarrhea Both in Children in Areas of Endemicity and in Travelers

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea among children in developing countries and in travelers to areas of ETEC endemicity. ETEC strains isolated from humans may produce a heat-labile enterotoxin (LT) and two types of the heat-stable enterotoxin STa, called STh and STp, encoded by the estA gene. Two commonly used assay methods for the detection of STa, the infant mouse assay or different enzyme-linked immunosorbent assays, are unable to distinguish between the two subtypes of ST. Different genotypic methods, such as DNA probes or PCR assays, may, however, allow such discrimination. Using gene probes, it has recently been reported that ETEC strains producing STp as the only enterotoxin are not associated with diarrhea. In this study, we have used highly specific PCR methods, including newly designed primers for STh together with previously described STp primers, to compare the relative distribution of STh and STp in ETEC isolated from children with diarrhea in three different geographically distinct areas, i.e., Bangladesh, Egypt, and Guatemala, and from travelers to Mexico and Guatemala. It was found that ETEC strains producing STp were as commonly isolated from cases of diarrhea as strains producing STh both in Egypt and Guatemala, whereas STp strains were considerably less common in Bangladesh. No difference was found in the relative distribution of STh and STp in ETEC strains isolated from travelers with diarrhea and from asymptomatic carriers. Irrespective of ST genotype, the disease symptoms were also similar in both children and travelers.

Attempted transmission of Candidatus Mycoplasma haemominutum and Mycoplasma haemofelis by feeding cats infected Ctenocephalides felis

To determine whether Mycoplasma haemofelis (Mhf) and Candidatus Mycoplasma haemominutum (Mhm) can be transmitted by ingestion of Mycoplasma-infected Ctenocephalides felis and by-products (feces, larvae, and eggs). 10 cats. 3 cats were carriers of Mhf, and 1 was a carrier of Mhm. Six cats had negative results of PCR assay for Mhf and Mhm DNA. A chamber containing 100 C felis was bandaged to 2 Mhf carrier cats. Five days later, fleas and by-products were analyzed for Mycoplasma spp DNA. The remaining fleas and a sample of by-products were fed to 2 Mycoplasma-naïve cats. A chamber containing 200 C felis was bandaged to the Mhm carrier cat. Five days later, fleas and by-products were analyzed for Mycoplasma spp DNA. The remaining fleas and a sample of by-products were fed to 2 Mycoplasma-naïve cats. A chamber containing 200 C felis was bandaged to an Mhf carrier cat and Mhm-carrier cat. Three days later, fleas and by-products were analyzed for Mycoplasma spp DNA. The remaining fleas and a random sample of by products were fed to 4 Mycoplasma-naïve cats. All cats were monitored for infection for >or=7 weeks. Uptake of Mhf and Mhm DNA into fleas and by-products was detected. None of the naïve cats became infected. Results suggested that ingestion of Mycoplasma-infected C felis or by-products is not an important means of transmission for Mhf or Mhm.

Evaluation of an automated and highly sensitive real-time PCR assay for CMV DNA in whole blood samples

Evaluation of an automated and highly sensitive real-time PCR assay for CMV DNA in whole blood samples

Breast milk as a source for acquisition of cytomegalovirus (HCMV) in a premature infant with sepsis syndrome: Detection by real-time PCR

Symptomatic disease due to human cytomegalovirus (HCMV) has been increasingly recognized in low birth weight (LBW) premature infants. Breast milk has been identified as a potential source for these infections. At approximately 60 days of life a LBW, premature infant was diagnosed with HCMV sepsis syndrome, prompting further investigation of the source for HCMV acquisition. The infant had received a combination of both fresh and frozen breast milk from his mother throughout his first 2 months of life. We utilized a real-time PCR assay for HCMV DNA to retrospectively examine viral genome copy number in serial samples of stored, frozen breast milk. This analysis indicated an increase in breast milk viral load over the first 45 days of life, heralding the HCMV sepsis syndrome that was observed clinically. Real-time PCR may be a useful tool in the evaluation of HCMV viral load in breast milk.

Association of Cytomegalovirus with Infantile Hepatitis

Infantile hepatitis is occasionally seen in apparently healthy children. In most cases, the etiology of the infection is uncertain. However, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus-6 (HHV-6), human herpesvirus-7 (HHV-7), human parvovirus B19, and TT virus (TTV) are considered to be associated with hepatitis in children. The objective of this study was to investigate the correlations between these viruses and infantile hepatitis. Twenty-six children from 1 to 24 months old (median age, 7 months) who had liver dysfunction of unknown etiology were enrolled in this study. Plasma samples were examined by a real-time PCR assay for CMV, EBV, HHV-6, HHV-7, parvovirus B19, and TTV DNA. The DNA of CMV was detected in the plasma of four patients (15.4%) and was detected significantly more often in the patient group than in the control group. The CMV-infected patients were 1 to 3 months old, which was significantly younger than the remaining patients. The serological findings did not always correlate with the results of the real-time PCR assay. The DNA of TTV was detected in four patients (15.4%), while human parvovirus B19 DNA was detected in three (11.5%). However, the detection frequencies of these viral DNAs were not significantly different from those in the control groups, and some of these patients had co-infections. These results indicate that CMV might be one of the major pathogens responsible for infantile hepatitis; however, serological tests have limited utility for the diagnosis of CMV infection in young children.

Coccidioidomycosis and blastomycosis: Advances in molecular diagnosis

Clinical isolates of Coccidioides spp. and Blastomyces dermatitidis can be identified by chemiluminescent DNA probes and PCR assays targeting multicopy genes. In fixed tissue samples, cells of the two fungi are specified by in situ hybridization and PCR assays targeting 18S rDNA but sequencing of the products is mandatory. Nested PCR assays targeting genes encoding species- or genus-specific proteins like proline rich antigen of Coccidioides spp. and B. dermatitidis adhesin facilitate amplification of specific DNA from fixed tissue samples. The value of DNA amplification from native specimens of suspected cases of coccidioidomycosis or blastomycosis still needs to be determined.

Proteomic Analysis of Protein Expression and Oxidative Modification in R6/2 Transgenic Mice

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by motor, psychiatric, and cognitive symptoms. The genetic defect responsible for the onset of the disease, expansion of CAG repeats in exon 1 of the gene that codes for huntingtin on chromosome 4, has been unambiguously identified. On the other hand, the mechanisms by which the mutation causes the disease are not completely understood yet. However, defects in energy metabolism of affected cells may cause oxidative damage, which has been proposed as one of the underlying molecular mechanisms that participate in the etiology of the disease. In our effort to investigate the extent of oxidative damage occurring at the protein level, we used a parallel proteomic approach to identify proteins potentially involved in processes upstream or downstream of the disease-causing huntingtin in a well established HD mouse model (R6/2 transgenic mice). We have demonstrated that the expression levels of dihydrolipoamide S-succinyltransferase and aspartate aminotransferase increase consistently over the course of disease (10-week-old mice). In contrast, pyruvate dehydrogenase expression levels were found to be decreased in 10-week-old HD transgenic mice compared with young (4-week-old) mice. Our experimental approach also led to the identification of oxidatively modified proteins. Six proteins were found to be significantly oxidized in old R6/2 transgenic mice compared with either young transgenic mice or non-transgenic mice. These proteins are alpha-enolase, gamma-enolase (neuron-specific enolase), aconitase, the voltage-dependent anion channel 1, heat shock protein 90, and creatine kinase. Because oxidative damage has proved to play an important role in the pathogenesis and the progression of Huntington disease, our results for the first time identify specific oxidatively modified proteins that potentially contribute to the pathogenesis of Huntington disease.

A follow up study of cytomegalovirus infection in a group of Turkish renal transplant recipients using molecular assays

The clinical value of an in-house cytomegalovirus nested polymerase chain reaction (CMV-PCR) and a commercial molecular assay hybrid capture CMV DNA assay (HCA) was evaluated in monitoring a group of renal transplant patients for six months follow up. In this study, the sensitivity, specificity, positive predictive value, and negative predictive value of nested CMV DNA PCR assay and HCA at the beginning of the study were 70, 42.9, 46.7, 66.7, and 60, 78.6, 66.7, and 73.3% respectively. After six months, they were 80, 66.7, 80, 66.7 for CMV PCR and 73.3, 88.9, 91.7, 66.7% for HCA respectively. These results indicate that in monitoring and predicting CMV infections in renal transplant recipients, not only qualitative but also quantitative assays must be used together in order to decide the preemptive strategies.

Hypoglycorrhachia in herpes zoster associated encephalitis of an immunocompetent young male

Sirs: A thirty-one year old male was admitted to the Department of Medicine in Yan Chai Hospital with fever and headache. He had history of fever and sore throat for one week. His family doctor made a diagnosis of acute pharyngitis and gave him a course of amoxicillin. Few days later, he developed bilateral hearing loss and increasing early morning headache, exacerbated by cough. His temperature was 38 °C. There was no sign of meningeal irritation. Scattered blisters were noted over the left side of his neck along the distribution of cervical nerve C3 and C4. Bilateral hearing loss was present. Audiometry test revealed neurosensory hearing loss in both ears. His complete blood count, renal and liver functions, chest radiograph, electrocardiogram and CT of the brain were normal. Lumbar puncture revealed clear CSF with an opening pressure of 200 mm H2O, white cell count 240 WBC per mm3 (polymorphs 10 %, lymphocyte 90 %), protein 1.8 g/L, glucose 1.9 mmol/L. Simultaneous serum glucose was 6.9 mmol/L. CSF bacterial antigen test, cryptococcal antigen, fungal culture, PCR for TB and herpes simplex virus (HSV) DNA were negative. Brain MRI showed nonenhancing signal change in the lower pons and medulla, which was hypodense on T1-weighted images, hyperintense on T2-weighted and FLAIR images. The features were compatible with rhomboencephalitis (Fig. 1). IV acyclovir 10 mg/kg q8h was administered for 14 days. His fever came down on day 3 after the treatment began. His blisters and headache resolved gradually. Follow-up lumbar puncture showed normalization of CSF sugar with improvement in CSF protein and white cell counts. CSF viral titer revealed a 4-fold rise for IgG against varicella (pre-treatment titer 1: < 4, follow-up titer 1:16). PCR varicella-zoster DNA in his CSF before treatment was positive. He had mild residual neurosensory hearing loss and reported occasional tinnitus six months after the encephalitis. This case is a reminder that despite being a rare entity, a strikingly low CSF glucose can occur in herpes zoster-associated encephalitis. It can be potentially confusing to the attending clinician by mimicking other common infectious disease such as tuberculosis. Among 2 case reports in the past [1, 2], anti-tuberculous therapy was given to one patient before serological confirmation of varicella zoster meningoencephalitis. With the help of PCR assays, an early diagnosis can be achieved through detection of VZV DNA in CSF as soon as 24 hours after the onset of symptoms [3]. Compared with antibody indices [4], the intrathecal production of antibody is rarely detected before day 5 after onset of neurological symptoms, limiting the use of antibody index for early diagnosis of CNS infection. Currently available VZV DNA PCR assay methods have high specificity and do not cross-react with herpes simplex infection. The diagnostic sensitivity, however, ranges from 29 % to 100 % [5]. Therefore, we suggest that simultaneous analysis of CSF for both varicella-zoster virus DNA and antibody level should be studied in all suspected cases of herpes-zoster associated encephalitis. The presence of either or both in CSF in the typical clinical setting helps the clinician to establish the diagnosis of herpes-zoster associated encephalitis. This approach is particularly useful in Asian regions such as Hong Kong where the prevalence of mycobacterium tuberculous infection is high. Our case illustrated that, though rare, hypoglycorrhachia can occur in herpes zoster associated encephalitis, which has a mortality rate of 4 % to 30 % [6, 7] and residLETTER TO THE EDITORS

Role of the T cell receptor alpha chain in the development and phenotype of naturally arising CD4 +CD25 + T cells

The T cell receptor alpha chain repertoire and the possible influence of the alpha chain on the development and phenotype of naturally arising mouse CD4 +CD25 + T cells have not been extensively analysed. We used all available Vα-specific monoclonal antibodies and a sensitive multiplex genomic DNA PCR assay to study the Vα repertoire of CD4 +CD25 + T cells in normal mice. To address whether CD4 +CD25 + T cells express two TCR alpha chains, we have carried out four-colour flow cytometry using combinations of the available anti-Vα reagents in mice where one allele of the TCRA locus had been inactivated. Results indicate that the Vα repertoire of CD4 +CD25 + T cells is as diverse as their CD25 − partners. In addition, CD4 +CD25 + T cells develop normally in Tcrα +/− mice and we show for the first time that despite expressing only one TCRα chain, they retain their characteristic CD4 low, CD3 low, TCRβ low, CD5 high, CD45RB low and cytoplasmic CD152 high phenotype.

CEBPα mutations in childhood acute myeloid leukemia

CEBPalpha: mutations have been described in adult acute myeloid leukemia (AML) and conferred a favorable prognosis. However, CEBPalpha mutation has not been reported in children. We investigated 117 children with de novo AML using DNA PCR assay followed by sequencing for each PCR product. CEBPalpha mutations were detected in seven patients, four had FAB M2, two M1 and one M4. CEBPalpha mutations only occurred in patients with intermediate cytogenetics and not in 56 children with AML1-ETO, CBFbeta-MYH11, PML-RARalpha or MLL rearrangements. Five patients had mutations occurred in both N-terminal part and basic-leucine zipper (bZIP) domain, one had an N-terminal frameshift mutation and the remaining one had an inframe insertion in the bZIP domain. Cloning analysis on five samples carrying more than one mutations demonstrated one homozygous combined mutations and four heterozygous biallelic mutations. Four of seven CEBPalpha mutation(+) patients had cooperating mutations with FLT3-ITD or N-ras mutations compared to 27 in 109 CEBPalpha mutation(-) patients. Our results showed that CEBPalpha mutations occurred in 6% of childhood AML and most exhibited combined mutations in both N-terminal part and bZIP domain.

Re-examination of feline leukemia virus: host relationships using real-time PCR

The mechanisms responsible for effective vs. ineffective viral containment are central to immunoprevention and therapies of retroviral infections. Feline leukemia virus (FeLV) infection is unique as a naturally occurring, diametric example of effective vs. ineffective retroviral containment by the host. We developed a sensitive quantitative real-time DNA PCR assay specific for exogenous FeLV to further explore the FeLV–host relationship. By assaying p27 capsid antigen in blood and FeLV DNA in blood and tissues of successfully vaccinated, unsuccessfully vaccinated, and unvaccinated pathogen-free cats, we defined four statistically separable classes of FeLV infection, provisionally designated as abortive, regressive, latent, and progressive. These host–virus relationships were established by 8 weeks post-challenge and could be maintained for years. Real-time PCR methods offer promise in gaining deeper insight into the mechanisms of FeLV infection and immunity.

FLT3 Mutations in Normal Karyotype Acute Myeloid Leukemia in First Complete Remission Treated with Autologous Peripheral Blood Stem Cell Transplantation.

Two types of activating FLT3 mutations have been described in AML. FLT3/ITDs can be detected in 20% to 30% of patients with AML. Point mutations of FLT3/D835 have been described in 7% of adult AML patients. Activating FLT3 mutations has been associated with the leukocytosis and poor prognosis. We retrospectively analyzed the significance of FLT3 mutations in AML patients of normal karyotype treated with autologous peripheral blood stem cell transplantation (auto-PBSCT).METHOD: We evaluated 34 consecutive patients with first CR AML of normal karyotype who received myeloablative therapy and auto-PBSCT to analyze clinical features and outcomes between patients with and without FLT3/ITDs and FLT3/D835. There were 16 males and 18 females with a median age of 41.5 years ranging 15–74 years. Cytogenetic G-banding analysis was performed with standard method. The distribution of morphologic types of AML according to the FAB classification was as follows; one patient was M0, twelve M1, eight M2, seven M4 and six M5. The pre-transplant conditioning regimen was G-CSF combined with the high-dose chemotherapy consisting of busulfan (16 mg/kg), etoposide (40 mg/kg) and Ara-C (3 g/m2x4) (BEA regimen). DNA PCR assay was used to detect FLT3/ITDs. All abnormal longer products by agarose gel electrophoresis were subsequently sequenced. DNA PCR assay followed by direct sequence were used to detect FLT3/D835.RESULT: FLT3/ITDs were detected in 8 of 34 patients (23.5 %). FLT3 D835 mutation was detected in 2 of 34 patients (5.9%). To define clinical differences between patients with and without FLT3/ITDs, clinical variables at the diagnosis were compared. WBC (p=0.013), LDH (p=0.0147), the percentage of PB and BM blasts (p=0.0422 and P=0.0021) were significantly higher in the FLT3/ITDs patients. Other clinical parameters such as age, sex, FAB classification, Hb, Plt, remission induction therapy, interval from diagnosis to auto-PBSCT, number of infused CD34+ cells and hematological recovery after auto-PBSCT are not associated with the presence or absence of FLT3/ITDs. We analyzed the clinical significance of FLT3/ITDs mutations. OS and DFS was similar in patients with or without FLT3/ITDs (5 years OS, 71.4% vs 78.9%, p=0.5746; 5 years DFS, 72.9% vs 68.6%, p=0.9273 by the log-rank test).DISCUSSION: As far as we know, this is the first report to describe the significance of FLT3 mutations in AML patients of normal karyotype treated with auto-PBSCT. We show that FLT3 mutations have no prognostic impact in autotransplanted AML 1CR patients of normal karyotype. Our data suggest dose escalation of chemotherapy may conquer the poor prognostic implications of FLT3 mutations. The prognostic significance of activating FLT3 mutations in AML patients with normal karyotype should be evaluated in relation with post-remission therapeutic modalities.

Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA.

An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples.

Comprehensive analysis of risk factors associating with Hepatitis B virus (HBV) reactivation in cancer patients undergoing cytotoxic chemotherapy.

For cancer patients with chronic hepatitis B virus (HBV) infection, who receive cytotoxic chemotherapy, HBV reactivation is a well-described complication, which may result in varying degrees of liver damage. Several clinical features and the pre-chemotherapy HBV viral load have been suggested to be associated with an increased risk of developing the condition: (1) to assess the clinical and virological factors in a comprehensive manner and thereby identify those that are associated with the development of HBV reactivation; (2) to develop a predictive model to quantify the risk of HBV reactivation. In all, 138 consecutive cancer patients who were HBV carriers and undergoing chemotherapy were studied, of which 128 patients had sera available for real-time PCR HBV DNA measurement. They were followed up throughout their course of chemotherapy and the HBV reactivation rate was determined. The clinical and virological features between those who did and did not develop viral reactivation were compared. These included age, sex, baseline liver function tests, HBeAg status and viral load (HBV DNA) prior to the chemotherapy, and the use of specific cytotoxic agents. In all, 36 (26%) developed HBV reactivation. Multivariate analysis revealed pre-chemotherapy HBV DNA level, the use of steroids and a diagnosis of lymphoma or breast cancer to be significant factors. Based on real-time HBV DNA PCR assay, detectable baseline HBV DNA prior to the administration of cytotoxic chemotherapy, the use of steroids and a diagnosis of lymphoma or breast cancer are predictive factors for the development of HBV reactivation. A predictive model was developed from the current data, based on a logistic regression method.

Detection of transgenes in soybean via a polymerase chain reaction and a simple bioluminometric assay based on a universal aequorin-labeled oligonucleotide probe

The recombinant photoprotein aequorin was used as a reporter in highly sensitive and automatable hybridization assays for the analysis of transgenic sequences in genetically modified organisms (GMO). The terminator of the nopaline synthase gene (NOS) from Agrobacterium tumefaciens and the 35S promoter sequence were detected in genetically modified soybean. The endogenous, soybean-specific, lectin gene was also detected for confirmation of the integrity of extracted DNA. A universal detection reagent was produced through conjugation of aequorin to the oligonucleotide (dA)(30). Biotinylated (through PCR) products for the three target sequences were captured onto streptavidin-coated wells, and one strand was removed by NaOH treatment. The immobilized single-stranded DNAs were then hybridized with oligonucleotide probes consisting of a target-specific segment and a poly(dT) tail. This allowed the subsequent determination of all hybrids through the use of the (dA)(30)-aequorin conjugate as a universal reagent. The bound aequorin was measured by adding Ca(2+) and integrating the light emission for 3 s. As low as 2 pM (100 amol per well) of amplified DNA was detectable for all three targets, with a signal-to-background ratio of about 2. The analytical range extended up to 2000 pM. As low as 0.05% GMO content in soybean can be detected with a signal-to-background ratio of 8.2. The overall repeatability of the proposed assay, including DNA extraction, PCR, and hybridization assay, ranged from 7.5-19.8%. The use of a (dA)(30)-aequorin conjugate renders the assay configuration general for any target DNA, provided that the specific probe carries a poly(dT) tail.