TaqMan-based Quantitative PCR Research Articles

Acute responses of circulating microRNAs to low-volume sprint interval cycling.

Low-volume high-intensity interval training is an efficient and practical method of inducing physiological responses in various tissues to develop physical fitness and may also change the expression of circulating microRNAs (miRNAs). The purpose of the present study was to examine whether miRNAs for muscle, heart, somatic tissue and metabolism were affected by 30-s intervals of intensive sprint cycling. We also examined the relationship of these miRNAs to conventional biochemical and performance indices. Eighteen healthy young males performed sprint interval cycling. Circulating miRNAs in plasma were detected using TaqMan-based quantitative PCR and normalized to Let-7d/g/i. In addition, we determined the levels of insulin-like growth factor-I, testosterone and cortisol, and anaerobic capacity. Compared to plasma levels before exercise muscle-specific miR-1 (0.12 ± 0.02 vs. 0.09 ± 0.02), miR-133a (0.46 ± 0.10 vs. 0.31 ± 0.06), and miR-133b (0.19 ± 0.02 vs. 0.10 ± 0.01) decreased (all P < 0.05), while miR-206 and miR-499 remained unchanged. The levels of metabolism related miR-122 (0.62 ± 0.07 vs. 0.34 ± 0.03) and somatic tissues related miR-16 (1.74 ± 0.27 vs. 0.94 ± 0.12) also decreased (both P < 0.05). The post-exercise IGF-1 and cortisol concentrations were significantly increased, while testosterone concentrations did not. Plasma levels of miR-133b correlated to peak power (r = 0.712, P = 0.001) and miR-122 correlated to peak power ratio (r = 0.665, P = 0.003). In conclusion sprint exercise provokes genetic changes for RNA related to specific muscle or metabolism related miRNAs suggesting that miR-133b and miR-122 may be potential useful biomarkers for actual physiological strain or anaerobic capacity. Together, our findings on the circulating miRNAs may provide new insight into the physiological responses that are being performed during exercise and delineate mechanisms by which exercise confers distinct phenotypes and improves performance.

A TaqMan-based qPCR assay for quantitative detection of the biocontrol agents Bacillus subtilis strain QST713 and Bacillus amyloliquefaciens subsp. plantarum strain D747

Biological control agents (BCAs) play an important role in crop protection. They can improve sustainability, prevent resistance to fungicides in target pathogens and reduce fungicide residues on produce. Bacillus subtilis strain QST713 (Serenade Max; Bayer CropScience, Leverkusen, Germany) and Bacillus amyloliquefaciens subsp. plantarum strain D747 [Amylo-X; CBC Europe S.r.l.—Biogard Division, Nova Milanese (MB), Italy] are two commercially available BCAs that are used against grey mould on table grape and other crops. TaqMan-based quantitative (q)PCR assays were developed and validated for quantitative and specific detection of the two BCAs on grape bunches following field sprays. Specific molecular markers were developed for each BCA, and TaqMan probes were designed and tested in qPCR on DNA extracted from washing water of grape berries. The assay proved specific and sensitive for both BCAs allowing detection at density as low as three colony-forming units (CFU) per gram of berries. The method will be useful to investigate the population dynamics of the two BCAs following their applications in vineyards.

Multimerin-1 (MMRN1) As Novel Adverse Prognostic Marker in Acute Myeloid Leukemia: A Report from the Children’s Oncology Group

Background: Both cytogenetic and molecular abnormalities have been identified in acute myeloid leukemia (AML) that provide the framework for diagnostic classification and risk-stratification schemes. However, there are currently only a small number of informative prognostic markers, and it is a recurrent clinical observation that this limited battery fails to accurately predict outcome for many patients. This highlights the need for additional and refined tools to characterize disease risk in AML.Patients and Methods: For gene discovery, gene expression array data from 211 pediatric patients with AML were queried. Subsequently, expression of the gene of interest was quantified in cryopreserved pretreatment specimens from patients enrolled on the AAML03P1 and AAML0531 trials: AAML03P1 was a pilot phase 3 study investigating the feasibility of combining gemtuzumab ozogamicin (GO) with intensive chemotherapy in pediatric patients up to age 30, whereas AAML0531 was a phase 3 study on >1,000 patients testing the value of GO addition to chemotherapy in a randomized fashion. Taqman-based quantitative reverse-transcriptase PCR was used to quantify mRNA expression of the gene of interested and the housekeeping gene, b-glucuronidase (GUSB). Cytogenetics and molecular prognostic markers were used for risk classification as follows: low risk (mutation in core-binding factor, NPM1, or CEBPa), high risk (-5/5q-, monosomy 7, or FLT3-ITD with high allelic ratio), or standard risk (all other patients with cytogenetic/molecular data).Results: Multimerin-1 (MMRN1), a member of the elastin microfibrillar interface protein (EMILIN)/multimerin family that may mediate cellular adhesion via integrin receptors, was identified in gene expression array data as a gene whose expression varied widely, with expression levels possibly related to patient outcomes. To study this possible relationship further, we quantified MMRN1 mRNA in 183 participants of AAML03P1 and correlated expression levels with clinical outcome. MMRN1 expression varied >80,000-fold relative to GUSB. Patients with the highest MMRN1 expression (4th quartile, corresponding to a relative MMRN1 expression of >0.5) indeed had inferior event-free survival (EFS; P<0.002) and higher relapse risk (P<0.004), suggesting that high MMRN1 expression could serve as adverse prognostic factor in pediatric AML. To further validate these findings, we quantified MMRN1 expression in 750 participants of AAML0531, and correlated expression levels with clinical outcome and disease characteristics. In 740 of the 750 patient specimens, MMRN1 was detectable, varying >130,000-fold relative to GUSB. Using a cut-off for high MMRN1 expression as defined in the AAML03P1 cohort (i.e. relative MMRN1 <0.5 [n=590] vs ≥0.5 [n=160]), we found that patients with high MMRN1 expression had a lower response rate after the first course of induction therapy (67% vs. 77%, p=0.013) and more likely minimal residual disease after this initial chemotherapy cycle (43% vs. 26%, p=0.001). They also had inferior 5-year overall survival (OS; 44±9% vs. 69±4%, p<0.0001), lower 5-year EFS (32±8% vs. 54±4%, p<0.0001), and higher 5-year relapse risk (57±10% vs. 35±5%, p<0.0001). At least partially, these differences could be attributed to associations between MMRN1 expression and disease characteristics. Specifically, patients with high MMRN1 expression were less likely to have low-risk disease (P<0.001), and more likely had standard-risk (P<0.001) or high-risk (P<0.001) disease compared to those with low MMRN1 expression. However, after adjustment for disease risk, age, and treatment arm, high MMRN1 expression remained statistically significantly associated with inferior OS (hazard ratio [HR]=1.37 [95% confidence interval: 1.05-1.86] p=0.022), and a trend toward lower EFS (HR=1.25 [0.98-1.59] p=0.077) in multivariate cox models.Conclusion: This study identifies high MMRN1 expression as a novel adverse prognostic factor in pediatric AML. Although the association between adverse outcome and high MMRN1 expression is at least partly due to the higher proportion of adverse-risk features among AML patients with high MMRN1 expression, high MMRN1 expression continued to be associated with inferior survival even after adjustment of disease risk. DisclosuresNo relevant conflicts of interest to declare.

Development of TaqMan-based quantitative PCR for sensitive and selective detection of toxigenic Clostridium difficile in human stools.

Background Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.MethodsTaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC).ResultsThe analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA+TcdB+, TcdA−TcdB+, and TcdA−TcdB− types), TcdB-producing strains (TcdA+TcdB+ and TcdA−TcdB+ types), and TcdA-producing strains (TcdA+TcdB+ type), respectively, with a lower detection limit of 103 cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA+TcdB+ strain in six specimens and TcdA−TcdB− strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 104.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.ConclusionOur qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.

Relative abundance and treatment reduction of viruses during wastewater treatment processes — Identification of potential viral indicators

Waterborne pathogenic viruses discharged from wastewater treatment plants (WWTPs) pose potential public health risks. In the present study, we investigated the relative abundance, occurrence, and reduction of eleven different viruses at two WWTPs in southern Arizona over a 12-month period, from August 2011 to July 2012. Influent and effluent samples from the two WWTPs were collected monthly. Viruses were concentrated using an electronegative filter method and quantified using TaqMan-based quantitative PCR (qPCR) assays for each of the virus types (i.e., genogroup I, II and IV noroviruses, sapovirus, enterovirus, group A rotavirus, Aichi virus, pepper mild mottle virus, adenovirus, and JC and BK polyomaviruses), with murine norovirus internal control for the monitoring of extraction-RT-qPCR efficiencies. The pepper mild mottle virus, a plant virus, was found to be the most prevalent virus in both influent and effluent wastewater (annual mean concentration of 3.7–4.4×106copies/L and 4.6–6.3×105 copies/L in influent and effluent wastewater, respectively), showing a low reduction by the treatment processes (0.76–0.99 annual mean log10 reduction), and no significant seasonal change in concentration. Aichi virus, a human enteric virus, was also found in greater abundance, and showed lower reduction during wastewater treatment than other human enteric viruses. Our results suggest that these viruses could be used as potential indicators of wastewater reclamation system performance, with respect to virus occurrence and removal.

Rapid TaqMan-Based Quantification of Chlorophyll d -Containing Cyanobacteria in the Genus Acaryochloris

Reports of the chlorophyll (Chl) d-containing cyanobacterium Acaryochloris have accumulated since its initial discovery in 1996. The majority of this evidence is based on amplification of the gene coding for the 16S rRNA, and due to the wide geographical distribution of these sequences, a global distribution of Acaryochloris species was suggested. Here, we present a rapid, reliable, and cost-effective TaqMan-based quantitative PCR (qPCR) assay that was developed for the specific detection of Acaryochloris species in complex environmental samples. The TaqMan probe showed detection limits of ~10 16S rRNA gene copy numbers based on standard curves consisting of plasmid inserts. DNA from five Acaryochloris strains, i.e., MBIC11017, CCMEE5410, HICR111A, CRS, and Awaji-1, exhibited amplification efficiencies of >94% when tested in the TaqMan assay. When used on complex natural communities, the TaqMan assay detected the presence of Acaryochloris species in four out of eight samples of crustose coralline algae (CCA), collected from temperate and tropical regions. In three out of these TaqMan-positive samples, the presence of Chl d was confirmed via high-performance liquid chromatography (HPLC), and corresponding cell estimates of Acaryochloris species amounted to 7.6 × 10(1) to 3.0 × 10(3) per mg of CCA. These numbers indicate a substantial contribution of Chl d-containing cyanobacteria to primary productivity in endolithic niches. The new TaqMan assay allows quick and easy screening of environmental samples for the presence of Acaryochloris species and is an important tool to further resolve the global distribution and significance of this unique oxyphototroph.

No association of the SLC1A2 rs3794087 allele with risk for essential tremor in the Spanish population

A recent genome-wide association study and other replication studies have suggested that the rs3794087 single nucleotide polymorphism in the solute carrier family 1 - glial affinity glutamate transporter-member 2 (SLC1A2) gene is associated with an increased risk for essential tremor (ET), and a replication study in an Asian cohort has shown a decreased risk for ET associated with the rs3794087T allele. We tried to replicate this association in a White Spanish population. We analyzed the distribution of allelic and genotypic frequencies of rs3794087 in 202 patients with familial ET and 308 healthy controls using a TaqMan-based quantitative PCR assay. Genotypic and allelic frequencies of rs3794087 did not differ significantly between patients with ET and controls and were unrelated with the age at onset of tremor or sex. Our study suggests that SLC1A2 rs3794087 is not associated with the risk for developing familial ET in the Spanish population, thus subtracting relevance to SLC1A2 rs3794087 as a risk biomarker for ET.

Correlation between Preferentially Expressed Antigen of Melanoma and Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand Gene Expression in Different Types of Leukaemia Patients

Introduction: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) down-regulation by preferentially expressed antigen of melanoma (PRAME) is a general phenomenon in different types of solid tumours, but research on the correlation between PRAME and TRAIL gene expression in leukaemia patients is rare. Method: PRAME and TRAIL expression was detected in bone marrow samples from 80 newly diagnosed acute leukaemia (AL) patients and 40 chronic myeloid leukaemia (CML) patients using TaqMan-based real-time quantitative PCR methods, and a linear correlation analysis was performed on their levels of expression. A total of 15 normal bone marrow samples from individuals with non-malignant haematological diseases served as normal controls. Results: PRAME expression was higher in both AL and CML patients compared to controls (both p < 0.001). CML patients in both blast crisis (BC) and the accelerated phase (AP) had significantly higher PRAME levels than CML patients in the chronic phase (CP) (p = 0.006 and 0.0461, respectively). TRAIL expression was higher in both the acute myeloid leukaemia (AML) group and the acute lymphoblastic leukaemia (ALL) group than in the controls (p = 0.039 and 0.047, respectively). In contrast, CML patients had lower TRAIL levels than controls (p = 0.043), and TRAIL expression in CML patients in the advanced phases (BC and AP) was significantly lower than in CML-CP patients (p = 0.006). In CML patients, there was a significant inverse correlation (Spearman's R = -0.6669, p < 0.0001) between PRAME and TRAIL gene expression, while a greater significant inverse correlation was found in patients in the advanced phases (BC and AP) (R = -0.6764). In addition, no correlation was observed in AML and ALL patients. Conclusion: The simultaneous detection of PRAME and TRAIL gene expression may be helpful to monitor condition changes in leukaemia patients and evaluate therapeutic effects in clinical practice, particularly in CML patients.

The analytical comparison of phage-based magnetoelastic biosensor with TaqMan-based quantitative PCR method to detect Salmonella Typhimurium on cantaloupes

A phage-based magnetoelastic (ME) biosensor method for the detection of Salmonella Typhimurium was compared with a TaqMan-based quantitative real-time PCR (qPCR) method on cantaloupes. The selectivity of a qPCR primer set was investigated and different DNA extraction methods were compared. The limits of detection (LOD) of both detection methods were determined by serial inoculation of S. Typhimurium suspensions on cantaloupe surfaces. The repeatability of both detection methods was determined over three-day periods. Among 74 bacteria tested, only S. Typhimurium exhibited positive amplification, and other bacterial strains did not show any positive amplification. The DNeasy tissue kit showed the best performance for DNA purification due to the significantly superior threshold cycle (Ct) values, compared with either direct applications of qPCR without any extraction procedure or after employing a simple boiling method for extraction. The LOD and repeatability of qPCR and ME biosensor methods for S. Typhimurium detection were determined to be 1.35 ± 0.07 and 2.47 ± 0.50 log CFU/2 mm2 surface of cantaloupe, and 2.41 and 6.28%, respectively. The results demonstrate that the ME biosensor method is one of promising and practical on-site detection methods for S. Typhimurium on fresh produce.

Evaluation of phage-based magnetoelastic biosensors for direct detection of Salmonella Typhimurium on spinach leaves

Abstract For a phage-based magnetoelastic (ME) biosensor method to become accepted for evaluating foodborne contamination, the method of use must be objectively compared, evaluated and validated against more widely accepted standard methods. In this study, a ME biosensor method was evaluated by comparison with TaqMan-based quantitative PCR (qPCR) for the detection of Salmonella Typhimurium on spinach leaves. Limit of detection (LOD) was determined after serial inoculation of S. Typhimurium on spinach leaves. The LOD for the ME biosensor was 2.17 and 1.94 log CFU/spinach for adaxial and abaxial surface, respectively. The LOD for qPCR was 1.37 log CFU/spinach. The repeatability of both methods was measured over a three day period by inoculation of S. Typhimurium on spinach leaves. The repeatability of the ME biosensor method was determined to be 6.38%, competitive with qPCR at 1.92%. For the direct comparison of both methods, 3 log CFU/spinach of S. Typhimurium was grown on two groups of 25 spinach leaves for 24 h and the number of S. Typhimurium was quantified by both methods. After confirmation of the growth, S. Typhimurium was positively detected and the quantified numbers were 5.79 ± 0.88 and 6.11 ± 0.26 log CFU/spinach for the ME biosensor and the qPCR method, respectively. This study demonstrated that the ME biosensor method was competitive and promising as an on-site and in-field detection method for the detection of pathogens.

Anal human papillomavirus DNA in women at a colposcopy clinic

To describe the type-specific prevalence of anal and cervical human papillomavirus (HPV) infections and the cytology in HIV-negative women without a history of cervical cancer, attending a colposcopy clinic. To examine if an HPV positive anal smear is related to anal pathology and consequently indicative for further examinations (high resolution anoscopy, anal biopsy). From 149 consecutive women an anal swab and a cervical swab were taken, using the Cervex-Brush. The presence of 18 different HPV genotypes was determined using TaqMan-based real-time quantitative PCR targeting type-specific sequences of viral genes. From the fluid containing the cellular material, a liquid-based cytology sample was prepared of both collections with the robotic BD PrepStain Slide Processor. All slides were pre-screened by BD FocalPoint system and categorized from quintiles 1 to 5 and afterwards screened using targeted microscopic interpretation of selected suspicious fields using FocalPoint guided screening review stations. The 2001 Bethesda System Terminology was used for the anal slides. Ninety-six anal samples and all 149 cervical samples were adequate. Overall presence of HPV in the anus was 56.3% and in the cervix 53.7%. Overall, cytological abnormalities were found in 10.8% of anal smears and in 32.8% of cervical smears. HPV genotypes were identified in 47 samples on both sites: partial or complete concordance was found in 85.1%. HPV types 6, 16 and 18 were found in 27.9% and in 26.6% of the anal and cervical samples, respectively. The top three HPV types in the anus were 16, 51 and 39; in the cervix 16, 39, 51 and 56 (a shared 3(rd) place). HPV type 11 was not found. The presence of HPV genotypes is clearly multifocal in this study population of women attending a colposcopy clinic, with high concordance of genotypes. The number of anal HPV infections is high. Although cytological abnormalities are rare, the presence of HPV may lead to anal lesions later in life. From this perspective, complementary medical history and clinical examination of the anal region are advised.

Antimicrobial Drug Resistance in Corynebacterium diphtheriae mitis

Antimicrobial Drug Resistance in Corynebacterium diphtheriae mitis

Effects of an immunomodulatory feed additive on the development of mastitis in a mouse infection model using four bovine-origin isolates

The goal of this study was to examine the ability of a commercially available feed additive (OmniGen-AF) to reduce mammary infections caused by a single strain of mastitic pathogens (Streptococcus uberis, Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae) and to examine the effects of the additive on markers of mammary immunity. Four experiments were completed using a murine model of bovine mastitis. Infection progression was examined using Sybr-green- and TaqMan-based quantitative PCR assays of 16S ribosomal DNA. Infection of the mammary gland with all pathogens caused rapid (24 to 48 h) appearance of pathogen DNA in mammary tissue. Provision of the feed additive for 2 weeks before infection significantly (P < 0.05) reduced the extent of pathogen DNA accumulation in models of S. uberis, E. coli and S. aureus infection. The additive was ineffective in reducing mammary infections caused by K. pneumoniae. We examined mechanisms of action of the additive through assessment of mammary concentrations of mammary myeloperoxidase (MPO), major histocompatibility complex 2 class II (MHC) and macrophage inflammatory protein-1α (MIP) messenger RNA (mRNA) concentrations and by examining serum complement C3 concentration. Infection of the mammary gland increased concentrations of MPO and MHC mRNAs (P < 0.05). Ability of the pathogen to elicit changes in mammary MPO and MHC gene expression was enhanced by the provision of the additive for 2 weeks before infection. These data imply that the additive increased the mammary inflammatory response and increased antigen presentation during a mammary infection. Value of the additive in preventing mastitis in cattle awaits additional studies using a bovine model and further evaluation of additional strains of the pathogens used in this study.

High responders to resistance exercise training demonstrate differential regulation of skeletal muscle microRNA expression

MicroRNAs (miRNA), small noncoding RNA molecules, may regulate protein synthesis, while resistance exercise training (RT) is an efficient strategy for stimulating muscle protein synthesis in vivo. However, RT increases muscle mass, with a very wide range of effectiveness in humans. We therefore determined the expression level of 21 abundant miRNAs to determine whether variation in these miRNAs was able to explain the variation in RT-induced gains in muscle mass. Vastus lateralis biopsies were obtained from the top and bottom ∼20% of responders from 56 young men who undertook a 5 day/wk RT program for 12 wk. Training-induced muscle mass gain was determined by dual-energy X-ray absorptiometry, and fiber size was evaluated by histochemistry. The expression level of each miRNA was quantified using TaqMan-based quantitative PCR, with the analysis carried out in a blinded manner. Gene ontology and target gene profiling were used to predict the potential biological implications. Of the 21 mature miRNAs examined, 17 were stable during RT in both groups. However, miR-378, miR-29a, miR-26a, and miR-451 were differentially expressed between low and high responders. miR-378, miR-29a, and miR-26a were downregulated in low responders and unchanged in high responders, while miR-451 was upregulated only in low responders. Interestingly, the training-induced change in miR-378 abundance was positively correlated with muscle mass gains in vivo. Gene ontology analysis of the target gene list of miR-378, miR-29a, miR-26a, and miR-451, from the weighted cumulative context ranking methodology, indicated that miRNA changes in the low responders may be compensatory, reflecting a failure to "activate" growth and remodeling genes. We report, for the first time, that RT-induced hypertrophy in human skeletal muscle is associated with selected changes in miRNA abundance. Our analysis indicates that miRNAs may play a role in the phenotypic change and pronounced intergroup variation in the RT response.

Imatinib Plasma Levels Correlate with Molecular Response in CML Patients

Measurement of imatinib plasma levels is recommended for CML patients on Glivec therapy with suboptimal response or failure. Recently, reports suggesting that imatinib plasma levels correlate both with cytogenetic and molecular response of the patient and may be used for routine management of the treatment were published. We have introduced the examination of imatinib plasma levels into our laboratory follow-up of CML patients in 2007. All patients have signed informed consent before sampling. New capillary electrophoretic method was developed for determination of imatinib plasma levels. Plasma samples were deproteinated by methanol, evaporated, resuspended and directly analyzed. Separation was performed in buffer consisting of 50 mmol/L citrate adjusted with γ-amino-n-butyric acid to pH 4.0. Imatinib was quantified by UV detection in the range 250 – 270 nm (limit of detection is 10 nmol/L). BCR-ABL transcript levels were monitored by TaqMan-based real-time quantitative PCR, the baseline value of BCR-ABL/ABL% was 60%. Here we report comparison of plasma drug levels in CML patients with optimal and suboptimal molecular response. Total 238 samples from 100 CML patients were examined. Patients with major and complete molecular response (n = 68) and patients with suboptimal molecular response (n = 35) were chosen for the final comparison. The actual sample was eligible for analysis providing patient had been treated with standard dose 400 mg daily for at least one year, there was no other evident cause of the suboptimal response (presence of additional cytogenetic abnormalities or BCR/ABL mutation) and the sample was taken 24±6 hours after the ingestion of the drug. Other medication, the actual duration of the therapy (providing it was longer than one year), time interval between the dose and the meal ingestion, weight, body-mass index, body surface area and co-morbidity of patients were not taken into the account. All patients in the suboptimal group have achieved the complete cytogenetic response. Three patients in the suboptimal group have progressed, while there were any such patients in the optimal group. The results showed considerable inter-individual variability between the patients. However, the imatinib plasma levels found in the optimal group significantly higher (median of 2.34 umol/l; range of 0.46 – 6.23) than in the suboptimal group (median of 1.75 umol/l; range of 0.84 – 4.63) p < 0.003. Moreover, patients with negativity in real-time quantitative PCR testing showed a trend for even higher drug plasma levels (median of 3.18 umol/l; range of 0.46 – 6.23, n = 36) p < 0.001. We can conclude that higher imatinib plasma levels are associated with better molecular response to therapy and laboratory measurements may be used for modification of the imatinib dosage.

Internally Controlled Triplex Quantitative PCR Assay for Human Polyomaviruses JC and BK

We have developed a triplex TaqMan-based quantitative PCR assay for the human polyomaviruses JC (JCPyV) and BK (BKPyV). The assay simultaneously detects and quantifies both JCPyV and BKPyV in human clinical samples, and it includes an internal amplification control consisting of murine polyomavirus (MuPyV) plasmid DNA. We developed the assay for the Roche LightCycler 480 platform with the reporter dyes VIC, 6-FAM, and Cy5 for MuPyV, BKPyV, and JCPyV, respectively. The assay had a high specificity for BKPyV and JCPyV when either viral genome was present alone or in mixed samples over a range of 10(1) to 10(7) copy numbers per reaction. The analytical sensitivity was 50 copies for BKPyV and 10 copies for JCPyV. The use of the MuPyV internal control ensured monitoring of the quality of the extraction and of PCR inhibition, even in samples such as cerebrospinal fluid and plasma in which controls based on host genes cannot be effectively used. In addition, we developed a similar assay using a different dye configuration (6-FAM, VIC, and NED) that could be used on an ABI 7500 Fast platform. This assay had sensitivities similar to those of the LightCycler 480 configuration for BKPyV and JCPyV when either viral genome was present alone, but the sensitivity of detection of BKPyV was greatly decreased when an excess of JCPyV (>100-fold) was present in the sample. This internally controlled combined assay offers greater convenience and cost-effectiveness compared to separate assays for each virus and can also detect unexpected PyV activations by testing for both viruses in all samples.

Partial duplication of the PRLR and SPEF2 genes at the late feathering locus in chicken

BackgroundOne of the loci responsible for feather development in chickens is K. The K allele is partially dominant to the k+ allele and causes a retard in the emergence of flight feathers at hatch. The K locus is sex linked and located on the Z chromosome. Therefore, the locus can be utilized to produce phenotypes that identify the sexes of chicks at hatch. Previous studies on the organization of the K allele concluded the integration of endogenous retrovirus 21 (ev21) into one of two large homologous segments located on the Z chromosome of late feathering chickens. In this study, a detailed molecular analysis of the K locus and a DNA test to distinguish between homozygous and heterozygous late feathering males are presented.ResultsThe K locus was investigated with quantitative PCR by examining copy number variations in a total of fourteen markers surrounding the ev21 integration site. The results showed a duplication at the K allele and sequence analysis of the breakpoint junction indicated a tandem duplication of 176,324 basepairs. The tandem duplication of this region results in the partial duplication of two genes; the prolactin receptor and the gene encoding sperm flagellar protein 2. Sequence analysis revealed that the duplication is similar in Broiler and White Leghorn. In addition, twelve late feathering animals, including Broiler, White Leghorn, and Brown Layer lines, contained a 78 bp breakpoint junction fragment, indicating that the duplication is similar in all breeds. The breakpoint junction was used to develop a TaqMan-based quantitative PCR test to allow distinction between homozygous and heterozygous late feathering males. In total, 85.3% of the animals tested were correctly assigned, 14.7% were unassigned and no animals were incorrectly assigned.ConclusionThe detailed molecular analysis presented in this study revealed the presence of a tandem duplication in the K allele. The duplication resulted in the partial duplication of two genes; the prolactin receptor and the gene encoding sperm flagellar protein 2. Furthermore, a DNA test was developed to distinguish between homozygous and heterozygous late feathering males.

Detection of Fusobacterium necrophorum subsp. funduliforme in tonsillitis in young adults by real-time PCR

Throat swabs from 61 patients, aged 18-32 years, with non-streptococcal tonsillitis (NST) and 92 healthy controls were examined for the presence of Fusobacterium necrophorum DNA using a novel TaqMan-based real-time quantitative PCR assay for F. necrophorum subspecies. The assay was based on the gyrB subunit gene, and detected F. necrophorum DNA in 48% of patients with NST and in 21% of controls (p <0.001). F. necrophorum subsp. funduliforme was the only subspecies found in both patients and controls. The load of F. necrophorum DNA on swabs from patients with NST was significantly higher than that on swabs from controls (p <0.001). Furthermore, patients with recurrent NST had a significantly higher load of F. necrophorum DNA compared to patients with acute NST (p 0.04). In addition, 26 patients with tonsillitis and group C streptococci (GCS) had a significantly higher load of F. necrophorum DNA compared to the NST group (p <0.001). It was concluded that F. necrophorum subsp. funduliforme is present in small numbers as part of the normal human throat flora, and that F. necrophorum in large quantities may cause tonsillitis, especially recurrent tonsillitis. In addition, the study suggests that the concomitant presence of GCS may aggravate F. necrophorum tonsillitis.

Prognostic significance of circulating tumor cells in bone marrow or peripheral blood detected by qualitative and quantitative PCR in pediatric NPM-ALK positive anaplastic large cell lymphoma

9559 Background: Clinical and histopathological characteristics have limited prognostic value for children with anaplastic large cell lymphoma (ALCL). We evaluated the presence, extent and prognostic impact of circulating tumor cells in bone marrow (BM) and peripheral blood (PB) of children and adolescents with NPM-ALK positive ALCL by real-time quantitative PCR for NPM-ALK. Methods: Qualitative and TaqMan-based quantitative PCR assays targeting NPM-ALK were developed with a sensitivity to detect 1 NPM-ALK positive cell among 105 cells. Numbers of NPM-ALK transcripts were normalized to 104 copies ABL (NCN). BM was analyzed from 80 and PB from 52 German patients registered into the subsequent protocols NHL-BFM95 and ALCL99. Results: BM was positive for NPM-ALK in 47.5% of patients, and positivity was significantly correlated with clinical stage, mediastinal or visceral involvement, microscopic BM involvement, and histological subtype, but not with skin or CNS involvement. Qualitative and quantitative PCR results in BM and PB strongly correlated. BM PCR was associated with the cumulative incidence of relapses (CI-R): CI-R was 50±10% for 38 PCR-positive and 15±7% for 42 PCR-negative patients (p10 NCN NPM-ALK in BM had a CI-R of 71±14% compared to a CI-R of 18±6% for 59 patients with =10 NCN (p10 NCN NPM-ALK in BM, clinical risk factors (skin, mediastinal or visceral involvement) and atypical histological subtype, only &gt;10 NCN NPM-ALK remained a significant poor prognostic factor with a risk ratio of 4.74 (1.57–14.3; p&lt;0.006). Conclusions: The detection of NPM-ALK positive cells by PCR in BM is associated with advanced stage disease, visceral involvement and atypical histology. Quantitative PCR in BM or PB allows identification of 20% of patients experiencing 60% of all relapses with an event-free survival of 20%. No significant financial relationships to disclose.

Improved endocervical sampling and HPV viral load detection by Cervex‐Brush® Combi

Liquid-based cytology (LBC) for cervical screening is becoming increasingly used. Together with SurePath LBC, various collecting devices can be utilized, among which the Cervex-Brush is the most widely used. The new Rovers Cervex-Brush Combi combines the advantages of the Cervex-Brush with the EndoCervex-Brush increasing sampling of the endocervical canal. The objective of this study was to analyse and to compare the Cervex-Brush Combi with the Cervex-Brush for the collection of squamous and endocervical cells, human papillomavirus (HPV) typing/quantification and disease detection in SurePath LBC. Using either the Cervex-Brush or the Cervex-Brush Combi 100 consecutive SurePath LBC samples were collected using each brush type. All 200 slides were read by the FocalPoint and screened by guided screening using slide wizards. The viral load of HPV type 16 E7, 18 E7, 31 E6, 33 L1, 33 E6, 35 E4, 39 E7, 45 E7, 51 E6, 52 L1, 52 E7, 53 E6, 56 E7, 58 L1, 58 E6, 59 E7, 66 E6 and 68 E7 was determined using a TaqMan-based real-time quantitative PCR analysis. The mean number of sampled squamous cells did not differ between the two brush types (54 963 versus 54 595 cells). The use of the Cervex-Brush Combi, however, resulted in a two- to threefold increase in the number of sampled endocervical cells (P < 0.00001). Using the Cervex-Brush Combi slightly more lesions were detected (three versus two low-grade squamous intraepithelial lesions), and resulted in the detection of more atypical squamous cells of undetermined significance (six versus three). In the Cervex-Brush group, 60% (3/5) of abnormal smears were positive for oncogenic HPV types, whereas 66.7% (6/9) of abnormal smears in the Cervex-Brush Combi group tested positive. The median HPV viral load for samples taken with the Cervex-Brush Combi was 0.1825 copies/cell and was significantly higher than in samples taken with the Cervex-Brush (0.0042 copies/cell) (P = 0.02). Sampling with the Cervex-Brush Combi resulted in the collection of the same amount of squamous cells, but in a two to threefold harvest of endocervical cells. This led to the detection of a higher viral load for oncogenic HPV and an increase in the number of detected abnormal smears.