TaqMan Quantitative PCR Assay Research Articles

Does the survival motor neuron copy number variation play a role in the onset and severity of sporadic amyotrophic lateral sclerosis in Malians?

IntroductionSpinal muscular atrophy (SMA) and sporadic amyotrophic lateral sclerosis (SALS) are both motor neuron disorders. SMA results from the deletion of the survival motor neuron (SMN) 1 gene. High or low SMN1 copy number and the absence of SMN2 have been reported as risk factors for the development or severity of SALS.ObjectiveTo investigate the role of SMN gene copy number in the onset and severity of SALS in Malians.Material and MethodsWe determined the SMN1 and SMN2 copy number in genomic DNA samples from 391 Malian adult volunteers, 120 Yoruba from Nigeria, 120 Luyha from Kenya and 74 U.S. Caucasians using a Taqman quantitative PCR assay. We evaluated the SALS risk based on the estimated SMA protein level using the Veldink formula (SMN1 copy number + 0.2 ∗ SMN2 copy number). We also characterized the disease natural history in 15 ALS patients at the teaching hospital of Point G, Bamako, Mali.ResultsWe found that 131 of 391 (33.5%) had an estimated SMN protein expression of ≤ 2.2; 60 out of 391 (15.3%) had an estimated SMN protein expression < 2 and would be at risk of ALS and the disease onset was as early as 16 years old. All 15 patients were male and some were physically handicapped within 1–2 years in the disease course.ConclusionBecause of the short survival time of our patients, family histories and sample DNA for testing were not done. However, our results show that sporadic ALS is of earlier onset and shorter survival time as compared to patients elsewhere. We plan to establish a network of neurologists and researchers for early screening of ALS.

A Quantitative Real-Time RT-PCR Assay for the Detection of Venezuelan equine encephalitis virus Utilizing a Universal Alphavirus Control RNA.

Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV.

CCN2 - Exploring a New Biomarker in Myelofibrosis

BACKGROUNDBone marrow fibrosis in myelofibrosis (MF) is the result of a complex and poorly understood interaction between megakaryocytes, fibroblasts, endothelial cells, cytokines and marrow stroma. Preclinical studies support a pathobiological role of TGF-β. It is overexpressed by megakaryocytes in MF and the TGF-β signature is upregulated and has been targeted in MF animal models. However TGF-β is a pleiotropic cytokine implicated in many cellular processes. CCN proteins are a group of 6 matricellular proteins important in fibrotic diseases and injury repair. CCN1 (CYR61) and CCN2 (CTGF) are transcriptionally activated by mitogenic growth factors such as PDGF, FGF2 and TGF-β. Recombinant CCN2 induces differentiation of human bone marrow mesenchymal stem cells into fibroblasts (Lee et al. JCI 2010). Levels of CCN2 in biologic fluids correlate with severity of fibrosis in scleroderma, liver cirrhosis and nephropathy. Given the proven role of CCN2 as a measurable serum biomarker in pro-fibrotic diseases and a downstream effector of TGF-β, in this retrospective study we examined CCN2 expression in myelofibrosis. We studied its correlation with clinical response following allogeneic stem cell transplant (ASCT), the only therapeutic modality to date which can consistently reverse fibrosis. METHODSPatients diagnosed with MF at our institution from 1998 to 2015 were identified by diagnostic code (IRB#2013-0896). Bone marrow (BM) specimens at diagnosis and 1 year following ASCT were retrieved. Staging bone marrows from lymphoma patients, read as normal, were used as negative controls. CCN2 localization and expression was assessed by IHC (Abcam 5097). The slides were scored from 0-100% for megakaryocyte cytoplasmic staining by a blinded hematopathologist. RNA was extracted and reverse transcribed from frozen fixed paraffin embedded (FFPE) BM samples using the Qiagen RNeasy FFPE RNA Purification Kit. Assays for CCN1, CCN2 and CCN3 (NOV) and endogenous controls RPLPO and GAPDH were performed using Taqman quantitative PCR assays. mRNA data were analyzed using the software package DataAssist (v3.01; Life Technologies). RESULTSCCN2 expression is upregulated in myelofibrosis compared to healthy bone marrow. mRNA analysis showed a 27 fold increase (p=0.19) in CCN2 mRNA expression in patients with MF when compared to healthy controls. IHC data also showed increased expression in megakaryocytes of MF patients compared to controls (63% vs 40%, p=0.28). Exploratory analysis showed a 36 fold increase in CCN2 mRNA expression in JAK2 negative (n=3) MF patients compared to JAK2 positive (n=7) patients (p=0.06), suggesting expression may depend on the molecular profile. CCN2 expression in the bone marrow of myelofibrosis patients is significantly downregulated at 1 year after allogeneic stem cell transplant. 13 pre-treatment cases of MF were compared with 6 post-transplant cases. The mean percentage of megakaryocyte with cytoplasmic expression of CCN2 by IHC in MF at diagnosis was 63%, compared to 22% in post-transplant specimens, (p= 0.01). mRNA extracted from the post-transplant marrows showed 0.01 fold expression of CCN2 compared to pre-transplant (p= 0.18). This decrease in CCN2 correlated with clinical and pathologic resolution of disease. The average DIPSS score pre-transplant was 2 (range 0-5) which improved to 0 for all patients post-transplant. The mean BM cellularity pre-transplant was 82% (range 50%-95%) and post-transplant was 48% (range 35%-65%). The average decrease in cellularity after transplant was 29%. Reticulin fibrosis ranged from grade 2-3 in the pre-transplant MF bone marrow samples which improved to grade 0-1 post-transplant. CONCLUSIONSCCN2 is a downstream effector of TGF-β and a measurable biomarker of fibrosis in fibroproliferative diseases. We show that CCN2 expression in myelofibrosis decreases significantly after ASCT, suggesting its role as a biomarker of fibrosis in this disease. This paralleled clinical and pathologic resolution of disease. This will need to be validated in a larger number of paired patient samples where in addition to bone marrow expression, serum CCN2 levels will be measured by ELISA. DisclosuresNo relevant conflicts of interest to declare.

Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India

Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 109 copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 109 to 10 copies) with coefficient of regression (R2) > 0.99, calculated average slope −3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and −3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV.

Monitoring the dynamics of syntrophic β-oxidizing bacteria during anaerobic degradation of oleic acid by quantitative PCR.

The ecophysiology of long-chain fatty acid-degrading syntrophic β-oxidizing bacteria has been poorly understood due to a lack of quantitative abundance data. Here, TaqMan quantitative PCR (qPCR) assays targeting the 16S rRNA gene of the known mesophilic syntrophic β-oxidizing bacterial genera Syntrophomonas and Syntrophus were developed and validated. Microbial community dynamics were followed using qPCR and Illumina-based high-throughput amplicon sequencing in triplicate methanogenic bioreactors subjected to five consecutive batch feedings of oleic acid. With repeated oleic acid feeding, the initial specific methane production rate significantly increased along with the relative abundances of Syntrophomonas and methanogenic archaea in the bioreactor communities. The novel qPCR assays showed that Syntrophomonas increased from 7 to 31% of the bacterial community 16S rRNA gene concentration, whereas that of Syntrophus decreased from 0.02 to less than 0.005%. High-throughput amplicon sequencing also revealed that Syntrophomonas became the dominant genus within the bioreactor microbiomes. These results suggest that increased specific mineralization rates of oleic acid were attributed to quantitative shifts within the microbial communities toward higher abundances of syntrophic β-oxidizing bacteria and methanogenic archaea. The novel qPCR assays targeting syntrophic β-oxidizing bacteria may thus serve as monitoring tools to indicate the fatty acid β-oxidization potential of anaerobic digester communities.

Taqman quantitative PCR for detection of Indian arecanut yellow leaf disease phytoplasma

Phytoplasmas belonging to 16SrXI-B subgroup are associated with the Indian arecanut yellow leaf disease (YLD). Efficient detection protocol is essential in disease management for both rouging the diseased palms and screening the seedlings used for planting. A Taqman quantitative PCR assay targeting the YLD phytoplasmas was developed. The assay was optimized and used for testing arecanut spindle leaf samples collected from plants growing in the disease endemic areas of Sullia, Karnataka. Thirteen out of 18 samples from symptomatic plants gave positive amplification. The healthy arecanut samples and negative control produced no amplification signal. Amplicon sequence confirmation ensured the reliability of the assay.

Development of rapid canine fecal source identification PCR-based assays.

The extent to which dogs contribute to aquatic fecal contamination is unknown despite the potential for zoonotic transfer of harmful human pathogens. We used genome fragment enrichment (GFE) to identify novel nonribosomal microbial genetic markers potentially useful for detecting dog fecal contamination with PCR-based methods in environmental samples. Of the 679 sequences obtained from GFE, we used 84 for the development of PCR assays targeting putative canine-associated genetic markers. Twelve genetic markers were shown to be prevalent among dog fecal samples and were rarely found in other animals. Three assays, DG3, DG37, and DG72, performed best in terms of specificity and sensitivity and were used for the development of SYBR Green and TaqMan quantitative PCR (qPCR) assays. qPCR analysis of 244 fecal samples collected from a wide geographic range indicated that marker concentrations were below limits of detection in noncanine hosts. As a proof-of-concept, these markers were detected in urban stormwater samples, suggesting a future application of newly developed methods for water quality monitoring.

Enhanced antiretroviral therapy in rhesus macaques improves RT-SHIV viral decay kinetics.

Using an established nonhuman primate model, rhesus macaques were infected intravenously with a chimeric simian immunodeficiency virus (SIV) consisting of SIVmac239 with the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase from clone HXBc2 (RT-SHIV). The impacts of two enhanced (four- and five-drug) highly active antiretroviral therapies (HAART) on early viral decay and rebound were determined. The four-drug combination consisted of an integrase inhibitor, L-870-812 (L-812), together with a three-drug regimen comprising emtricitabine [(-)-FTC], tenofovir (TFV), and efavirenz (EFV). The five-drug combination consisted of one analog for each of the four DNA precursors {using TFV, (-)-FTC, (-)-β-D-(2R,4R)-1,3-dioxolane-2,6-diaminopurine (amdoxovir [DAPD]), and zidovudine (AZT)}, together with EFV. A cohort treated with a three-drug combination of (-)-FTC, TFV, and EFV served as treated controls. Daily administration of a three-, four-, or five-drug combination of antiretroviral agents was initiated at week 6 or 8 after inoculation and continued up to week 50, followed by a rebound period. Plasma samples were collected routinely, and drug levels were monitored using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Viral loads were monitored with a standard TaqMan quantitative reverse transcriptase PCR (qRT-PCR) assay. Comprehensive analyses of replication dynamics were performed. RT-SHIV infection in rhesus macaques produced typical viral infection kinetics, with untreated controls establishing persistent viral loads of >10(4) copies of RNA/ml. RT-SHIV loads at the start of treatment (V0) were similar in all treated cohorts (P > 0.5). All antiretroviral drug levels were measureable in plasma. The four-drug and five-drug combination regimens (enhanced HAART) improved suppression of the viral load (within 1 week; P < 0.01) and had overall greater potency (P < 0.02) than the three-drug regimen (HAART). Moreover, rebound viremia occurred rapidly following cessation of any treatment. The enhanced HAART (four- or five-drug combination) showed significant improvement in viral suppression compared to the three-drug combination, but no combination was sufficient to eliminate viral reservoirs.

Evidence for free-living Bacteroides in Cladophora along the shores of the Great Lakes

Bacteroides is assumed to be restricted to the alimentary canal of animals and humans and is considered to be non-viable in ambient environments. We hypothesized that Bac- teroides could persist and replicate within beach-stranded Cladophora glomerata mats in south- ern Lake Michigan, USA. Mean Bacteroides concentration (per GenBac3 Taqman quantitative PCR assay) during summer 2012 at Jeorse Park Beach was 5.2 log calibrator cell equivalents (CCE) g �1 dry weight (dw), ranging from 3.7 to 6.7. We monitored a single beach-stranded mat for 3 wk; bacterial concentrations increased by 1.6 log CCE g �1 dw and correlated significantly with ambient temperature (p = 0.003). Clonal growth was evident, as observed by >99% nucleotide sequence similarity among clones. In in vitro studies, Bacteroides concentrations increased by 5.5 log CCE g �1 after 7 d (27°C) in fresh Cladophora collected from rocks. Partial sequencing of the 16S rRNA gene of 36 clones from the incubation experiment showed highly similar genotypes (≥97% sequence overlap). The closest enteric Bacteroides spp. from the National Center for Biotechnology Information database were only 87 to 91% similar. Genomic similarity, clonality, growth, and persistence collectively suggest that putative, free-living Bacteroides inhabit Cladophora mats of southern Lake Michigan. These findings may have important biological, med- ical, regulatory, microbial source tracking, and public health implications.

JAK2 V617F allele burden quantified by real time quantitative polymerase chain reaction and competitive polymerase chain reaction in patients with chronic myeloproliferative neoplasia

Assessing the clinical significance of JAK2 V617F mutant allele burden is complicated by a myriad of techniques reported to detect and quantify the mutation. As a consequence, the level of sensitivity and how the data is reported vary. Harmonization of well-defined molecular studies would permit evaluation of the clinical significance of measuring allele burden and rapid determination of the efficacy of novel agents for the treatment of chronic myeloproliferative neoplasia via multicenter clinical trials, at the subclinical level. Here we report a comparison between the widely available TaqMan quantitative real time polymerase chain reaction (Q-PCR) and competitive PCR (C-PCR) assays. We found that the tumor load was invariably greater when measured by C-PCR compared to that recorded by Q-PCR. Furthermore, none of the samples converted from undetectable to detectable when the enriched granulocyte (GR) fraction was tested. While a difference in the V617F allele levels was detected between GR fraction and whole blood, this was not statistically significant.

Abstract 4180: miRNA-mediated anticancer properties of the PPARα-activator fenofibrate on glioblastoma cell growth and migration.

Abstract Background: Glioblastoma is an aggressive tumor which originates from the glial cells and accounts for nearly 50% of adult brain tumors. These tumors are characterized by rapid proliferation, aggressive metastatic potential, and resistance to radio- and chemotherapy. MicroRNAs (miRNAs) are 22-nucleotides long RNAs that regulate gene expression post-transcriptionally. Fenofibrate is a potent agonist of peroxisome proliferator activated receptor alpha (PPARα) that is used for the treatment of cardiovascular disease and various metabolic disorders. In addition, treatment with Fenofibrate results in the proliferation of peroxisomes and a marked reduction in the growth of Glioblastoma cells. Methods: To evaluate the effects of treatment on miRNA expression, we used TaqMan quantitative real-time PCR assays from Life Technologies (Grand Island, NY). miRNAs were cloned into BLOCK-iT Pol II miR RNAi Expression Vectors (Life Technologies). Cell growth was evaluated using Guava ViaCount Reagent (Guava Technologies, Hayward, CA). Migration of miRNA-expressing cells was assessed 24h after plating on 8 μm membranes (BD Biosciences, San Jose, CA). Results: In this study, we utilized the LN229 Glioblastoma cell line and found that among the miRNAs up-regulated by Fenofibrate treatment, miR-634 and miR-136 targeted CYR61, a protein involved in migration and proliferation of cancer cells. We additionally found that miR-136 was up-regulated by Fenofibrate treatment while miR-155, another miRNA previously shown to target CYR61, was down-regulated by Fenofibrate. All three miRNAs were predicted and validated to directly target the 3’-UTR of Cyr61. Over-expression of miR-136 and miR-634 miRNAs negatively affected proliferation, but not migration, of LN229 cells. Forced expression of miR-155 reduced migration and addition of recombinant Cyr61 reversed this impairment in migration. Investigation of the signaling pathways regulated by miR-634, revealed an increased phosphorylation of p70 S6 kinase (Thr389), suggesting an induction of the mammalian target of rapamycin (mTOR) complex 1 pathway. Induction of the mTOR pathway was dependent on Akt and Erk activation. In miR-634 overexpressing cells, TSC2, a negative regulator of mTOR signaling, was found to be decreased. Further studies on the differential roles of miRs-136, -155, and -634 in controlling cell growth, cell size, and migration will provide important information on the molecular mechanisms involved in the anticancer properties of Fenofibrate. Citation Format: Duane Jeansonne, Marco Pacifici, Adam Lassak, Krzysztof Reiss, Francesca Peruzzi. miRNA-mediated anticancer properties of the PPARα-activator fenofibrate on glioblastoma cell growth and migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4180. doi:10.1158/1538-7445.AM2013-4180

TaqMan Real-Time Quantitative PCR Assay for Detection of Fluoroquinolone-Resistant Neisseria gonorrhoeae

It is noted that more than 99 % of fluoroquinolone resistance in Neisseria gonorrhoeae (QRNG) specimens have been shown to have the mutation of Ser91/Phe in the gyrA gene. In order to detect QRNG isolates as quickly as possible, the real-time TaqMan quantitative PCR assay was established for detection of the point mutation of Ser91/Phe in gyrA gene. The standard curve was generated automatically on ABI Prism PE7500. The correlation coefficient (r) of the standard curve was -0.9984 (R(2) = 0.9968), indicating a quietly precise log-linear relationship between the concentration of target DNA and the Ct value. Presently, correlated, cultured antimicrobial susceptibility testing of N. gonorrhoeae isolates continues to be the gold standard method for the detection of antimicrobial resistance. Comparison to the correlated, cultured antimicrobial susceptibility testing, the sensitivity and specificity of the established TaqMan assay for the detection of the QRNG specimens were 100 and 99 %, respectively. The TaqMan assay also allows for rapid detection of QRNG isolates without complex laboratory techniques. Therefore, real-time TaqMan quantitative PCR assay is a rapid, simple, highly sensitive, highly specific, and easy-to-perform method for the detection of the QRNG specimens. It can be applied as a quick screening method for QRNG isolates to help clinical determination of optimal treatment prescription.

EBV Copy Number Variation in Twins Discordant for Young Adult Hodgkin Lymphoma

Abstract 2631 Background:Epstein-Barr Virus (EBV) is a risk factor for some types of Hodgkin lymphoma (HL). Host response to the virus may play a role in pathogenesis and is in part determined by genetic factors. Therefore, using only traditional unrelated controls may not be appropriate. Methods:We compared EBV DNA copy number in peripheral blood mononuclear cells (PBMCs) collected from 63 young adult HL patients in remission and their unaffected co-twins, and 43 spouse controls. Viral load was measured with TaqMan quantitative PCR assays, tested in triplicate and reported as viral copies per million cells. Samples positive in all 3 replicates were classified as positive, and those positive in 1–2 samples were classified as qualitative positive (QP). EBV copy number was log-transformed for some analyses. A binomial proportion test was used to compare EBV copy number in cases and their unaffected co-twins (n=50 like-sex pairs). Conditional (case vs. unaffected co-twin) and unconditional logistic regression (case and unaffected co-twin vs. spouse controls) were used to estimate the effect of EBV copy number on risk as a categorical (positive, QP, or negative) or continuous variable (SAS 9.1). Unconditional logistic regression analyses were adjusted for sex and age. Results:26% of case, 18% of unaffected co-twins and 9% of spouse controls had positive EBV copy number measurements. Like-sex young adult HL cases had higher EBV copy numbers compared to their unaffected co-twins (P = 0.04). When EBV copy number was considered as a categorical variable, both cases and monozygotic unaffected co-twins were more likely to be positive compared to controls (cases v. controls: Odds Ratio [OR] = 3.2, 95% confidence intervals [CI] = 0.9, 11.1; unaffected co-twins v. controls: OR= 3.6, 95% CI= 0.9, 14.6). However, there was little difference between dizygotic unaffected co-twins and controls (OR= 0.7, 95% CI = 0.5, 5.5). Conclusions:That young adult HL cases had higher EBV DNA copies than their unaffected co-twins implies that an acquired pre-disease factor or disease affect altered the host response to EBV. However, that both cases and unaffected co-twins had higher EBV DNA copies compared to controls suggests that there is also a heritable component to the host response to EBV.HL cases > co-twins (acquired or disease effect)HL cases and co-twins > controls (heritable effect) [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Quantitative PCR demonstrates a positive correlation between a Rickettsia‐like organism and severity of strawberry disease lesions in rainbow trout, Oncorhynchus mykiss (Walbaum)

Strawberry disease (SD) is an inflammatory skin disorder in rainbow trout, Oncorhynchus mykiss (Walbaum). The aetiology of SD is unknown although the 16S rDNA sequence of a Rickettsia-like organism (RLO) has been associated with SD lesions using a nested PCR assay. In this study, we developed a Taqman quantitative PCR assay (qPCR) that targeted the RLO 16S rDNA sequence to examine the distribution of RLO relative to lesion status. We compared 18 lesion samples from 13 fish representing high or low lesion severity as judged by gross examination. QPCR results showed that there was a higher number of RLO sequences in high severity lesions (mean of 12,068 copies) compared with fewer copies of RLO sequence in low severity lesions (mean of 3287 copies, P = 0.012). Grossly normal skin samples (n = 13) from SD-affected fish were all negative by qPCR except two samples (121 and 139 copies). The qPCR assay described herein is a useful tool to investigate the role of RLO in SD in the absence of a culture system for RLO. Our results demonstrate a positive correlation between copy number and lesion severity consistent with the hypothesis that the RLO is the aetiologic agent of SD.

Development of a real-time PCR targeting the yidC gene for the detection of Mycoplasma hominis and comparison with quantitative culture

Mycoplasma hominis is an opportunistic human mycoplasma species that can be either commensal or pathogenic. Its detection by culture is considered to comprise the reference technique. Previously reported PCR techniques target the 16S rRNA or the gap gene, although sequence variations among clinical isolates may lead to variations in clinical sensitivity. The present study aimed to develop a specific TaqMan quantitative real-time PCR assay, targeting a gene conserved in all M. hominis isolates, and to compare it with quantitative culture. With the knowledge of the M. hominis PG21 genome sequence, the yidC gene, encoding a membrane protein translocase, was chosen as target. Its intraspecies heterogeneity was checked at the nucleotide level using 31 reference or clinical strains. The limit of detection, the analytical specificity and the reproducibility of the assay were assessed. Moreover, PCR and culture results were compared using 153 urogenital specimens. The limit of detection was seven copies/μL. The analytical specificity was 100%, with good inter- and intra-assay reproducibility. Among the 153 urogenital specimens, the yidC PCR and culture allowed detection of 55 and 45 M. hominis-positive samples, respectively. Comparison of the bacterial load among the 45 specimens found to be M. hominis-positive by both techniques revealed a statistically significant association between the quantitative results obtained. In conclusion, we developed a specific, sensitive and reproducible real-time PCR to detect all M. hominis clinical isolates. This PCR was shown to have higher sensitivity than culture, although both methods were correlated for quantification of M. hominis loads in urogenital specimens.

Aging and microRNA expression in human skeletal muscle: a microarray and bioinformatics analysis

A common characteristic of aging is loss of skeletal muscle (sarcopenia), which can lead to falls and fractures. MicroRNAs (miRNAs) are novel posttranscriptional modulators of gene expression with potential roles as regulators of skeletal muscle mass and function. The purpose of this study was to profile miRNA expression patterns in aging human skeletal muscle with a miRNA array followed by in-depth functional and network analysis. Muscle biopsy samples from 36 men [young: 31 ± 2 (n = 19); older: 73 ± 3 (n = 17)] were 1) analyzed for expression of miRNAs with a miRNA array, 2) validated with TaqMan quantitative real-time PCR assays, and 3) identified (and later validated) for potential gene targets with the bioinformatics knowledge base software Ingenuity Pathways Analysis. Eighteen miRNAs were differentially expressed in older humans (P < 0.05 and >500 expression level). Let-7 family members Let-7b and Let-7e were significantly elevated and further validated in older subjects (P < 0.05). Functional and network analysis from Ingenuity determined that gene targets of the Let-7s were associated with molecular networks involved in cell cycle control such as cellular proliferation and differentiation. We confirmed with real-time PCR that mRNA expression of cell cycle regulators CDK6, CDC25A, and CDC34 were downregulated in older compared with young subjects (P < 0.05). In addition, PAX7 mRNA expression was lower in older subjects (P < 0.05). These data suggest that aging is characterized by a higher expression of Let-7 family members that may downregulate genes related to cellular proliferation. We propose that higher Let-7 expression may be an indicator of impaired cell cycle function possibly contributing to reduced muscle cell renewal and regeneration in older human muscle.

Identification, Molecular Characterization, and Occurrence of Two Bovine Hemoplasma Species in Swiss Cattle and Development of Real-Time TaqMan Quantitative PCR Assays for Diagnosis of Bovine Hemoplasma Infections

Concomitantly with an outbreak of fatal anaplasmosis in a cattle herd in Switzerland in 2002, we detected two bovine hemoplasma species in diseased animals: Mycoplasma wenyonii (formerly Eperythrozoon wenyonii) and a second, novel bovine hemoplasma species later designated "Candidatus Mycoplasma haemobos" (synonym, "Candidatus Mycoplasma haemobovis"). The second species was characterized by a shorter 16S rRNA gene. The aims of the present study were to provide a detailed molecular characterization of this species, to develop specific quantitative real-time PCR assays for the two bovine hemoplasma species, and to apply these assays in order to evaluate the prevalence and clinical significance of the hemoplasmas. Sequencing of the near-complete 16S rRNA gene of the second hemoplasma revealed that it was 94% identical to that of Mycoplasma haemofelis, an anemia-inducing feline hemoplasma species, but less than 85% identical to that of the bovine hemoplasma M. wenyonii. Using the newly developed assays, a total of 159 animals from the anaplasmosis outbreak were reexamined. In addition, we tested 57 clinically ill and 61 healthy Swiss cattle, as well as 47 calves. Both hemoplasmas were highly prevalent in adult cattle but occurred rarely in calves. Animals from the herd with the fatal anemia outbreak were more frequently infected with M. wenyonii and exhibited higher M. wenyonii blood loads than animals with unrelated diseases and healthy animals. Coinfections may increase the pathogenicity and clinical significance of bovine hemoplasmosis.

Evolutionary divergence and biogeography of sympatric niche-differentiated bacterial populations

Using multiple lines of evidence from denaturing gradient gel electrophoresis, environmental sequences and TaqMan quantitative PCR assays targeting a functional gene for sulfate respiration (dsr) affiliated with the geochemically important genus Desulfobulbus, we revealed strongly restricted distributions of specific genotypes and populations correlated with sampling position along an estuarine gradient free of dispersal barriers. Evidence of evolutionary divergence of populations was provided by three complementary analyses. First, analysis of molecular variance rejected the null hypothesis that genetic diversity within each sampling site was not significantly different than that of all sites pooled together (P<0.0001). Second, UniFrac and Parsimony tests showed phylogenetic clustering of sampling sites was highly significant (P<0.001). Third, pairwise F(ST) statistics showed significant evolutionary divergence of populations based on the location in the estuary. To test the hypothesis that environmental niche-driven evolutionary divergence can create and maintain microbial biogeography, we used both statistical inference and an experimental manipulation to assess the independent effects of environment and geography. Significant effects of each on genotype distributions and population divergence supported the hypothesis. Our data are consistent with both sympatric and parapatric models of speciation, and suggest niche partitioning can contribute to evolutionary divergence and observable biogeographic patterns in microbial communities even among closely related taxa at limited spatial scales without significant barriers to dispersal.

Qualitative and Quantitative Detection of Chlamydophila pneumoniae DNA in Cerebrospinal Fluid from Multiple Sclerosis Patients and Controls

A standardized molecular test for the detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid (CSF) would assist the further assessment of the association of C. pneumoniae with multiple sclerosis (MS). We developed and validated a qualitative colorimetric microtiter plate-based PCR assay (PCR-EIA) and a real-time quantitative PCR assay (TaqMan) for detection of C. pneumoniae DNA in CSF specimens from MS patients and controls. Compared to a touchdown nested-PCR assay, the sensitivity, specificity, and concordance of the PCR-EIA assay were 88.5%, 93.2%, and 90.5%, respectively, on a total of 137 CSF specimens. PCR-EIA presented a significantly higher sensitivity in MS patients (p = 0.008) and a higher specificity in other neurological diseases (p = 0.018). Test reproducibility of the PCR-EIA assay was statistically related to the volumes of extract DNA included in the test (p = 0.033); a high volume, which was equivalent to 100 µl of CSF per reaction, yielded a concordance of 96.8% between two medical technologists running the test at different times. The TaqMan quantitative PCR assay detected 26 of 63 (41.3%) of positive CSF specimens that tested positive by both PCR-EIA and nested-PCR qualitative assays. None of the CSF specimens that were negative by the two qualitative PCR methods were detected by the TaqMan quantitative PCR. The PCR-EIA assay detected a minimum of 25 copies/ml C. pneumoniae DNA in plasmid-spiked CSF, which was at least 10 times more sensitive than TaqMan. These data indicated that the PCR-EIA assay possessed a sensitivity that was equal to the nested-PCR procedures for the detection of C. pneumoniae DNA in CSF. The TaqMan system may not be sensitive enough for diagnostic purposes due to the low C. pneumoniae copies existing in the majority of CSF specimens from MS patients.

Comparison of Automated Microarray Detection with Real-Time PCR Assays for Detection of Respiratory Viruses in Specimens Obtained from Children

Respiratory virus infections are a major health concern and represent the primary cause of testing consultation and hospitalization for young children. We developed and compared two assays that allow the detection of up to 23 different respiratory viruses that frequently infect children. The first method consisted of single TaqMan quantitative real-time PCR assays in a 96-well-plate format. The second consisted of a multiplex PCR followed by primer extension and microarray hybridization in an integrated molecular diagnostic device, the Infiniti analyzer. Both of our assays can detect adenoviruses of groups A, B, C, and E; coronaviruses HKU1, 229E, NL63, and OC43; enteroviruses A, B, C, and D; rhinoviruses of genotypes A and B; influenza viruses A and B; human metapneumoviruses (HMPV) A and B, human respiratory syncytial viruses (HRSV) A and B; and parainfluenza viruses of types 1, 2, and 3. These tests were used to identify viruses in 221 nasopharyngeal aspirates obtained from children hospitalized for respiratory tract infections. Respiratory viruses were detected with at least one of the two methods in 81.4% of the 221 specimens: 10.0% were positive for HRSV A, 38.0% for HRSV B, 13.1% for influenzavirus A, 8.6% for any coronaviruses, 13.1% for rhinoviruses or enteroviruses, 7.2% for adenoviruses, 4.1% for HMPV, and 1.5% for parainfluenzaviruses. Multiple viral infections were found in 13.1% of the specimens. The two methods yielded concordant results for 94.1% of specimens. These tests allowed a thorough etiological assessment of respiratory viruses infecting children in hospital settings and would assist public health interventions.