Reverse transcription-PCR Method Research Articles

Molecular detection of human parechovirus type 1 in stool samples from children with diarrhea

Human parechovirus type 1 (HPeV-1) appears to be associated with gastrointestinal and respiratory infections, and occasionally with central nervous system symptoms. Since there is no accurate information about the incidence and epidemiology of HPeV-1 in Iran, this study was designed to describe the frequency of HPeV-1 in stool samples from children under 4 years of age with gastroenteritis. RNA was isolated from stool suspensions and cDNA was prepared and amplified using a specific nested reverse transcription PCR (RT-PCR). We also compared this RT-PCR method with a cell culture procedure. Out of 472 samples, 112 (23.7%) were HPeV-1-positive. The incidence and prevalence of this virus was highest in children under 1 year old with diarrhea (p=0.036), and was higher in the spring and autumn than in summer and winter (p<0.001). More males than females were HPeV-1-positive (p<0.001). This is the first systematic study of the incidence and epidemiology of HPeV-1 among children with acute gastroenteritis in Iran. The results revealed that RT-PCR is a more practical, sensitive, and rapid technique for the detection of HPeV-1 directly from clinical samples than cell culture, which makes this a valuable method for epidemiology. Also, the rapid detection of virus can decrease both the unnecessary use of antibiotics and costs in clinical practice.

Detection of viable potato cyst nematodes (Globodera rostochiensis and G. pallida) by reverse transcription PCR

Currently plant infectivity assays (bioassays) seem most reliable to determine viability of potato cyst nematodes (Globodera rostochiensis and G. pallida), but they are time consuming and labour-intensive. Hence, the objective of the present investigation was to establish a reliable and time-saving reverse transcription PCR (RT-PCR) assay for determining viability of potato cyst nematodes. Sensitivity of the developed test was very high enabling the detection of the gpd1 mRNA of one untreated second-stage juvenile (J2) in 200 μl of water in 5 out of 10 tested samples. The adopted RT-PCR protocol was used to study the thermo-sensitivity of G. rostochiensis and G. pallida between 35 and 55°C as well as the fate during anaerobic digestion at 38°C. The test proved to be very robust concerning PCR inhibiting factors, mRNA detection of ten untreated J2 in 200 μl of digester substrate was always successful. Results of the RT-PCR assay and the bioassay congruently showed that 4 days of anaerobic digestion were suitable to decontaminate plant material containing cysts of G. rostochiensis or G. pallida. Whereas usefulness of the RT-PCR to estimate the viability of heat treated cysts was limited to long incubation times or temperature treatments above 45°C, because mRNA persisted unexpectedly long at short incubation times or temperature treatments below 45°C. Nevertheless the presented RT-PCR method may be useful for selected applications that require a fast and sensitive determination of the vitality of potato cyst nematodes.

Development of Early Humoral and Cell-Mediated Immunity in Piglets with Experimentally Induced Subclinical Swine Influenza

Abstract Development of early immune response in piglets with subclinical swine influenza was investigated. Fourteen, seronegative piglets were used. Ten of them were infected intranasally with swine influenza virus (SIV) H1N1 subtype. Temperature and clinical signs were assessed daily. Leukocyte proportions and concentrations were analysed on a haematology analyser. Antibodies against SIVs were measured by haemagglutination inhibition assay. To measure influenza-specific cell-mediated immunity (CMI), the proliferation assay was performed. The real time reverse transcription PCR method was used for detection of SIV. No relevant respiratory or systemic clinical signs were observed. The presence of SIV RNA in nasal swabs from all infected piglets was confirmed between 2 and 5 dpi. The overall number of leukocytes did not differ during the study. The number of medium-sized cells (MID) was significantly higher on 2 and 4 dpi, as compared to day 0 level. The percentage of lymphocytes decreased from 74% on day 0 to 67.06% on 4 dpi, while the percentage of MID significantly increased at the same time. In control pigs no significant changes were observed. All infected pigs exhibited specific antibodies between 7 and 10 dpi. Specific CMI was observed before specific antibodies were present. Results of our research indicate that kinetics of the humoral and CMI response during subclinical infection is similar to that observed in clinical form of the disease.

Atorvastatin delays progression of pancreatic lesions to carcinoma by regulating PI3/AKT signaling in p48Cre/+ LSL-KrasG12D/+ mice.

Pancreatic cancer is the one of most common causes of cancer deaths and has the worst prognosis. Clinical observational studies suggest that statins may reduce the risk of pancreatic cancer. The chemopreventive efficacy of the statin atorvastatin (Lipitor(®)) and the role of the phosphatidyl-inositol 3-kinase (PI3/AKT) signaling pathway were evaluated for the progression of pancreatic intraepithelial neoplasms (PanINs) to pancreatic ductal adenocarcinoma (PDAC) in conditional p48(Cre/+) -LSL-Kras(G12D/+) transgenic mice. Six-week-old male p48(Cre/+) -LSL-Kras(G12D/+) (20/group) mice were fed AIN-76A diets containing 0, 200 and 400 ppm atorvastatin for 35 weeks. At termination, pancreata were evaluated histopathologically for PanINs and PDAC, and for various PI3/AKT signaling markers, and inflammatory cytokines, by immunohistochemistry/immunohistoflourscence, ELISA, Western blotting and/or reverse transcription-PCR methods. Control diet-fed mice showed 85% incidence of PDAC; whereas, mice fed with atorvastatin showed PDAC incidence of 65 and 35%, respectively (p < 0.0001). Similarly, significant suppression of PanIN-3 (22.6%) was observed in mice fed 400 ppm atorvastatin. Importantly, pancreata from atorvastatin-treated mice were ∼68% free from ductal lesions. Furthermore, pancreas of mice administered with atorvastatin had significantly reduced expressions levels of PCNA, p2X7, p-ERK, RhoA, cyclin D1, survivin, Akt, pAKT, β-catenin, cyclin E, cdK2 and caveolin-1. Also, atorvastatin-treated mice had shown dose-dependent suppression of inflammatory cytokines and a significant increase in tunnel-positive cells, p21 and PARP expression levels in pancreas. Atorvastatin significantly delays the progression of PanIN-1 and -2 lesions to PanIN-3 and PDAC by modulating PI3/AKT signal molecules in a preclinical model, suggesting potential clinical benefits of statins for high-risk pancreatic cancer patients.

Specific TaqMan probed real-time quantitative RT-PCR methods and their application to differentiate the transcripts of duplicated BF or BLB genes in chicken MHC

BF and BLB genes of chicken major histocompatibility complex (MHC) are responsible for classical antigen processing and presentation; therefore they play a central role in determining the genetic resistance or susceptibility of different MHC-B haplotypes to some infectious diseases. In this study, we developed specific TaqMan probed real-time quantitative reverse transcription PCR (TaqMan qRT-PCR) methods based on the diagnostic nucleotide polymorphisms present in duplicated BF or BLB genes in B2 and B19 haplotypes. The results showed very similar amplification efficiency but no cross-reaction between the duplicated BF or BLB genes of the same haplotype. Spleen mRNA samples of B2 and B19 chickens were used to validate these TaqMan qRT-PCR methods. We observed that BF2 or BLB2 gene was dominantly transcribed in all B2 and B19 chickens. Our findings verified the impact of diversified promoter sequences on the function of duplicated BF or BLB genes. Hence the principles adopted to establish these specific TaqMan qRT-PCR methods in this study can be applied to differentiate the transcripts of duplicated BF or BLB genes of other MHC-B haplotypes in chicken.

Characterization of Edwardsiella tarda rpoN: roles in σ70 family regulation, growth, stress adaption and virulence toward fish

Edwardsiella tarda EIB202, a Gram-negative pathogen with strong virulence, is an opportunistic pathogen capable of causing edwardsiellosis with high mortality to fish. Alternative sigma factor 54 (RpoN) is an important regulator of virulence and stress resistance genes in many bacterial species and mainly responsible for transcription of genes in nitrogen utilization. In this study, the in-frame rpoN deletion mutant was constructed to analyze the function of RpoN in Edwardsiella tarda firstly. Compared to the wild-type and complemented strain rpoN (+), the ΔrpoN was impaired in terms of the ability to survive under oxidative stress, osmotic stress and acid resistance, as well as the growth in Luria-Bertani medium, demonstrating essential roles of RpoN in stress resistance and nitrogen utilization. In addition, the ΔrpoN displayed markedly decreased biofilm formation and chondroitinase activity and was attenuated in virulence reflected in the increased median lethal dose value and extended infection cycle. Real-time polymerase chain reaction revealed that the expression levels of σ(70) class changed in varying degrees in the rpoN mutant. Especially, the expression levels of rpoS and fliA were down-regulated 4.1-fold and 7.9-fold in stationary phase in comparison with the wild type, respectively. Furthermore, two differential expression genes, znuA and flhC, were detected in the wild type and ΔrpoN using the method of differential display reverse transcription PCR.

Serum amyloid A triggers the mosodium urate -mediated mature interleukin-1β production from human synovial fibroblasts

BackgroundMonosodium urate (MSU) has been shown to promote inflammasome activation and interleukin-1β (IL-1β) secretion in monocyte/macrophages, but the cellular pathway and nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation in synovial tissues, remain elusive. In this study, we investigated the effects of MSU on synovial fibroblasts to elucidate the process of MSU-mediated synovial inflammation.MethodsHuman synovial fibroblasts were stimulated with MSU in the presence or absence of serum amyloid A (SAA). The cellular supernatants were analyzed by immunoblotting using anti-IL-1β or anti-caspase-1 antibodies. IL-1β or NLRP3 mRNA expressions were analyzed by real-time PCR or reverse transcription-PCR (RT-PCR) method.ResultsNeither SAA nor MSU stimulation resulted in IL-1β or interleukin-1α (IL-1α) secretions and pro-IL-1β processing in synovial fibroblasts. However, in SAA-primed synovial fibroblasts, MSU stimulation resulted in the activation of caspase-1 and production of active IL-1β and IL-1α. The effect of SAA on IL-1β induction was impaired in cells by silencing NLRP3 using siRNA or treating with caspase-1 inhibitor. In addition, SAA induced the secretion of cathepsin B and NLRP3 mRNA expression in synovial fibroblasts.ConclusionsOur data demonstrate that exposure of human synovial fibroblasts to SAA promotes MSU-mediated caspase-1 activation and IL-1β secretion in the absence of microbial stimulation. These findings provide insight into the molecular processes underlying the synovial inflammatory condition of gout.

Detection of Negative-Sense RNA in Packaged Hepatitis E Virions by Use of an Improved Strand-Specific Reverse Transcription-PCR Method

Current hepatitis E virus (HEV) negative-sense RNA detection assays have the drawback of false positivity. cDNA synthesis using tag-based primer and Superscript RT-III followed by exonuclease I treatment increased the specificity. Assays could detect as few as 10 copies of negative-sense RNA and could be used in detecting low levels of HEV replication in cells. Virus particles in stool samples of hepatitis E patients showed encapsidation of negative-sense RNA along with HEV genomic RNA.

Serotype Specific Primers and Gel-Based RT-PCR Assays for ‘Typing’ African Horse Sickness Virus: Identification of Strains from Africa

African horse sickness is a devastating, transboundary animal disease, that is ‘listed’ by the Office International des Epizooties (OIE). Although attenuated, inactivated and subunit vaccines have been developed for African horse sickness virus (AHSV), these are serotype-specific and their effective deployment therefore relies on rapid and reliable identification of virus type. AHSV serotype is controlled by the specificity of interactions between neutralising antibodies, and components of the outer-capsid, particularly protein VP2 (encoded by AHSV genome segment 2 (Seg-2)). We report the development and evaluation of novel gel based reverse transcription-PCR (RT–PCR) assays targeting AHSV Seg-2, which can be used to very significantly increase the speed and reliability of detection and identification (compared to virus neutralisation tests) of the nine serotypes of AHSV. Primer sets were designed targeting regions of Seg-2 that are conserved between strains within each of the AHSV serotype (types 1 to 9). These assays were evaluated using multiple AHSV strains from the orbivirus reference collection at IAH (www.reoviridae.org/dsRNA_virus_proteins/ReoID/AHSV-isolates.htm). In each case the Seg-2 primers showed a high level of specificity and failed to cross-amplify the most closely related heterologous AHSV types, or other related orbiviruses (such as bluetongue virus (BTV), or equine encephalosis virus (EEV)). The assays are rapid and sensitive, and can be used to detect and type viral RNA in blood, tissue samples, or cultivated viral suspensions within 24 h. They were used to identify AHSV strains from recent outbreaks in sub-Saharan African countries. These methods also generate cDNAs suitable for sequencing and phylogenetic analyses of Seg-2, identifying distinct virus lineages within each virus-type and helping to identify strain movements/origins. The RT-PCR methods described here provide a robust and versatile tool for rapid and specific detection and identification of AHSV serotypes 1 to 9.

Three years surveillance of viral etiology of acute lower respiratory tract infection in children from 2007 to 2010

Viruses are common pathogens of acute lower respiratory tract infection (ALRTI) in children. There are few studies on consecutive monitoring of viral pathogens of ALRTI in a larger cohort during the past several years. The aim of this study was to investigate the viral pathogens of ALRTI in children of different age groups and to outline the epidemic feature of different viruses. (1) Totally 1914 (1281 male and 709 female) children with clinical diagnosis of ALRTI during the period of March 2007 to March 2010 were recruited into this study. These patients were hospitalized patients in department of internal medicine or outpatients in emergency department in Beijing Children's Hospital. The patients were divided into four groups, including 1072 patients < 1 year old, 326 patients 1- < 3 years old, 158 patients 3- < 6 years old, 358 patients ≥ 6 years old. One nasopharyngeal aspirate specimen was collected from each patient. Reverse transcription (RT) PCR methods were applied to detect common respiratory viruses including respiratory syncytial virus (RSV), human rhinovirus (HRV), influenza virus type A, B and C (IFA, IFB, IFC), parainfluenza virus (PIV) type 1-4, adenovirus (ADV), enterovirus (EV), human coronavirus (HCOV), human metapneumovirus (HMPV) and human bocavirus (HBOV). (1) The total positive rate of viruses was 70.3%. The positive rate was 83.0% (890/1072) in the group of < 1 year old, and 80.1% (261/326) in group of 1- < 3 years old, 60.8% (96/158) in group of 3- < 6 years old and 27.7% (99/358) in group of ≥ 6 years old, respectively. There was a significant difference in the positive rate among different age groups (χ² = 2213.5, P = 0.000). The top three viruses were RSV, HRV and PIV; and the positive rates were 50.9%, 36.2% and 12.0% respectively in group of < 1 year old. (2) The epidemic seasons of RSV and HRV were winter and spring, and PIV infection was epidemic in spring and summer. (3) The detection rates of 2 or more viruses were 38.2%, 36.4%, 30.2% and 15.2% in groups of < 1 year old, 1- < 3 years old, 3- < 6 years old and ≥ 6 years old, respectively. There was a significant difference in the mixed infection rate among different age groups (χ² = 1346.00, P = 0.000). RSV, HRV and PIV were the most predominant pathogens in younger children with ALRTI. Different viral infections had different seasonal features. Mixed infections with two or more viruses were detected in substantial proportion of patients with ALRTI, but further studies are needed to explore the clinical significance of mixed infection with viruses in patients with ALRTI.

Comparison of a Newly Developed Automated and Quantitative Hepatitis C Virus (HCV) Core Antigen Test with the HCV RNA Assay for Clinical Usefulness in Confirming Anti-HCV Results

Hepatitis C virus (HCV) is a global health care problem. Diagnosis of HCV infection is mainly based on the detection of anti-HCV antibodies as a screening test with serum samples. Recombinant immunoblot assays are used as supplemental tests and for the final detection and quantification of HCV RNA in confirmatory tests. In this study, we aimed to compare the HCV core antigen test with the HCV RNA assay for confirming anti-HCV results to determine whether the HCV core antigen test may be used as an alternative confirmatory test to the HCV RNA test and to assess the diagnostic values of the total HCV core antigen test by determining the diagnostic specificity and sensitivity rates compared with the HCV RNA test. Sera from a total of 212 treatment-naive patients were analyzed for anti-HCV and HCV core antigen both with the Abbott Architect test and with the molecular HCV RNA assay consisting of a reverse transcription-PCR method as a confirmatory test. The diagnostic sensitivity, specificity, and positive and negative predictive values of the HCV core antigen assay compared to the HCV RNA test were 96.3%, 100%, 100%, and 89.7%, respectively. The levels of HCV core antigen showed a good correlation with those from the HCV RNA quantification (r = 0.907). In conclusion, the Architect HCV antigen assay is highly specific, sensitive, reliable, easy to perform, reproducible, cost-effective, and applicable as a screening, supplemental, and preconfirmatory test for anti-HCV assays used in laboratory procedures for the diagnosis of hepatitis C virus infection.

Evaluation of third-generation ELISA and a rapid immunochromatographic assay for the detection of norovirus infection in fecal samples from inpatients of a German tertiary care hospital

The analytical accuracy of the RIDASCREEN Norovirus 3rd Generation ELISA assay and the rapid immunochromatographic RIDAQUICK Norovirus assay were determined in comparison to PCR. In a prospective study 410 consecutive samples were collected from inpatients of a tertiary care hospital in Germany. All samples were tested with the two antigen detection assays, as well as with three different real-time reverse transcription PCR methods as the reference standard. A sample was considered true-positive if at least 2 out of 3 PCR methods yielded a positive signal (137 positive samples, >99% genogroup II). Compared with the PCR-based reference the overall diagnostic sensitivities of the ELISA and the immunochromatographic assay were 77% and 69% and the diagnostic specificities were 96% and 97% respectively. Both assays allow the rapid and economic screening of large numbers of samples and thus are useful diagnostic tools for the detection of suspected norovirus infections.

Reverse transcription-PCR methods significantly impact richness and composition measures of expressed fungal cellobiohydrolase I genes in soil and litter

The importance of soil fungi in complex carbon degradation and the recent identification of genes involved in this process have sparked considerable interest in examining fungal gene expression in situ. Expression of target eukaryotic genes is commonly examined using reverse transcription (RT)-PCR, during which single-stranded (ss) complementary DNA (cDNA) is synthesized from an oligo (dT) primer and the gene of interest is subsequently amplified by PCR using gene specific primers. Another method that is being increasingly employed in environmental gene expression studies is SMART PCR, which generates and amplifies double-stranded (ds) complementary DNA (cDNA) from sscDNA using PCR, prior to gene-specific PCR. We performed a replicated comparison of these two methods using RNA extracted from forest soil and litter to determine if the two approaches yielded comparable results. Richness, composition and reproducibility of gene expression profiles of the fungal glycosyl hydrolase family 7 (GH7) cellobiohydrolase I gene ( cbhI) were examined when amplified from sscDNA or dscDNA synthesized using SMART PCR. In the dscDNA libraries from soil or litter samples, richness was significantly reduced and the composition was altered relative to sscDNA libraries. Library composition was significantly more reproducible among replicate sscDNA libraries than among parallel dscDNA libraries from litter. In sum, the reduced richness and altered composition produced in the dscDNA libraries could substantially influence ecological interpretations of the data. Defining the factors underpinning the methodological biases will potentially aid in optimizing the design of gene expression studies in soils and other complex environmental samples.

Rapid Simultaneous Detection of Enterovirus and Parechovirus RNAs in Clinical Samples by One-Step Real-Time Reverse Transcription-PCR Assay

Enteroviruses (EVs) are recognized as the major etiological agent in meningitis in children and young adults. The use of molecular techniques, such as PCR, has substantially improved the sensitivity of enterovirus detection compared to that of virus culture methods. PCR-based methods also can detect a much wider range of EV variants, including those within species A, as well as human parechoviruses (HPeVs) that often grow poorly in vitro and which previously have been underdiagnosed by traditional methods. To exploit these developments, we developed a real-time one-step reverse transcription-PCR (RT-PCR) for the rapid and sensitive detection of EV and HPeV in clinical specimens. Two commercially available RT-PCR kits were used (method I, Platinum one-step kit; method II, Express qPCR one-step kit) with primers and probes targeting the EV and HPeV 5'-untranslated regions (5'UTR). Amplification dynamics (threshold cycle [C(T)]values and efficiencies) of absolutely quantified full-length RNA transcripts representative of EV species A to D and HPeV were similar, demonstrating the effectiveness of both assays across the range of currently described human EV and HPeV variants. Probit analysis of multiple endpoint replicates demonstrated comparable sensitivities of the assays for EV and HPeV (method I, approximately 10 copies per reaction for both targets; method II, 20 copies per reaction). C(T) values were highly reproducible on repeat testing of positive controls within assays and between assay runs. Considering the sample turnaround time of less than 3 h, the multiplexed one-step RT-PCR method provides rapid diagnostic testing for EV and HPeV in cases of suspected central nervous system infections in a clinically relevant time frame.

DLBS3233 increases glucose uptake by mediating upregulation of PPARγ and PPARδ expression

DLBS3233 is a standardized extract combination containing Lagerstroemia speciosa and Cinnamomum burmannii. The effect on glucose uptake, adiponectin secretion, and insulin signaling of DLBS3233 (1–5 μg/ml) was examined in this study. 3T3 Swiss albino preadipocytes and adipocytes were used to investigate gene expressions which were detected using reverse transcription PCR method. DLBS3233 was found to enhance the expression of genes associated with increased insulin signaling and sensitivity such as PPARγ and PPARδ. In addition, the expressions of PI3 kinase, Akt, as well as GLUT4 genes were seen to be increased. This extract combination also increased glucose uptake, increased adiponectin secretion, and decreased resistin secretion significantly relative to control cells. Moreover, DLBS3233 administered in insulin resistant Wistar strain rats ( Rattus norvegicus strain Wistar), showed a property to control blood glucose, insulin and cholesterol levels. Therefore, DLBS3233 is a promising combination of herbal extracts which conveys medical utilities in term of type II diabetes treatment, and, possibly also in diabetes disease prevention.

Design and assessment of a real time reverse transcription-PCR method to genotype single-stranded RNA male-specific coliphages (Family Leviviridae)

A real-time, reverse transcription-PCR (RT-qPCR) assay was developed to differentiate the four genogroups of male-specific ssRNA coliphages (FRNA) (family Leviviridae). As FRNA display a trend of source-specificity (human sewage or animal waste) at the genogroup level, this assay provides a tool to help identify the origin of fecal contamination. Primers and probes were designed using complete genomic sequences from 29 FRNA phages. The final selection of primer/probe sets were based on (i) ability to amplify a single, specific product, (ii) genogroup specificity, (iii) lack of cross-reactivity, and (iv) experimental reproducibility and sensitivity over a range of target concentrations. Assay time was reduced by using heat-released viral RNA rather than purified RNA. For quality assurance, a custom RNA molecule was employed as an internal, non-competitive control. The usefulness of this method to identify sources of fecal contamination was tested on a total of 49 FRNA phages isolated from various warm-blooded animals, sewage and combined sewage overflow. FRNA phages from animal wastes were genotyped as 86% I, 4% III Q-like and 9% IV. Two sewage isolates typed to genogroup I and combined sewage overflow isolates genotyped as 40% II and 52% III. Primer specificity designed from this comprehensive sequence database may better discriminate FRNA from different sources.

Microfluidic-Based Multiplex qRT-PCR Identifies Diagnostic and Prognostic microRNA Signatures in the Sera of Prostate Cancer Patients

Recent prostate-specific antigen-based screening trials indicate an urgent need for novel and noninvasive biomarker identification strategies to improve the prediction of prostate cancer behavior. Noncoding microRNAs (miRNA) in the serum and plasma have been shown to have potential as noninvasive markers for physiologic and pathologic conditions. To identify serum miRNAs that diagnose and correlate with the prognosis of prostate cancer, we developed a multiplex quantitative reverse transcription PCR method involving the purification of multiplex PCR products followed by uniplex analysis on a microfluidics chip to evaluate 384 human miRNAs. Using Dgcr8 and Dicer knockout (small RNA-deficient) mouse ES cells as the benchmark, we confirmed the validity of our technique and uncovered a considerable lack of accuracy in previously published methods. Profiling 48 sera from healthy men and untreated prostate cancer patients with differing CAPRA scores, we identified miRNA signatures that allow us to diagnose cancer patients and correlate with a prognosis. These serum signatures include oncogenic and tumor-suppressive miRNAs, suggesting functional roles in prostate cancer progression.

Quantification of Norwalk virus inocula: Comparison of endpoint titration and real‐time reverse transcription‐PCR methods

Human noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis. In order to fully characterize features such as persistence and infectious dose, precise quantification of virus concentration is necessary. The purpose of this study was to compare two methods [endpoint titration RT-PCR and quantitative RT-PCR (RT-qPCR)] with respect to quantification of Norwalk virus (NV) in inocula made from purified stock suspensions of human fecal specimens. A full-length NV RNA transcript was developed to facilitate quantification using RT-qPCR and provided log linear detection in the range of 49-4.9 x 10(4) genome equivalent copies (GEC) per reaction. Endpoint titration RT-PCR was used to estimate PCR detection units, and RT-qPCR was used to estimate genome copies in two NV inocula (8fIIa and 8fIIb) used in previous human challenge studies. Overall, RT-qPCR was 1.1-1.6 log(10) more sensitive (lower detection limit) than endpoint titration RT-PCR when the same RNA release method, PCR primers and thermocycle program were used. These findings have important implications for many experimental interpretations, not the least of which is estimating the median infectious dose in human challenge studies.

Plasma MicroRNA 499 as a Biomarker of Acute Myocardial Infarction

MicroRNAs (miRNAs) are endogenous small RNAs 21-25 nucleotides in length. Recently, we reported that miRNA 208 (miR-208) is produced exclusively in the rat myocardium and that plasma miR-208 is a biomarker of myocardial injury in rats. In the present study, we assessed the hypothesis that plasma concentrations of myocardial-specific miRNAs can be used to diagnose myocardial injury in humans. We used array analysis of miRNA production in various human tissues to identify heart-specific miRNAs. We assessed the plasma concentrations of miR-499 in 14 individuals with acute coronary syndromes, 15 individuals with congestive heart failure, and 10 individuals without cardiovascular diseases. Plasma miR-499 concentrations were measured with a real-time reverse-transcription PCR method that used an artificial small RNA as an internal calibrator. The miRNA array analysis of various human tissues indicated that miR-499 was produced almost exclusively in the heart. Plasma miR-499 concentrations were measurably increased in all individuals with acute myocardial infarction but were below the limit of detection for all individuals in the other patient groups. The plasma concentration of miR-499 may be a useful biomarker of myocardial infarction in humans.

Comparison of virus isolation using the Vero E6 cell line with real-time RT-PCR assay for the detection of human metapneumovirus

BackgroundThe use of cell culture for the diagnosis of human metapneumovirus (hMPV) infection is uncommon at present and molecular method such as reverse-transcription PCR (RT-PCR) has been widely and most commonly used as the preferred test. We aimed to compare the results of virus isolation using Vero E6 cells with real-time RT-PCR for the detection of hMPV, since such a comparison data is not available.MethodsBetween December 2007 and July 2008, we obtained 224 nasopharyngeal swab specimens from patients with acute respiratory infection and tested by the two methods.ResultsForty-three (19.2%) were found positive by cell culture and 62 (27.7%) by real-time RT-PCR. Cell cultures were positive for 42 of 62 specimens found positive by real-time RT-PCR (67.7% sensitivity) and for 1 of 162 specimens found negative by real-time RT-PCR (99.4% specificity), respectively. The sensitivity of the cell culture was 76.2-87.5% (mean 81.8%) when specimens were collected within 3 days after the onset of symptoms, and the sensitivity decreased to 50% or less thereafter. Among specimens collected within 3 days after symptom onset, all of the real-time RT-PCR positive specimens having a viral load of more than 1.25×105 copies/ml were found positive by cell culture.ConclusionsCell culture using Vero E6 cell line has 81.8% sensitivity compared with the real-time RT-PCR method, when specimens are collected within 3 days after the onset of symptoms. Thus, this method is a useful method for epidemiological and virological research even in facilities with minimal laboratory resources.