The value of urinary exosomal microRNA-21 in the early diagnosis and prognosis of bladder cancer.

Bladder cancer (BC) poses high morbidity and mortality, with urinary exosomal microRNA (miR)-21 showing potential value in its diagnosis and prognosis, and we probed its specific role. We prospectively selected 116 BC patients and 116 healthy volunteers as the BC and control groups, respectively. BC urinary exosomal miR-146a-5p, miR-93-5p, miR-663b, miR-21, and miR-4454 relative expression levels were assessed. The correlations between clinical indexes and urinary exosomal miR-21, prognostic value of miR-21, and diagnostic value of the five candidate miRNAs, urine cytology, and miRNA joint diagnostic panel for BC and urinary exosomal miR-21, miR-4454, and urine cytology for Ta-T1 and T2-T4 stage BC were analyzed. Urinary exosomal miR-146a-5p, miR-93-5p, miR-663b, miR-21, and miR-4454 were highly expressed in BC patients. miR-146a-5p, miR-93-5p, miR-663b, miR-21, miR-4454, miRNA combined diagnostic panel, and urine cytology had certain diagnostic value for BC, with miR-21, miR-4454, and miRNA co-diagnostic panel showing the highest diagnostic value. Collectively, urinary exosomal miR-21 was closely related to Tumor-Node-Metastasis staging and grading in BC patients. Urinary exosomal miR-21 had high diagnostic value for BC and Ta-T1 and T2-T4 stage BC, and had high predictive value for BC poor prognosis, providing an effective indicator for the occurrence, development, and prognostic assessment of BC.

Role of Oncostatin M in the prognosis of inflammatory bowel disease: A meta-analysis.

BACKGROUND: Oncostatin M (OSM) is a pleiotropic cytokine which is implicated in the pathogenesis of inflammatory bowel disease (IBD). AIM: To evaluate the prognostic role of OSM in IBD patients. METHODS: Literature search was conducted in electronic databases (Google Scholar, Embase, PubMed, Science Direct, Springer, and Wiley). Studies were selected if they reported prognostic information about OSM in IBD patients. Outcome data were synthesized, and meta-analyses were performed to estimate standardized mean differences (SMDs) in OSM levels between treatment responders and non-responders and to seek overall correlations of OSM with other inflammatory biomarkers. RESULTS: Sixteen studies (818 Crohn's disease and 686 ulcerative colitis patients treated with anti-tumor necrosis factor-based therapies) were included. OSM levels were associated with IBD severity. A meta-analysis found significantly higher OSM levels in non-responders than in responders to therapy [SMD 0.80 (0.33, 1.27); P = 0.001], in non-remitters than in remitters [SMD 0.75 (95%CI: 0.35 to 1.16); P < 0.0001] and in patients with no mucosal healing than in those with mucosal healing [SMD 0.63 (0.30, 0.95); P < 0.0001]. Area under receiver operator curve values showed considerable variability between studies but in general higher OSM levels were associated with poor prognosis. OSM had significant correlations with Simple Endoscopic Score of Crohn's disease [r = 0.47 (95%CI: 0.25 to 0.64); P < 0.0001], Mayo Endoscopic Score [r = 0.35 (95%CI: 0.28 to 0.41); P < 0.0001], fecal calprotectin [r = 0.19 (95%CI: 0.08 to 0.3); P = 0.001], C-reactive protein [r = 0.25 (95%CI: 0.11 to 0.39); P < 0.0001], and platelet count [r = 0.28 (95%CI: 0.17 to 0.39); P < 0.0001]. CONCLUSION: OSM is a potential candidate for determining the severity of disease and predicting the outcomes of anti-tumor necrosis factor-based therapies in IBD patients.

Comparison of the chronic and multigenerational toxicity of racemic glufosinate and l-glufosinate to Caenorhabditis elegans at environmental concentrations.

Glufosinate-ammonium, the second largest transgene crop resistant herbicide, is classified as a mobile persistent pollutant by the U.S. Environmental Protection Agencybecause of its slow decomposition and easy mobile transfer in a water environment. The chronic and multigeneration toxicity of this compound to environmental organisms are alarming. In this study, racemic glufosinate-ammonium and the effective isomer, l-glufosinate-ammonium, were used as the test agents. The developmental, neurotoxic and reproductive toxicities of Caenorhabditis elegans to their parents and progeny were studied by continuous exposure in water at concentrations of 0.1, 1, 10 and 100 µg/L. The causes of toxicity differences were analysed from oxidative stress and transcription levels. Through oxidative stress of C. elegans, racemic glufosinate-ammonium and l-glufosinate-ammonium both mediated the developmental toxicity (shortened developmental cycle, reduced body length and width, promoted ageingand decreased longevity), neurotoxicity (inhibited head swinging, body bending frequency and acetylcholinesterase [AchE] activity) and reproductive toxicity (significant reductions in the number of eggs and offspring in vivo and induced apoptosis of gonadal cells). These phenomena caused oxidative damage (protein and membrane lipid peroxidation) and further induced apoptosis. The changes in various indicators caused by racemic glufosinate-ammonium exposure were more significant than those caused by l-glufosinate-ammonium exposure, and the reproduction-related indicators were more significant than the developmental and neurological indicators. A continuous accumulation of toxicity was observed after multiple generations of continuous exposure. These research results provide a data reference for the ecotoxicological evaluation and risk assessment of glufosinate-ammonium and contribute to the revision and improvement of the related environmental policies of glufosinate-ammonium.

[Spatiotemporal Distribution of Ammonia Emissions from Poultry Farming in the Yangtze River Delta Based on Online Monitoring Derived Local Emission Factors].

In order to obtain the ammonia emission level and space-time distribution characteristics of the poultry production industry in the Yangtze River Delta, an online high-resolution monitoring system was used to continuously monitor the atmospheric ammonia concentration in the breeding house and compost shed in a typical large-scale layer farm. By obtaining the ammonia emission level and emission factor during each growth stage, we established the localized ammonia emission inventory for the poultry production industry in the Yangtze River Delta. The results showed that the average daily ρ(NH3) in the breeding house and compost shed for spring, summer, autumn, and winter were (1.85±0.38), (4.58±0.33), (3.87±0.12), and (2.83±0.47) mg·m-3 and (2.04±0.50), (4.04±1.04), (2.51±0.67), and (1.55±0.16) mg·m-3 respectively. Ammonia emissions showed a significant daily hourly change trend. The highest hourly ammonia concentration in the layer house appeared from 13:00-14:00 in the afternoon, and the minimum appeared from 01:00-03:00 in the morning. The highest hourly ammonia concentration in the compost shed occurred between 16:00-19:00 in summer and autumn, whereas the diurnal changes in spring and winter were not significant. Hourly changes in ammonia emissions during the day were mainly affected by daily temperature, poultry activities, and manure management. Ammonia concentrations at different growth stages of laying hens showed significant differences. ρ (NH3) from young chickens, laying hens, and pre-eliminated chickens were (1.85±0.38), (2.83±0.47), and (1.61±0.32) mg·m-3, respectively. The ammonia emission rate from laying hens reached 1.53 times and 1.65 times that of young chickens and pre-eliminated chickens, respectively. Metabolism levels and feed intake at different growth stages were the main reasons for the differences in ammonia emissions. Ammonia emission factors for the layer house and compost shed in spring, summer, autumn, and winter were (0.13±0.02), (0.54±0.01), (0.39±0.01), and (0.17±0.01) g·(bird·d)-1 and (0.07±0.01), (0.17±0.02), (0.08±0.01), and (0.04±0.01) g·(bird·d)-1, respectively. Annual ammonia emission factors reached (0.11±0.06) kg·(bird·a)-1 and (0.03±0.02) kg·(bird·a)-1, respectively. Our results suggest that ambient temperature, ventilation mode, chicken house type, and manure removal frequency were the main influencing factors of ammonia emissions from poultry production. The uncertainty ranges of the ammonia emission coefficients reached±122%,±79%, and±74%, and±56%, respectively. Great uncertainties were generated when empirical emission factors were used for emission inventory establishment. Based on the results of online monitoring, model simulation, and literature analysis, we established an ammonia emission inventory for the poultry production industry within the Yangtze River Delta region by adopting the emission factors of (0.16±0.08) kg·(bird·a)-1. In 2019, the total ammonia emission from poultry production was (108.81±54.41) kt. In terms of spatial distribution, ammonia emission intensities in the northern regions were significantly higher than those in the southern parts. The ammonia emission intensities during summer were 3.38-3.56 times higher than those in spring and winter.

[Emission Characteristics, Transformation Mechanism, and Reduction Potential of Ammonia Emissions from a Crop Rotation System in Yangtze River Delta].

To study the characteristics and reduction potential of the ammonia emissions of a crop rotation system in the Yangtze River Delta, we monitored and compared the ammonia fluxes from two rotation systems:a conventional rice/winter wheat rotation system and a rice-shrimp cultivation/Chinese milk vetch rotation system. This study was conducted through closing chamber methods to investigate the influencing factors and transformation mechanism of ammonium emissions between the two studied cultivation patterns. Additionally, we established the temporal-spatial emission inventory by sorting out the local ammonia emission factors of farmland in the Yangtze River Delta in the last ten years. The emission reduction effects under different ammonia emission reduction paths were also obtained. The results showed that, the cumulative amount of ammonia emissions throughout the whole monitoring year for the conventional rice/winter wheat rotation system (CR-W) and the rice-shrimp cultivation/Chinese milk vetch rotation system (RS-C) were 65.95 and 20.31 kg·hm-2, respectively, whereas the ammonia loss rates of CR-W and RS-C were 10.86% and 9.20%, respectively. Field surface water NH4+-N, field surface water pH, and topsoil NH4+-N were the major internal factors of ammonia emissions from paddy fields, whereas topsoil NH4+-N and atmospheric temperature had an important impact on ammonia emissions in the wheat season. The ammonia flux/field NH4+-N ratio (ARN) of field surface water under the CR and RS modes in the rice season reached 0.35±0.27 and 0.14±0.19, respectively, which was 10-25 times that of topsoil in the wheat season, such that the ammonia emission flux in the rice season was significantly higher than that in the wheat season. Under the conditions of high field water pH (8.0-9.0), atmospheric temperature (>28℃), and wind speed (>5.0 m·s-1), the ammonia flux/field NH4+-N ratios (ARN) were around 1.6-4.6 times that under low pH, temperature, and wind speed conditions, indicating that those three factors were the main factors affecting the conversion of NH4+-N from farmland to atmospheric NH3. Fertilization types also had significant effects on ARN; under different conditions, the ARN of urea was 1.5-5.5 times that of organic fertilizer. In 2019, the ammonia emission flux of rice and wheat under a conventional planting pattern in the Yangtze River Delta were (49.2±17.6) kg·hm-2 and (16.0±13.5) kg·hm-2, respectively, whereas the ammonia loss rates of rice and wheat were (20.1±5.7)% and (5.9±3.6)%, respectively. The ammonia emission loss rate of the former was about three times that of the latter. The ammonia emission inventory built by local factors shows that the total ammonia emissions of the farmland rotation system in the Yangtze River Delta reached (400.3±206.4) kt in 2019, which was mainly concentrated in the central and northern regions of Anhui province and Jiangsu province, and the ammonia emission intensity reached (1.33±1.39) t·km-2. The selection of different emission factors had a relatively large impact on the change range of the inventory results, reaching the standard of -51.6%~51.6%. Through combing and analyzing the six main paths of ammonia emission reduction in farmland, it was found that nitrogen fertilizer synergism was the best way to reduce ammonia emissions, with the efficiency of (30.9±51.4)%; however, the grain yield increase rate was (-4.2±17.4)%, with great uncertainty. The ammonia emission reduction effect of adding soil additives was relatively poor (-5.4±45.1)%; however, the grain yield increase rate was the highest among those of the six emission reduction paths, reaching (6.8±23.9)%. The ammonia emission reduction effect and grain yield increase rate of the ecological planting and breeding mode were (22.3±15.1)% and (5.6±3.8)%, respectively, which had the advantages of reducing ammonia emissions and increasing crop yield.

Xanthotoxin induced photoactivated toxicity, oxidative stress and cellular apoptosis in Caenorhabditis elegans under ultraviolet A.

Xanthotoxin (XAT) is widely present in many kinds of plants. Caenorhabditis elegans, a typical model organism, was used to study the effects of XAT on C. elegans developmental toxicity, neurotoxicity, reproductive toxicity induced under ultraviolet A (UVA), oxidative stress and apoptosis in C. elegans. The results showed that after XAT exposure treatment, the hatchability of C. elegans decreased significantly as the concentration increased; the body length and width increased markedly, the external morphology was swollen; the brood sizes had been decreased; and the frequencies of head thrashes and body bend decreased significantly. At 80 and 100 mg/L, XAT reduced the activities of mitochondrial complex enzymes I and III, resulting in the excessive production of ROS, and inhibited SOD and CAT so that the ROS could not be eliminated over time. ROS accumulation in the bodies further caused the contents of MDA, protein carbonyl and lipofuscin to increase significantly, the mitochondrial membrane potential to be severely damaged, apoptosis to occur, and the apoptosis genes ced-3 and ced-4 to be significantly upregulated. Thus, XAT showed photoactivated toxicity to C. elegans under UVA, which will help people to make full and rational use of plants containing XAT.

A REPORT OF RADIATION RISKS DURING AND AFTER PROCESSING MINERAL PLACER FROM SOUTHEAST AFRICA.

Recently, high levels of radioactivity were found in products from Chinese mineral processing industries that handle mineral placer from Southeast Africa. The findings led to public panic. The aim of this work is to provide radiological data for the government, workers and the public. In this work, activity concentrations of 238U,232Th,226Ra and40 K in raw ore from Southeast Africa were analysed in the laboratory. Products like monazite and building material were analysed. High concentrations of 238U,232 Th and 226Ra in products were found to be at a level of 104  Bq/kg. Around the South China Mineral Processing Industry, radioactivity in soil and groundwater was analysed. Absorbed dose rates in air and indoor radon concentrations at workplaces were monitored. Annual effective dose to workers and the public was calculated and found to exceed Chinese dose criterion. This report might be an alert for mining and mineral processing in Southeast Africa.

Collective topo-epitaxy in the self-assembly of a 3D quantum dot superlattice.

Epitaxially fused colloidal quantum dot (QD) superlattices (epi-SLs) may enable a new class of semiconductors that combine the size-tunable photophysics of QDs with bulk-like electronic performance, but progress is hindered by a poor understanding of epi-SL formation and surface chemistry. Here we use X-ray scattering and correlative electron imaging and diffraction of individual SL grains to determine the formation mechanism of three-dimensional PbSe QD epi-SL films. We show that the epi-SL forms from a rhombohedrally distorted body centred cubic parent SL via a phase transition in which the QDs translate with minimal rotation (~10°) and epitaxially fuse across their {100} facets in three dimensions. This collective epitaxial transformation is atomically topotactic across the 103-105 QDs in each SL grain. Infilling the epi-SLs with alumina by atomic layer deposition greatly changes their electrical properties without affecting the superlattice structure. Our work establishes the formation mechanism of three-dimensional QD epi-SLs and illustrates the critical importance of surface chemistry to charge transport in these materials.

[Accumulation of Cd in Different Crops and Screening of Low-Cd Accumulation Cultivars].

Pot-culture experiments were carried out in Shanghai to screen crop varieties with low bioaccumulation properties with respect to cadmium (Cd). Eight common crops, such as green pepper, cucumber, cowpea, spinach, cauliflower, tomatoes, rice, and wheat, were planted in contaminated soil with different Cd concentrations of 0.23, 0.6, 1.2, 1.8, 2.4, and 3.0 mg·kg-1 to investigate the effects on biomass, Cd accumulation characteristics, and edible risk safety. The results indicated that:① With the increase in soil Cd content, the aboveground biomass of crops increased firstly and then decreased. The different crop types had different tolerance to Cd, with green pepper showed the strongest tolerance and spinach and tomato showed the least tolerance. ② The bioaccumulation factor of Cd in the edible parts of eight crops ranged in order of wheat > spinach > rice > green pepper > cauliflower > tomato > cucumber > cowpea. ③ Total Cd content in soil was significantly correlated with Cd content in the crops (P<0.05), and the order of the correlation coefficients was spinach > wheat > tomato > cucumber > green pepper > rice > cauliflower > cowpea. ④ The risk threshold value of Cd in soil based on the edible safety of different crops ranged in order of cowpea > cucumber > cauliflower > green pepper > tomato > rice > spinach > wheat. Cucumber, cowpea, and cauliflower were selected as the low-Cd-accumulating varieties according to their tolerance to soil Cd, bioaccumulation capacity, and edible risk threshold values.

Shortwave infrared fluorescence in vivo imaging of nerves for minimizing the risk of intraoperative nerve injury.

Here, we present a novel nerve specific imaging agent for preventing intraoperative nerve injuries based on SWIR QD-based in vivo imaging, which not only provides real-time and long-time SWIR images to intraoperatively identify nerves but can also markedly minimize the risk of iatrogenic nerve injuries during surgeries.

Classification of Tea Aromas Using Multi-Nanoparticle Based Chemiresistor Arrays.

Nanoparticle based chemical sensor arrays with four types of organo-functionalized gold nanoparticles (AuNPs) were introduced to classify 35 different teas, including black teas, green teas, and herbal teas. Integrated sensor arrays were made using microfabrication methods including photolithography and lift-off processing. Different types of nanoparticle solutions were drop-cast on separate active regions of each sensor chip. Sensor responses, expressed as the ratio of resistance change to baseline resistance (ΔR/R0), were used as input data to discriminate different aromas by statistical analysis using multivariate techniques and machine learning algorithms. With five-fold cross validation, linear discriminant analysis (LDA) gave 99% accuracy for classification of all 35 teas, and 98% and 100% accuracy for separate datasets of herbal teas, and black and green teas, respectively. We find that classification accuracy improves significantly by using multiple types of nanoparticles compared to single type nanoparticle arrays. The results suggest a promising approach to monitor the freshness and quality of tea products.

Heavy metals accumulation in soil after 4 years of continuous land application of swine manure: A field-scale monitoring and modeling estimation.

Land application of animal manure has been encouraged widely in China. This presents a risk of heavy metals (HMs) accumulation in the soil due to their high contents in the feeds and additives. A 4-year field-scale study was conducted to monitor and estimate HMs accumulation in the soil with land application of swine manure. The results show a clear tendency for As, Hg, Cr, Cu, Zn and Mn to increase gradually with the application duration, yielding an average annual increase of 0.57, 0.011, 6.20, 5.64, 22.58, and 23.45 mg kg-1, respectively, at the annual application rate of about 250 t ha-1 of swine manure. The estimation from the mass balance modeling indicates the environmental risk of Cd, Cu and Zn will exceed the threshold levels for agricultural soils in China in the next 10-50 years. Determination of a suitable application rate of animal manure would be the first consideration for mitigating the environmental risk of HMs currently.

Active Site Mapping of an Aspartic Protease by Multiple Fragment Crystal Structures: Versatile Warheads To Address a Catalytic Dyad.

Crystallography is frequently used as follow-up method to validate hits identified by biophysical screening cascades. The capacity of crystallography to directly screen fragment libraries is often underestimated, due to its supposed low-throughput and need for high-quality crystals. We applied crystallographic fragment screening to map the protein-binding site of the aspartic protease endothiapepsin by individual soaking experiments. Here, we report on 41 fragments binding to the catalytic dyad and adjacent specificity pockets. The analysis identifies already known warheads but also reveals hydrazide, pyrazole, or carboxylic acid fragments as novel functional groups binding to the dyad. A remarkable swapping of the S1 and S1' pocket between structurally related fragments is explained by either steric demand, required displacement of a well-bound water molecule, or changes of trigonal-planar to tetrahedral geometry of an oxygen functional group in a side chain. Some warheads simultaneously occupying both S1 and S1' are promising starting points for fragment-growing strategies.

High-Throughput Crystallography: Reliable and Efficient Identification of Fragment Hits.

Today the identification of lead structures for drug development often starts from small fragment-like molecules raising the chances to find compounds that successfully pass clinical trials. At the heart of the screening for fragments binding to a specific target, crystallography delivers structural information essential for subsequent drug design. While it is common to search for bound ligands in electron densities calculated directly after an initial refinement cycle, we raise the important question whether this strategy is viable for fragments characterized by low affinities. Here, we describe and provide a collection of high-quality diffraction data obtained from 364 protein crystals treated with diverse fragments. Subsequent data analysis showed that ∼25% of all hits would have been missed without further refining the resulting structures. To enable fast and reliable hit identification, we have designed an automated refinement pipeline that will inspire the development of optimized tools facilitating the successful application of fragment-based methods.

Experimental Active-Site Mapping by Fragments: Hot Spots Remote from the Catalytic Center of Endothiapepsin.

Successful optimization of a given lead scaffold requires thorough binding-site mapping of the target protein particular in regions remote from the catalytic center where high conservation across protein families is given. We screened a 361-entry fragment library for binding to the aspartic protease endothiapepsin by crystallography. This enzyme is frequently used as a surrogate for the design of renin and ß-secretase inhibitors. A hit rate of 20% was achieved, providing 71 crystal structures. Here, we discuss 45 binding poses of fragments accommodated in pockets remote from the catalytic dyad. Three major hot spots are discovered in remote binding areas: Asp81, Asp119, and Phe291. Compared to the dyad binders, bulkier fragments occupy these regions. Many of the discovered fragments suggest an optimization concept on how to grow them into larger ligands occupying adjacent binding pockets that will possibly endow them with the desired selectivity for one given member of a protein family.

High-speed ultrashort pulse fiber ring laser using charcoal nanoparticles.

A mode-locked erbium-doped fiber ring laser that is easy to set up is proposed and experimentally demonstrated to generate a high-repetition-rate optical pulse train with an ultrashort pulse width. The laser combines a rational harmonic mode-locking technique and charcoal nanoparticles as saturable absorbers. Compared to a solely active mode-locking scheme, the scheme with charcoal nanoparticles can remove the supermodes and narrow the pulse width by a factor of 0.57 at a repetition rate of 20 GHz. Numerical simulation of the laser performance is also provided, which shows good agreement with the experimental results.

Six Biophysical Screening Methods Miss a Large Proportion of Crystallographically Discovered Fragment Hits: A Case Study.

Fragment-based lead discovery (FBLD) has become a pillar in drug development. Typical applications of this method comprise at least two biophysical screens as prefilter and a follow-up crystallographic experiment on a subset of fragments. Clearly, structural information is pivotal in FBLD, but a key question is whether such a screening cascade strategy will retrieve the majority of fragment-bound structures. We therefore set out to screen 361 fragments for binding to endothiapepsin, a representative of the challenging group of aspartic proteases, employing six screening techniques and crystallography in parallel. Crystallography resulted in the very high number of 71 structures. Yet alarmingly, 44% of these hits were not detected by any biophysical screening approach. Moreover, any screening cascade, building on the results from two or more screening methods, would have failed to predict at least 73% of these hits. We thus conclude that, at least in the present case, the frequently applied biophysical prescreening filters deteriorate the number of possible X-ray hits while only the immediate use of crystallography enables exhaustive retrieval of a maximum of fragment structures, which represent a rich source guiding hit-to-lead-to-drug evolution.

DNA gold nanoparticle nanocomposite films for chemiresistive vapor sensing.

Chemiresistive vapor sensors combining functionalized gold nanoparticles and single-stranded DNA oligomers are investigated to enhance specificity in chemical sensing. Sensors are made by depositing DNA-functionalized gold nanoparticles onto microfabricated electrodes using four distinct sequences. Sensor performance is evaluated for response to relative humidity and exposure to vapor analytes including ethanol, methanol, hexane, dimethyl methylphosphonate, and toluene under different relative humidity. It is found that sensors display a nonmonotonic resistance change toward increasing humidity due to the combined effects of hydration induced swelling and ionic conduction. Responses to vapor analytes show sequence-dependent patterns as well as a strong influence of humidity. Overall, the findings are encouraging for using DNA oligomers to enhance specificity in chemical sensing.

Establishment of a fluorescent real-time quantitative RT-PCR assay for detection of genotype 4 hepatitis E virus in swine stools / 病毒学报

The primers and probes for the Real-time RT-PCR were designed based on the multiple sequence (swine and humans HEV strains) alignments of the ORF3 region of genotype 4 HEV. A rapid, sensitive and stable TaqMan Real-time RT-PCR assay was established, and its specificity and sensitivity were assessed, and comparison of the Real-time RT-PCR with conventional and nested RT-PCR was performed. The results found that the crossing points showed linearly proportional to the logarithm of the input copy number. The correlation coefficient (R2) and the slope value of the standard curves with plasmid DNA were 0.994 and -3.312, respectively. The efficiency (E) of the PCR was 100%. Coefficients of variation values of the different diluted plasmid DNA were low in the same or different repeated experimental group. In addition, the assay was able to correctly detect genotype 4 HEV RNA from swine fecal samples. The sensitivity of established assay was 100-fold higher than that of conventional RT-PCR and 10-fold higher than nested RT-PCR.