Development of a novel sequence based real-time PCR assay for specific and sensitive detection of Burkholderia pseudomallei in clinical and environmental matrices.

BACKGROUND: Melioidosis, caused by the category B biothreat agent Burkholderia pseudomallei, is a disease with a high mortality rate and requires an immediate culture-independent diagnosis for effective disease management. In this study, we developed a highly sensitive qPCR assay for specific detection of Burkholderia pseudomallei and melioidosis disease diagnosis based on a novel target sequence. METHODS: An extensive in-silico analysis was done to identify a novel and highly conserved sequence for developing a qPCR assay. The specificity of the developed assay was analyzed with 65 different bacterial cultures, and the analytical sensitivity of the assay was determined with the purified genomic DNA of B. pseudomallei. The applicability of the assay for B. pseudomallei detection in clinical and environmental matrices was evaluated by spiking B. pseudomallei cells in the blood, urine, soil, and water along with suitable internal controls. RESULTS: A novel 85-nucleotide-long sequence was identified using in-silico tools and employed for the development of the highly sensitive and specific quantitative real-time PCR assay S664. The assay S664 was found to be highly specific when evaluated with 65 different bacterial cultures related and non-related to B. pseudomallei. The assay was found to be highly sensitive, with a detection limit of 3 B. pseudomallei genome equivalent copies per qPCR reaction. The detection limit in clinical matrices was found to be 5 × 102 CFU/mL for both human blood and urine. In environmental matrices, the detection limit was found to be 5 × 101 CFU/mL of river water and 2 × 103 CFU/gm of paddy field soil. CONCLUSIONS: The findings of the present study suggest that the developed assay S664 along with suitable internal controls has a huge diagnostic potential and can be successfully employed for specific, sensitive, and rapid molecular detection of B. pseudomallei in various clinical and environmental matrices.

Development and validation of a 23-gene expression signature for molecular subtyping of medulloblastoma in a long-term Chinese cohort.

PURPOSE: Medulloblastoma is the most common childhood malignant brain tumor and is a leading cause of cancer-related death in children. Recent transcriptional studies have shown that medulloblastomas comprise at least four molecular subgroups, each with distinct demographics, genetics, and clinical outcomes. Medulloblastoma subtyping has become critical for subgroup-specific therapies. The use of gene expression assays to determine the molecular subgroup of clinical specimens is a long-awaited application of molecular biology for this pediatric cancer. METHODS: In the current study, we established a medulloblastoma transcriptome database of 460 samples retrieved from three published datasets (GSE21140, GSE37382, and GSE37418). With this database, we identified a 23-gene signature that is significantly associated with the medulloblastoma subgroups and achieved a classification accuracy of 95.2%. RESULTS: The 23-gene signature was further validated in a long-term cohort of 142 Chinese medulloblastoma patients. The 23-gene signature classified 21 patients as WNT (15%), 41 as SHH (29%), 16 as Group 3 (11%), and 64 as Group 4 (45%). For patients of WNT, SHH, Group 3, and Group 4, 5-year overall-survival rate reached 80%, 62%, 27%, and 47%, respectively (p < 0.0001), meanwhile 5-year progression-free survival reached 80%, 52%, 27%, and 45%, respectively (p < 0.0001). Besides, SHH/TP53-mutant tumors were associated with worse prognosis compared with SHH/TP53 wild-type tumors and other subgroups. We demonstrated that subgroup assignments by the 23-gene signature and Northcott's NanoString assay were highly comparable with a concordance rate of 96.4%. CONCLUSIONS: In conclusion, we present a novel gene signature that is capable of accurately and reliably assigning FFPE medulloblastoma samples to their molecular subgroup, which may serve as an auxiliary tool for medulloblastoma subtyping in the clinic. Future incorporation of this gene signature into prospective clinical trials is warranted to further evaluate its clinical.

Evaluation of the PneumoGenius® PCR assay for the diagnosis of Pneumocystis pneumonia and the detection of Pneumocystis dihydropteroate synthase mutations in respiratory samples.

Pneumocystis pneumonia (PCP) is the most frequent fungal opportunistic infection defining AIDS in HIV-infected patients, and is of growing importance in HIV-negative patients. In this latter category of patients, the diagnosis mainly relies on real-time polymerase chain reaction (qPCR) detection of Pneumocystis jirovecii (Pj) on respiratory samples. The PneumoGenius® kit (PathoNostics) allows the simultaneous detection of Pj mitochondrial large subunit (mtLSU) and dihydropteroate synthase (DHPS) polymorphisms, which could be of interest to anticipate therapeutic failure. This study aimed at evaluating its clinical performance on 251 respiratory specimens (239 patients), (i) for P. jirovecii detection in clinical samples, and (ii) for DHPS polymorphisms detection in circulating strains. Patients were classified according to modified European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria, as having proven PCP (n = 62), probable PCP (n = 87), Pneumocystis colonization (n = 37), and no PCP (n = 53). Compared with in-house qPCR, the sensitivity of PneumoGenius® assay for P. jirovecii detection reached 91.9% (182/198), the specificity was excellent (100%, 53/53) and the global concordance was 93.6% (235/253). A total of four diagnoses of proven/probable PCP were missed by the PneumoGenius® assay, reaching a 97.5% sensitivity (157/161) in this sub-group. The 12 other 'false-negative' results were obtained in patients diagnosed as colonized using the in-house PCR. DHPS genotyping was successful for 147/182 samples with PneumoGenius® and revealed dhps mutation in 8 samples, which were all confirmed by sequencing. In conclusion, PneumoGenius® assay missed the detection of low-burden PCP. This lower sensitivity for PCP diagnosis can be balanced by a higher specificity (P. jirovecii colonization less frequently detected) and the efficient detection of DHPS hot spot mutations.

The diagnosis of Pneumocystis pneumonia (PCP) relies on DNA detection of P. jirovecii in respiratory samples. In this study, we show that the commercial assay PneumoGenius® has a lower sensitivity than our in-house qPCR for PCP diagnosis, but provides accurate results for DHPS genotyping.

Novel Detection and Quantification Approach of Erwinia amylovora In Vitro and In Planta Using SYBR Green-Based Real-Time PCR Assay.

Fire blight, caused by the bacterial pathogen Erwinia amylovora, is a highly destructive disease of apple and pear. Because the apple tree gets systemically infected with E. amylovora and eventually dies, E. amylovora is a considerably important pathogen in the orchard that requires long-term management. In addition, it is crucial to prevent the spread of the pathogen by expeditious diagnosis. In this study, via comparative approaches to the genome sequences of the strains of various Erwinia spp., we designed specific primers targeting a hypothetical gene that is single copy and located in the chromosomal DNA of E. amylovora. This primer set specifically amplified the DNA of E. amylovora but no other bacteria, including E. pyrifoliae, Pectobacterium spp., Pantoea spp., and Dickeya chrysanthemi. Furthermore, the SYBR Green-based real-time PCR using the primer set allowed accurate estimation of the population of E. amylovora. Developing a rapid and accurate diagnostic method using the novel primer set enables effective defense against pathogen spread through continuous monitoring and quick response.

Development and application of a low-priced duplex quantitative PCR assay based on SYBR Green I for the simultaneous detection of porcine deltacoronavirus and porcine sapelovirus.

Porcine deltacoronavirus (PDCoV) and porcine sapelovirus (PSV) are two viruses that can cause diarrhoea in pigs and bring great economic loss to the pig industry. In this research, a duplex real-time quantitative polymerase chain reaction (qPCR) assay based on SYBR Green I was developed to simultaneously detect PDCoV and PSV. No specific melting peaks were found in other porcine diarrhoea-associated viruses, indicating that the method developed in this study had good specificity. The detection limits of PDCoV and PSV were 1.0 × 101 copies µl-1 and 1.0 × 102 copies µl-1, respectively. The duplex real-time qPCR assay tested two hundred and three (203) intestinal and faecal samples collected from diarrhoeal and asymptomatic pigs. The positive rates of PDCoV and PSV were 20.2% and 23.2%, respectively. The co-infection rate of PDCoV and PSV was 13.8%. To evaluate the accuracy of the developed method, conventional PCR and singular TaqMan real-time qPCR assays for PDCoV/PSV were also used to detect the samples. The results showed that the duplex real-time qPCR assay was consistent with the singular assays, but its sensitivity was higher than conventional PCR methods. This duplex real-time qPCR assay provides a rapid, sensitive and reliable method in a clinic to simultaneously detect PDCoV and PSV.

Molecular analysis of dominant paranasal sinus bacteria in patients with and without chronic rhinosinusitis.

Recent studies have established the possible role of microbiota in developing various diseases. In this regard, attention has shifted to the evaluation of microbiota changes in the paranasal sinuses and its relationship to chronic rhinosinusitis (CRS), especially CRS with nasal polyposis (CRSwNP). This study aimed to examine the bacterial communities of the sphenoidal sinus in Iranian patients with and without CRS. The investigation included 36 subjects, including 18 patients with CRSwNP who underwent Functional Endoscopic Sinus Surgery (FESS) and 18 non-CRS patients who underwent Endoscopic Endonasal Approach (EEA) for pituitary adenoma. The surgeries were performed under general anesthesia, and the sphenoidal sinus was sampled using sterile rayon-tipped swabs coated with a sheet. TaqMan quantitative real-time polymerase chain reaction (qPCR) method (the 16S rDNA gene from bacteria) was used for detection of bacterial communities in different samples. Staphylococcus haemolyticus and Pseudomonas aeruginosa were significantly more prevalent in CRS patients than non-CRS patients (P value ≤ 0.05). However, no significant difference in the frequency of Corynebacterium spp. and Staphylococcus aureus was observed between the two groups, and no Streptococcus pneumoniae or Haemophilus influenza species were isolated from any of the samples. The current study's findings indicated a significant difference in the frequency of certain bacterial species in patients with CRS vs. non-CRS patients. By establishing a link between microbial burden and CRS, it is possible to develop effective treatments or even prevent disorders in this body area.

Evaluation of the MIB-producing potential based on real-time qPCR in drinking water reservoirs.

Cyanobacteria release 2-methylisoborneol (MIB) as a secondary metabolite. Here, we propose a reverse transcription quantitative real-time PCR (RT-qPCR) based method to evaluate the MIB-producing potential in source water by detecting the MIB-synthesis gene (mic). A MIBQSF/R primer set was designed based on 35 mic gene sequences obtained from 12 pure-cultured MIB-producing strains and 23 sequences from the NCBI database. This primer set successfully identified all known 43 MIB-producing cyanobacterial strains (12 from this study and 31 from the NCBI database), belonging to different genera, showing a wider coverage than previous primer sets. The efficiency of the method was proved by the amplification efficiency (E = 91.23%), R2 of the standard curve (0.999), the limit of detection (LOD, 5.7 fg µL-1), and the limit of quantification (LOQ, 1.86 × 104 gene copies µL-1). Further, the method was verified by the correlation between the mic gene abundance and MIB concentration 50 field samples from different reservoirs (R2 = 0.614, p < 0.001) and one reservoir (R2 = 0.752, p < 0.001), suggesting its potential as an alternative warning tool to evaluate the risk of MIB problems in source water.

The Potential Role of Plastome Copy Number as a Quality Biomarker for Plant Products using Real-time Quantitative Polymerase Chain Reaction.

Background: Plastids are plant-specific semi-autonomous self-replicating organelles, containing circular DNA molecules called plastomes. Plastids perform crucial functions, including photosynthesis, stress perception and response, synthesis of metabolites, and storage. The plastome and plastid numbers have been shown to be modulated by developmental stage and environmental stimuli and have been used as a biomarker (identification of plant species) and biosensor (an indicator of abiotic and biotic stresses). However, the determination of plastome sequence and plastid number is a laborious process requiring sophisticated equipment. Methods: This study proposes using plastome copy number (PCN), which can be determined rapidly by real-time quantitative polymerase chain reaction (RT-qPCR) as a plant product quality biomarker. This study shows that the PCN log10 and range PCN log10 values calculated from RT-qPCR data, which was obtained for two years from leaves and lint samples of cotton and seed samples of cotton, rice, soybean, maize, and sesame can be used for assessing the quality of the samples. Results: Observation of lower range PCN log10 values for CS (0.31) and CR (0.58) indicated that the PCN showed little variance from the mean PCN log10 values for CS (3.81) and CR (3.85), suggesting that these samples might have encountered ambient environmental conditions during growth and/ or post-harvest storage and processing. This conclusion was further supported by observation of higher range PCN log10 values for RS (3.09) versus RP (0.05), where rice seeds in the RP group had protective hull covering compared to broken hull-less seeds in the RS group. To further support that PCN is affected by external factors, rice seeds treated with high temperatures and pathogens exhibited lower PCN values when compared to untreated seeds. Furthermore, the range PCN log10 values were found to be high for cotton leaf (CL) and lint (Clt) sample groups, 4.11 and 3.63, respectively, where leaf and lint samples were of different sizes, indicating that leaf samples might be of different developmental stage and lint samples might have been processed differently, supporting that the PCN is affected by both internal and external factors, respectively. Moreover, PCN log10 values were found to be plant specific, with oil containing seeds such as SeS (6.49) and MS (5.05) exhibiting high PCN log10 values compared to non-oil seeds such as SS (1.96). Conclusion: In conclusion, it was observed that PCN log10 values calculated from RT-qPCR assays were specific to plant species and the range of PCN log10 values can be directly correlated to the internal and external factors and, therefore might be used as a potential biomarker for assessing the quality of plant products.

Association of B-cell lymphoma 2/microRNA-497 gene expression ratio score with metastasis in patients with colorectal cancer: A propensity-matched cohort analysis.

BACKGROUND: Deregulated microRNAs (miRs) significantly impact cancer development and progression. Our in silico analysis revealed that miR-497 and its target gene B-cell lymphoma-2 (BCL2) could be related to poor cancer outcomes. PURPOSE: To investigate the BCL2/miRNA-497 expression ratio in colorectal cancer (CRC) and explore its association with the clinicopathological characteristics and CRC prognosis. METHODS: Archived samples from 106 CRC patients were enrolled. MiR-497 and BCL2 gene expressions were detected by Taq-Man Real-Time quantitative polymerase chain reaction in propensity-matched metastatic and nonmetastatic cohorts after elimination of confounder bias. RESULTS: B-cell lymphoma-2 gene was upregulated in metastatic samples (median = 1.16, 95%CI = 1.09-1.60) compared to nonmetastatic (median = 1.02, 95%CI = 0.89-1.25, p < 0.001). In contrast, lower levels of miR-495 were detected in specimens with distant metastasis (median = 0.05, 95%CI = 0.04-0.20) than nonmetastatic samples (median = 0.54, 95%CI = 0.47-0.58, p < 0.001). Estimated BCL2/miR-497 ratio yielded a significant differential expression between the two cohort groups. Higher scores were observed in metastasis group (median = 1.39, 95%CI = 0.9-1.51) than nonmetastatic patients (median = 0.29, 95%CI = 0.19-0.39, p < 0.001). Receiver operating characteristic curve analysis showed BCL2/miR-497 ratio score to have the highest predictive accuracy for metastasis at presentation. The area under the curve was 0.90 (95%CI = 0.839-0.964, p < 0.001) at cut-off of >0.525, with high sensitivity 81.1% (95%CI = 68.6%-89.4%) and specificity 92.5% (95%CI = 82.1%-97.0%). Also, the ratio score was negatively correlated with disease-free survival (r = -0.676, p < 0.001) and overall survival times (r = -0.650, p < 0.001). Kaplan-Meier curves showed lower survival rates in cohorts with high-score compared to low-score patients. CONCLUSION: The BCL2/miR497 expression ratio is associated with poor CRC prognosis in terms of metastasis and short survival.

Evaluation of real-time qPCR-based methods to detect the DNA of the three protozoan parasites Cryptosporidium parvum, Giardia duodenalis and Toxoplasma gondii in the tissue and hemolymph of blue mussels (M. edulis).

The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfish consumption. No standardized methods are available for their detection in these foods, and the performance of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performance criteria (e.g. sensitivity, estimated limit of detection (eLD95METH), parasite DNA recovery rates (DNA-RR)) of real-time qPCR based-methods for the detection of the three protozoa in mussel's tissues and hemolymph. Digestion of mussels tissues by trypsin instead of pepsin and the use of large buffer volumes was the most efficient for processing 50g-sample. Trypsin digestion followed by lipids removal and DNA extraction by thermal shocks and a BOOM-based technique performed poorly (e.g. eLD95METH from 30 to >3000 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performance (e.g. eLD95METH: 4-400 parasites/g, DNA-RR: 19-80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (e.g. eLD95METH: 10-1000 parasites/ml, DNA-RR ≤ 24%). The bead-beating DNA extraction based method is a rapid and simple approach to sensitively detect the three protozoa in mussels using tissues, that can be standardized to different food matrices. However, quantification in mussels remains an issue.

Blood RNA Sequencing Indicates Upregulated BATF2 and LY6E and Downregulated ISG15 and MT2A Expression in Children with Autism Spectrum Disorder.

Mutations in over 100 genes are implicated in autism spectrum disorder (ASD). DNA SNPs, CNVs, and epigenomic modifications also contribute to ASD. Transcriptomics analysis of blood samples may offer clues for pathways dysregulated in ASD. To expand and validate published findings of RNA-sequencing (RNA-seq) studies, we performed RNA-seq of whole blood samples from an Israeli discovery cohort of eight children with ASD compared with nine age- and sex-matched neurotypical children. This revealed 10 genes with differential expression. Using quantitative real-time PCR, we compared RNAs from whole blood samples of 73 Israeli and American children with ASD and 26 matched neurotypical children for the 10 dysregulated genes detected by RNA-seq. This revealed higher expression levels of the pro-inflammatory transcripts BATF2 and LY6E and lower expression levels of the anti-inflammatory transcripts ISG15 and MT2A in the ASD compared to neurotypical children. BATF2 was recently reported as upregulated in blood samples of Japanese adults with ASD. Our findings support an involvement of these genes in ASD phenotypes, independent of age and ethnicity. Upregulation of BATF2 and downregulation of ISG15 and MT2A were reported to reduce cancer risk. Implications of the dysregulated genes for pro-inflammatory phenotypes, immunity, and cancer risk in ASD are discussed.

Toxicological aspect of bioinsecticide pyrethrum extract and expressions of apoptotic gene levels in human hepotacellular carcinoma HepG2 cells.

Pyrethrum extract (PE), an important natural bioinsecticide, is extensively used across the world to control pest insects in homes and farms. The aim of this study was to evaluate the potential cytotoxic effect of PE using MTT assay and genotoxic effect using micronucleus (MN) assay. The changes in the expressions of the apoptosis genes in mRNA levels were also investigated using Real-Time qPCR analysis as well as the ratio of apoptotic/necrotic cells with AnnexinV-FITC/Propidium iodide (PI) assay in HepG2 cells. PE markedly suppressed the cell proliferation on HepG2 cells. It significantly increased the frequency of micronucleus (MN) at 500 and 1000 µg/mL. PE also induced the percentage of the cell population of late apoptotic/necrotic cells (FITC + PI+) and necrotic cells (FITC- PI+), especially at 4000 µg/mL analyzed by flow cytometry. PE caused significant fold changes in the expression of several apoptotic genes including APAF1, BIK, BAX, BAD, BID, MCL-1, CASP3, CASP1, CASP2, FAS, FADD and TNFRSF1A. In particular, the pro-apoptotic gene Hrk (Harakiri) remarkably and dose-dependently was overexpressed of the mRNA level. As a result, PE may exhibit cyto-genotoxic effects, especially at higher concentrations and lead to significant changes in the expression of mRNA levels in several apoptotic genes.HighlightsNatural bioinsecticide PE exhibited a cytotoxic effect in HepG2 cells.PE significantly induced the micronucleus (MN) frequency at 500 and 1000 µg/mL.This bioinsecticide induced cell death and it lead to significant fold changes in the expression of mRNA levels in several apoptotic genes in HepG2 cells.The highest increase of the expression of mRNA levels was determined in Hrk (Harakiri) at 4000 µg/mL.

Molecular Beacon DNA Probes with Fluorescein Bifluorophore.

An azido-derivative of a fluorescein bifluorophore was obtained and used for the synthesis of "molecular beacon"-type oligonucleotide fluorogenic probes for RT-PCR. Eight probe variants were synthesized based on an optimized sequence: with one or two quencher residues at the 3'-end, with a single or bifluorophore fluorescein label attached to 5'-end using modifying phosphoramidites (short linker) or "click reaction" (long linker). Comparison of probes in RT-PCR showed that probes with a doubled quencher (single fluorescein on a short linker) and doubled dye on a short linker (single dye) are somewhat superior in sensitivity to a standard probe (single quencher, single dye on a short linker) by the value of ΔCt = 1-2. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1134/S1068162021030055.

Downregulation of hsa-microRNA-204-5p and identification of its potential regulatory network in non-small cell lung cancer: RT-qPCR, bioinformatic- and meta-analyses.

BACKGROUND: Pulmonary malignant neoplasms have a high worldwide morbidity and mortality, so the study of these malignancies using microRNAs (miRNAs) has attracted great interest and enthusiasm. The aim of this study was to determine the clinical effect of hsa-microRNA-204-5p (miR-204-5p) and its underlying molecular mechanisms in non-small cell lung cancer (NSCLC). METHODS: Expression of miR-204-5p was investigated by real-time quantitative PCR (RT-qPCR). After data mining from public online repositories, several integrative assessment methods, including receiver operating characteristic (ROC) curves, hazard ratios (HR) with 95% confidence intervals (95% CI), and comprehensive meta-analyses, were conducted to explore the expression and clinical utility of miR-204-5p. The potential objects regulated and controlled by miR-204-5p in the course of NSCLC were identified by estimated target prediction and analysis. The regulatory network of miR-204-5p, with its target genes and transcription factors (TFs), was structured from database evidence and literature references. RESULTS: The expression of miR-204-5p was downregulated in NSCLC, and the downtrend was related to gender, histological type, vascular invasion, tumor size, clinicopathologic grade and lymph node metastasis (P<0.05). MiR-204-5p was useful in prognosis, but was deemed unsuitable at present as an auxiliary diagnostic or prognostic risk factor for NSCLC due to the lack of statistical significance in meta-analyses and absence of large-scale investigations. Gene enrichment and annotation analyses identified miR-204-5p candidate targets that took part in various genetic activities and biological functions. The predicted TFs, like MAX, MYC, and RUNX1, interfered in regulatory networks involving miR-204-5p and its predicted hub genes, though a modulatory loop or axis of the miRNA-TF-gene that was out of range with shortage in database prediction, experimental proof and literature confirmation. CONCLUSIONS: The frequently observed decrease in miR-204-5p was helpful for NSCLC diagnosis. The estimated target genes and TFs contributed to the anti-oncogene effects of miR-204-5p.

Detection of extended-spectrum beta-lactamase cefotaxime resistance and virulence genes in Escherichia coli by duplex quantitative real-time PCR and melt curve analysis.

Emerging virulent and antibiotic-resistant pathogens present a global public health risk. Routine monitoring of prevalence within the clinical, environmental and food production setting is vital. Quantitative real-time PCR (qPCR) coupled with melting curve analysis can rapidly and accurately characterize pathogens. We evaluated commercial qPCR mixes based on SYBR Green l and EvaGreen for developing an assay for simultaneously detecting antibiotic resistance (extended-spectrum beta-lactamase, ESBL and blaCTX-M ) and virulence (stx1, stx2 and eae) genes in Escherichia coli (n = 12) isolated from irrigation water and irrigated vegetables. SYBR Green and EvaGreen detected two amplicons (stx1 and blaCTX-M ) and (stx2 and eae) in a single reaction. A higher mean melting temperature (Tm ) separation between targeted amplicons and smoother melting curves were observed with the EvaGreen suggesting better performance when targeting multiple amplicons. Through simple stepwise optimization of DNA, cycling, primers, reaction volume and melting curve scanning rate, we adopted a conventional PCR assay for detection of large amplicons (375-1580 bp) for qPCR. This may facilitate development of cost-effective tailor-made assays for rapid and accurate monitoring of emerging foodborne and environmental pathogens in resource constrained regions.

Interspecific Competition for Colonization of Maize Plants Between Fusarium proliferatum and Fusarium verticillioides.

Fusarium proliferatum and F. verticillioides are mycotoxin-producing, seedborne pathogens of maize. They are often asymptomatic in seed, eluding symptom-based detection. Experiments were conducted in nonsterile soil to determine whether interspecific competition influenced establishment in maize plants of an introduced isolate of F. proliferatum or F. verticillioides. Hygromycin-resistant, green fluorescent protein (GFP) transformed (GFP-tagged) F. proliferatum (F. proliferatum-green) and hygromycin-resistant, monomeric red fluorescent protein (mRFP) transformed (mRFP-tagged) F. verticillioides (F. verticillioides-red) strains were developed to provide molecular markers to track fungal establishment. Heat-killed Fusarium-free maize seed, colonized with F. proliferatum-green or F. verticillioides-red by immersion in a spore suspension for 16 h, served as the source of inoculum. The ability of F. proliferatum-green and F. verticillioides-red to colonize viable maize plants already colonized by the other species was determined. Maize plants were retrieved from soil after 14 days and DNA was extracted from three consecutive root segments and three consecutive stem segments. A TaqMan multiplex real-time quantitative PCR protocol was developed to identify and quantify F. proliferatum-green and F. verticillioides-red from each plant segment from each treatment; the experiment was repeated three times. This experiment confirmed that F. proliferatum-green and F. verticillioides-red effectively colonized roots and stems of the maize plant already colonized with the other species. Prior colonization of maize tissues by F. verticillioides-red (P = 0.6749) and other seedborne microorganisms (P = 0.1910) reduced but did not prevent subsequent colonization by F. proliferatum-green. Similarly, prior colonization of maize tissues by F. proliferatum-green (P = 0.7032) and other seedborne microorganisms (P = 0.1447) reduced but did not prevent subsequent colonization by F. verticillioides-red.

Development and application of a real-time polymerase chain reaction method for quantification of Escherichia coli in oysters (Crassostrea gigas).

Oysters are important mariculture species worldwide. Because of their filter-feeding behaviors, oysters can accumulate microorganisms, including pathogens, from surrounding water and concentrate bacteria in high numbers. Rapid and suitable methods for quantification of Escherichia coli in oysters are necessary considering that oysters are perishable foods often consumed raw and some countries use E. coli as the regulatory limit. The objective of this study was to develop a qPCR method for quantification of E. coli in oysters. Additionally, different methods were evaluated for DNA extraction from oyster samples and the more reliable method was chosen. Primers and probe were designed targeting uidA gene of E. coli and shown to specifically amplify DNA from E. coli. Standard curves with bacterial DNA extracted from oysters samples artificially inoculated with E. coli were conducted. A good correlation was noticed when the qPCR method was compared to a culture method in oyster samples. This is the first report of a method exclusively developed for direct quantification of E. coli in oyster, the method showed to be suitable for quantification of E. coli in oysters and could be useful in routine analyses, as it requires less time than the culture method.

Anodic microbial community analysis of microbial fuel cells based on enriched inoculum from freshwater sediment.

The characterization of anodic microbial communities is of great importance in the study of microbial fuel cells (MFCs). These kinds of devices mainly require a high abundance of anode respiring bacteria (ARB) in the anode chamber for optimal performance. This study evaluated the effect of different enrichments of environmental freshwater sediment samples used as inocula on microbial community structures in MFCs. Two enrichment media were compared: ferric citrate (FeC) enrichment, with the purpose of increasing the ARB percentage, and general enrichment (Gen). The microbial community dynamics were evaluated by polymerase chain reaction followed by denaturing gradient gel electrophoresis (PCR-DGGE) and real time polymerase chain reaction (qPCR). The enrichment effect was visible on the microbial community composition both during precultures and in anode MFCs. Both enrichment approaches affected microbial communities. Shannon diversity as well as ß-Proteobacteria and γ-Proteobacteria percentages decreased during the enrichment steps, especially for FeC (p < 0.01). Our data suggest that FeC enrichment excessively reduced the diversity of the anode community, rather than promoting the proliferation of ARB, causing a condition that did not produce advantages in terms of system performance.

Indoor air pollution aggravates asthma in Chinese children and induces the changes in serum level of miR-155.

Indoor air pollution is associated with childhood asthma but the molecular mechanism remains unclear. We aimed to explore the relationship between indoor air pollution and pediatric asthma, and the potential molecular mechanism. The serum level of miR-155 was measured by real-time qPCR in 180 Chinese children with asthma caused by air pollution (an asthma group). Meanwhile, 180 healthy subjects were selected as a control group. HCHO, NO2, and particles (PM10, PM2.5, and PM1) were measured. Univariate and multivariate logistic regression were analyzed to assess the relationship between air pollutants and asthma risk. A rank correlation test was used to explore the relationship between serum level of miR-155 and the level of PM2.5 or HCHO. Serum level of miR-155 was higher in the asthma group than the control group (p < 0.001). The history of childhood allergy, breastfeeding, environmental tobacco smoke, PM2.5, and HCHO were significantly different between two groups (p < 0.05). Serum level of miR-155 was closely associated with the levels of indoor PM2.5 and HCHO in the asthma group (p < 0.05) but not in the control group (p > 0.05). Indoor air pollution aggravates the asthma in Chinese children and induces the changes in the serum level of miR-155. Abbreviation: DEP: Diesel exhaust particles; PAHs: Polycyclic aromatic hydrocarbons; THBS1: thrombospondin 1; ISAAC: International Study of Asthma and Allergies in Childhood; PFTs: Pulmonary Function Tests; FEV1: The first second of forced expiration; EDTA, ethylenediaminetetraacetic acid; RT-qPCR, Reverse transcription quantitative real-time PCR; ETS: environmental tobacco smoke; PAEs: phthalate esters.

A comprehensive fungi-specific 18S rRNA gene sequence primer toolkit suited for diverse research issues and sequencing platforms.

BACKGROUND: Several fungi-specific primers target the 18S rRNA gene sequence, one of the prominent markers for fungal classification. The design of most primers goes back to the last decades. Since then, the number of sequences in public databases increased leading to the discovery of new fungal groups and changes in fungal taxonomy. However, no reevaluation of primers was carried out and relevant information on most primers is missing. With this study, we aimed to develop an 18S rRNA gene sequence primer toolkit allowing an easy selection of the best primer pair appropriate for different sequencing platforms, research aims (biodiversity assessment versus isolate classification) and target groups. RESULTS: We performed an intensive literature research, reshuffled existing primers into new pairs, designed new Illumina-primers, and annealing blocking oligonucleotides. A final number of 439 primer pairs were subjected to in silico PCRs. Best primer pairs were selected and experimentally tested. The most promising primer pair with a small amplicon size, nu-SSU-1333-5'/nu-SSU-1647-3' (FF390/FR-1), was successful in describing fungal communities by Illumina sequencing. Results were confirmed by a simultaneous metagenomics and eukaryote-specific primer approach. Co-amplification occurred in all sample types but was effectively reduced by blocking oligonucleotides. CONCLUSIONS: The compiled data revealed the presence of an enormous diversity of fungal 18S rRNA gene primer pairs in terms of fungal coverage, phylum spectrum and co-amplification. Therefore, the primer pair has to be carefully selected to fulfill the requirements of the individual research projects. The presented primer toolkit offers comprehensive lists of 164 primers, 439 primer combinations, 4 blocking oligonucleotides, and top primer pairs holding all relevant information including primer's characteristics and performance to facilitate primer pair selection.