Specific Primer Sets Research Articles

Isolation of Crithidia spp. from lesions of immunocompetent patients with suspected cutaneous leishmaniasis in Iran.

Leishmania major has been considered as the main aetiological agent of cutaneous leishmaniasis in Iran. However, there are recent reports about the existence of Crithidia spp in cutaneous lesions in southern Iran. Therefore, this study was designed to decipher some morphological, biological and molecular aspects of this phenomenon. Clinical isolates were obtained from 167 patients with cutaneous ulcers. A set of specific primers based on GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) gene were used to distinguish between Crithidia and Leishmania genera. For molecular analysis, Pulsed Field Gel Electrophoresis and Mi-Seq Illumina platform were applied. Then, morphological analysis and some biological features (including potential growth at 37 °C and the ability of infecting mammalian macrophages) were studied. In 92.8% of clinical cases, L. major was the only causative microorganism isolated; in 5.4% of cases, co-infection of L. major and Crithidia spp. was identified; and in 1.8% of lesions, only Crithidia spp. were found. We isolated Crithidia spp. from clinical samples of patients suspected of cutaneous leishmaniasis in Iran, indicating that Crithidia spp. are capable of surviving at human body temperature and infecting macrophage cells. This raises questions on the influence of this phenomenon on pathogenicity, therapeutic outcome and disease control strategies.

Distribution of PAH-ring hydroxylating dioxygenase genes in bacteria isolated from two illegal oil refining sites in the Niger Delta, Nigeria

Abstract Polyaromatic hydrocarbons (PAHs) are commonly found pollutants in the Niger Delta. They are comparatively persistent in the environment. Also, they distort healthy microbial interaction and pose high risk to human health owing to their toxic, mutagenic and carcinogenic properties. This research investigated the distribution of naphthalene dioxygenase gene (nahAc) and PAH-ring hydroxylating dioxygenase alpha genes (PAH-RHDα-GP) amongst bacteria isolated from two disparate locations (Bomu in Gokana LGA and Ngia Ama in Degema LGA, in Rivers State, Nigeria) used as illegal modular refining sites. Spread plate method was used to isolate PAH-degrading bacteria followed by identification, characterisation and phylogenetic analysis. Polymerase chain reaction was used to detect the nahAc and PAH-RHDα-GPgene sequences (from chromosomal DNA and plasmid) of the α-subunit of PAH-ring hydroxylating dioxygenase, using specific primer sets. Genera of Enterobacter, Shewanella, Burkholderia, Pseudomonas, Bacillus, Acinetobacter, Exiguobacterium and Stenotrophomonas were isolated from the two study sites. nahAc genes were found in the genomes of both Gram-negative and Gram-positive bacteria while PAH-RHDα-GPgenes were detected in all the plasmids recovered from Bomu-isolates. These findings inferred that the catabolic potential detected in the PAH-degrading bacterial community could be contributing to the in-situ biodegradation of PAHs.

Minor groove binder probe real-time RT-PCR for detection of foot-and mouth disease virus in Egypt

Shorter, more specific minor groove binders (MGBs) probes are dsDNA-binding agents attached to the 3' end of TaqMan probes that could be designed strictly to invariant region. Application and assessing of a new trend for viral detection in Egypt depending on MGB probe real-time RT-PCR (rRT-PCR) was applied on local FMDV serotypes O, A, and SAT2. Moreover, FMDV O were detected using two serotype specific primer sets by SYBR Green real-time RT-PCR assaying rapid formats. The limit of detection of diluted RNAs using MGB probe rRT-PCR assay reached to ≤ 6 fg/ul. Besides, high specificity of the former assay was clear. In contrary, the employing of FMDV O specific primer pairs in SYBR Green real-time RT-PCR showed a relative less sensitivity and specificity, particularly one of them displayed a very poor performance. Lastly, the local financial cost of MGB probe is considered the obvious hinder.

Rapid diagnosis of Ditylenchus destructor by loop-mediated isothermal amplification assay based on 28S rRNA sequences

Ditylenchus destructor is an important plant pathogenic nematode in agricultural production and is listed as a quarantine pest by many countries and regions. Traditional identification depending on morphological characteristics requires taxonomic expertise and is difficult to apply for rapid identification of plant nematode species. Currently, with the development of molecular biology, many novel tools have been explored for the rapid identification of plant nematodes. In this study, loop-mediated isothermal amplification (LAMP) was used to conduct rapid detection of D. destructor from various origins. First, a set of new LAMP specific primers (DdBIP, DdFIP, DdF3, DdB3 and DdLF) were designed based on 28S rRNA sequences of D. destructor, and LAMP reaction conditions were optimized to use concentrations of 6.0 mmol L−1 Mg2+, 1.0 mmol L−1 dNTPs, and 0.2 U μL−1 Bst 2.0 DNA polymerase, performed at 65 °C for 50 min. Second, the specificity, stability and sensitivity of this method were confirmed using DNA from a single egg and juvenile, male and female nematodes of D. destructor and from D. destructor mixed with various nematode species and soil and tissue samples. The amplification products were detected by checking the ladder-like bands using electrophoresis and visualizing mixture colour changes using SYBR Green I dye. The results showed that the LAMP method could not only detect and identify a single egg and juvenile, female and male nematodes and different populations from different hosts and geographical origins of D. destructor but also directly detect D. destructor from mixed samples of various nematode species, plant tissue and soil. The sensitivity of LAMP amplification was a 1000-fold dilution of the template DNA from an individual female nematode. This report is the first to use LAMP to directly detect D. destructor from samples mixed with various nematode species and with plant tissue and soil.

Assessment of 16S rRNA gene primers for studying bacterial community structure and function of aging flue-cured tobaccos

Selection of optimal primer pairs in 16S rRNA gene sequencing is a pivotal issue in microorganism diversity analysis. However, limited effort has been put into investigation of specific primer sets for analysis of the bacterial diversity of aging flue-cured tobaccos (AFTs), as well as prediction of the function of the bacterial community. In this study, the performance of four primer pairs in determining bacterial community structure based on 16S rRNA gene sequences in AFTs was assessed, and the functions of genes were predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Results revealed that the primer set 799F–1193R covering the amplification region V5V6V7 gave a more accurate picture of the bacterial community structure of AFTs, with lower co-amplification levels of chloroplast and mitochondrial genes, and more genera covered than when using the other primers. In addition, functional gene prediction suggested that the microbiome of AFTs was involved in kinds of interested pathways. A high abundance of functional genes involved in nitrogen metabolism was detected in AFTs, reflecting a high level of bacteria involved in degrading harmful nitrogen compounds and generating nitrogenous nutrients for others. Additionally, the functional genes involved in biosynthesis of valuable metabolites and degradation of toxic compounds provided information that the AFTs possess a huge library of microorganisms and genes that could be applied to further studies. All of these findings provide a significance reference for researchers working on the bacterial diversity assessment of tobacco-related samples.

Transmission strategies in a chemosynthetic symbiosis: detection and quantification of symbionts in host tissues and their environment.

Transmission of bacteria vertically through host tissues ensures offspring acquire symbionts; however, horizontal transmission is an effective strategy for many associations and plays a role in some vertically transmitted symbioses. The bivalve Solemya velum and its gammaproteobacterial chemosynthetic symbionts exhibit evolutionary evidence of both transmission modes, but the dominant strategy on an ecological time scale is unknown. To address this, a specific primer set was developed and validated for the S. velum symbiont using a novel workflow called specific marker design (SMD). Symbionts were quantified in spawned eggs and sediment and seawater samples from S. velum habitats with qPCR. Each egg was estimated to contain 50-100 symbiont genomes. By contrast, symbiont DNA was found at low abundance/occurrence in sediment and seawater, often co-occurring with host mitochondrial DNA, obscuring its origin. To ascertain when eggs become infected, histological sections of S. velum tissues were labelled for symbiont 16S rRNA via in situ hybridization. This revealed symbionts in the ovary walls and mature oocytes, suggesting association in late oogenesis. These data support the hypothesis that S. velum symbionts are vertically transmitted every host generation, thus genetic signatures of horizontal transmission are driven by ecologically infrequent events. This knowledge furthers our understanding of vertical and horizontal mode integration and provides insights across animal-bacterial chemosynthetic symbioses.

Association between Clostridioides difficile ribotypes, restriction endonuclease analysis types, and toxin gene expression

ObjectivesClostridioides difficile strains cause severe infection. Previous studies suggested that the virulence of C. difficile is dependent on ribotype; however, this hypothesis is still controversial. We aim to investigate the relationship between ribotypes, restriction endonuclease analysis (REA) types, and toxin gene expression in C. difficile strains. MethodsWe utilized 53 clinical C. difficile strains. All strains were assigned a molecular strain type using PCR ribotyping and REA typing and classified into 17 ribotypes and six REA types. The expression of toxin genes (tcdA, tcdB, and cdtB) in C. difficile strains were quantified by real-time PCR using each specific primer set, and expression was normalized to that of the housekeeping gene rpoA. ResultsAll 53 strains expressed tcdB and four strains expressed cdtB. Five strains did not express tcdA. Most ribotype and REA type strains expressed tcdA and tcdB similar to the BAA-1870 strain. In cdtB-positive strains, the cdtB expression levels were similar to those in the BAA-1870 strain. tcdA and tcdB expression levels were similar in the cdtB-positive and cdtB-negative strains. ConclusionToxin gene expression was not associated with the ribotype. Production of binary toxin C. difficile transferase was not related to tcdA and tcdB expression levels.

11P - Expression of erythropoietin receptor variant forms is associated with lymphatic invasion and metastasis formation in breast cancer

Background: Anemia not only impairs the quality of life of patients, but it leads to tumor hypoxia, resistance to therapy and reduces survival. Although recombinant Epo has revolutionized the treatment of anemia, clinical trials suggested a potential adverse effect of Epo on tumor recurrence and patient survival. Previously we identified altered EpoR mRNA transcripts in breast cancer cells lacking ligand binding domain exon 1-3 sequences in vitro. Our hypothesis is that altered expression of these proteins may be associated with unique clinicopathologic features. Methods: The expression level of C-and N-terminal portion of EpoR were measured by quantitative RT-PCR in 157 retrospective cohort of archived breast cancers. In order to identify the variant EpoR mRNA forms we have established a SMART RACE PCR assay using EpoR specific primer sets producing large overlapping sequences. Total RNA was frozen sections of 20 primary breast cancers after the H&E diagnoses.The products of the SMART RACE and nested PCR reactions was subjected to sequence analysis. Results: Out of the 157 samples 95 (61%) showed more than 2-fold change between the expression levels of the C and N-terminal regions of EpoR by quantitative RT-PCR. Significant correlation was found between the expression levels of EpoR mRNA variants and lymphatic invasion of the tumors. Logistic regression model demonstrated the association of metastatic capacity and expression differences of extra and intracellular regions of EpoR in primary breast cancers (P < 0.05, greater than 2-fold change). Using a SMART RACE PCR assay a single EpoR mRNA species at ∼1500 kb, corresponding to the full length wild type EpoR mRNA, and several distinct bands at ∼1300 kb, ∼1000 kb and ∼800 kb, lacking exon 1-4 N-terminal ligand binding domain, were seen in the samples. Conclusions: We showed that different variant forms of EPOR present not only in vitro but also in the individual breast tumor samples. These altered forms of EpoR may be responsible for the observed differences in the Epo responsiveness of EpoR bearing breast cancers. Legal entity responsible for the study: Zsuzsa Rakosy. Funding: OTKA PD 116996, 2015-2018. Disclosure: The author has declared no conflicts of interest.

Effect of DA-9801 on the Expression of Drug-metabolizing Enzymes and Transporters in Human Hepatocytes

DA-9801, a mixed extract of Dioscoreae japonica and Dioscorea nipponica Makino, is awaiting phase 3 clinical study for the prevention or treatment of diabetic neuropathy in USA. To investigate whether DA-9801 acts as a perpetrator in drug interactions or not, we evaluated the mRNA induction of phase I and II drug metabolizing enzymes, including cyto-chrome P450 (CYP), UDP-glucuronosyltransferases (UGT) and sulfotransferases (SULT), and drug transporters. The mRNA levels of phase I and II enzymes and transporters were analyzed by real-time PCR using three different cryo-preserved human hepatocytes using specific primer and probe primer sets. We found that no significant alteration of the mRNA expression was observed in phase I enzymes including CYP1A2, CYP3A4, CYP2B6, CYP2C8, and CYP2C9, phase II enzymes such as UGT1A1, UGT1A4, UGT1A9, UGT2B7 and SULT2A1, and drug transporters including p-glycoprotein, MRP2, BCRP and OCT1 by DA-9801 treatment compared with vehicle treated control cells. Moreover, we found that CYP1A2-catalyzed phenacetin O-deethylase, CYP2B6-catalyzed bupropion hydroxylase, and CYP3A4-catalyzed midazolam 1’-hydroxylase activities were not interfered by DA-9801 in cryopreserved human hepatocytes. These results suggested that DA-9801 might not cause pharmacokinetic-based drug interactions with concurrent drugs which are the substrates of these drug metabolizing enzymes and transporters in humans.

Development of duplex fluorescence-based loop-mediated isothermal amplification assay for detection of Mycoplasma bovis and bovine herpes virus 1

Mycoplasma bovis (MB) and bovine herpes virus 1 (BHV-1) are two important pathogens that cause bovine respiratory disease in the beef feedlot and dairy industries. The aim of this study was to develop and validate a duplex fluorescence-based loop-mediated isothermal amplification (DLAMP) assay for simultaneous detection of MB and BHV-1. Two sets of specific primers for each pathogen were designed to target the unique sequences of the MB uvrC gene and the BHV-1 gB gene. The inner primer for BHV-1 was synthesized with the fluorophore FAM at the 5′ end to detect the BHV-1 gB gene, and the inner primer for MB was synthesized with the fluorophore CY5 at the 5′ end to detect the MB uvrC gene. The DLAMP reaction conditions were optimized for rapid and specific detection of MB and BHV-1. The DLAMP assay developed here could specifically detect MB and BHV-1 without cross-reaction with other known non-target bovine pathogens. The sensitivity of this DLAMP assay was as low as 2 × 102 copies for recombinant plasmids containing the MB and BHV-1 target genes. In a detection test of 125 clinical samples, the positive rates for MB, BHV-1 and co-infection were 44.8%, 13.6% and 1.6%, respectively. Furthermore, the sensitivity and specificity of DLAMP were determined as 95%–96.6% and 100%, respectively, of those of field sample detection by the real-time polymerase chain reaction (PCR) assay recommended by the World Organisation for Animal Health. Overall, DLAMP provides a rapid, sensitive and specific assay for the identification of MB and BHV-1 in clinical specimens and for epidemiological surveillance.

SEROLOGICAL AND MOLECULAR DETECTION OF VIRUSES INFECTING FIG TO IDENTIFY THE VIRUS-FREE PLANTS

Fig (Ficus carica L.) is one of the old and the most common fruit tree grown in all the Mediterranean countries, which is among those exposed to diseases and pathogens. A recent investigation indicated that fig mosaic disease was the main pathogenic agent. In this study, field surveys were conducted in different areas of Egypt. A total of 300 leaf samples were collected randomly from symptomatic and symptomless fig trees of six cultivars, and tested by RT-PCR for the presence of Fig mosaic virus (FMV), Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FMMaV-2), Fig mild mottle-associated virus (FMMaV) and Fig latent virus 1 (FLV-1), using specific sets of primers. About 84% of the samples tested were found to be infected by at least one virus. FLMaV-1 was the prevailing virus with a (78.3%) incidence followed by FMV (73.7%), FLMaV-2 (50%), FLV-1(40.3%) and FMMaV (35%). The highest infection rate was observed in Sultani, Asuani and Adasi-Ahmer (100%), followed by Kahramani (80%), Kadota (64%) and Black Mission (60%).Ultra thin section for infected fig plants showed a large double-membrane particles (DMPs) 100-150 nm in diameter were located in the cytoplasm of parenchyma cells. Fig mosaic virus-specific antiserum raised in rabbits, proved useful for its detection in fig tissues. The plantlets from meristem tip tissue culture were transplanted successfully. FMV could be detected through DAS-ELISA in the virus-free fig plantlets.

Molecular Verification of Bloom-forming Aphanizomenon flos-aquae and Their Secondary Metabolites in the Nakdong River.

Aphanizomenon spp. have formed harmful cyanobacterial blooms in the Nakdong River during spring, autumn, and now in winter, and the expansion of blooming period and area, associated with the global warming is predicted. The genus Aphanizomenon has been described to produce harmful secondary metabolites such as off-flavors and cyanotoxins. Therefore, the production of harmful secondary metabolites from the Aphanizomenon blooms in the Nakdong River needs to be monitored to minimize the risk to both water quality and public health. Here, we sampled the cyanobacterial blooms in the Nakdong River and isolated ten Aphanizomenon strains, morphologically classified as Aphanizomenon flos-aquae Ralfs ex Bornet et Flahault 1888. Phylogenetic analysis using 16S rRNA and internal transcribed spacer (ITS) region nucleotide sequences confirmed this classification. We further verified the harmful secondary metabolites-producing potential of A. flos-aquae isolates and water samples containing cyanobacterial blooms using PCR with specific primer sets for genes involved in biosynthesis of off-flavor metabolites (geosmin) and toxins (microcystins, saxitoxins and cylindrospermopsins). It was confirmed that these metabolite biosynthesis genes were not identified in all isolates and water samples containing only Aphanizomenon spp. Thus, it is likely that there is a low potential for the production of off-flavor metabolites and cyanotoxins in Aphanizomenon blooms in the Nakdong River.

Cloning of a putative sodium/calcium exchanger gene in the crayfish.

Crayfish is a common model animal for different experimental purposes. However, the lack of information about the genetic properties of the animal limits its use in comparison to other model animals. In the present study, a putative crayfish sodium/calcium exchanger gene has firstly been cloned in ganglia cDNA samples by conducting a series of PCR experiments, where a set of degenerate and specific primers and RACE method were used. The complete sequence is 2955bp, and the ORF is 2718bp in length. Molecular properties of the calculated peptide were similar to the sodium/calcium exchangers reported in the other species. Analysis of the qPCR data indicated that the putative gene has the highest expression level in the ganglia. However, an apparently elevated level of expression is observed in highly active tissues like heart, muscle and intestine, while the least expression level was observed in the stomach samples. It was proposed that the cloned gene may code the sodium/calcium exchanger protein in the crayfish.

Investigation of Wolbachia spp. and Spiroplasma spp. in Phlebotomus species by molecular methods

The aim of this study was to determine the presence of Wolbachia spp. and Spiroplasma spp. in natural populations of sand flies in Turkey by molecular methods. A total of 40 Phlebotomus specimens (19 female and 21 male) were used in this study. Genomic DNA from whole sand flies was isolated and Wolbachia spp. infection prevalence was investigated by using Wolbachia gene specific primer sets (wsp and GroEL). In addition, the DNA were analyzed for the presence of Spiroplasma infections utilizing bacterium specific 16 S rDNA PCR-amplification primers. Results of this analysis showed a Wolbachia infection prevalence of 70% (28/40). There was no sex-bias in infection prevalence, being 76% (16/21) and 63% (12/19) in males and females, respectively. Analysis of Spiroplasma infections indicated that 26% (5/19) of female sand flies were positive for infection, while none of the screened males (0/21) were positive. Of the 40 sand fly samples, only 2 were found to be positive for both Wolbachia spp. and Spiroplasma spp. The present study demonstrates the presence of Wolbachia and Spiroplasma infections in the natural sand fly populations in Turkey. This is the first report on Spiroplasma infection in the sand flies from Turkey.

Molecular Investigation of Tomato Yellow Leaf Curl Virus (TYLCV) and Identification its Isolates Disseminated in some Tomato Protected Culture in Al-Qadisiyah Province.

This study aimed at conducting a molecular investigation of some virus isolates associated with tomato yellow leaf curl disease (TYLCD). Ten tomato samples exhibiting TYLCD are collected from fields and tunnel at certain tomato growing area in Al-Qadisiyah Province (El-HamzaEl-Sharqi district, Al-Sadeer and Al-Sunnia).. Begomoviruses are investigated by Polymerase chain reaction (PCR) using Deng a genus specific primer set in symptomatic samples. The amplified DNA fragments of expected size are sequenced, analyzed and compared to equivalent Gene Bank sequences using MEGA6 software. PCR technique using Deng primers could detect begomoviruses when amplified the 540 bp DNA fragments from 6 out of 10 symptomatic tomato samples. Sequence analysis shows that sequences obtained shared 96 - 99.5% nucleotide (nt) sequence identities with Tomato yellow leaf curl virus (TYLCV) equivalent Gene bank coat protein CP sequences from Cuba (AJ223505), USA (AY530931) and Israel (AB110217). Neighbor-Joining phylogenetic tree constructed from partial coat protein region confirmed the relatedness when grouped all sequences obtained to equivalent sequences belong to TYLCV from Gen Bank but not to other begomovirus species. Sequence comparison shows that TYLCV isolates are variable when share nt sequence 91-100% (approximated)to each other’s, which indicates that sequences isolated may belong to different TYLCV strains. The high nucleotide sequence identities suggests that TYLCV may have been introduced to Al-Qadisiyah Provence through infected plant materials, however full length sequence comparisons and biological assays are required to confirm this relatedness and resolve the possible origin of TYLCV isolates in Al-Qadisiyah Province..

In situ loop-mediated isothermal amplification (LAMP) for identification of Plasmodium species in wide-range thin blood smears

BackgroundFive species of Plasmodium are known to infect humans. For proper treatment of malaria, accurate identification of the parasite species is crucial. The current gold standard for malaria diagnosis is microscopic examination of Giemsa-stained blood smears. Since the parasite species are identified by microscopists who manually search for the parasite-infected red blood cells (RBCs), misdiagnosis due to human error tends to occur in case of low parasitaemia or mixed infection. Then, molecular methods, such as polymerase chain reaction or loop-mediated isothermal amplification (LAMP), are required for conclusive identification of the parasite species. However, since molecular methods are highly sensitive, false-positive results tend to occur due to contamination (carry over) or the target gene products may be detected even after clearance of the parasites from the patient’s blood. Therefore, accurate detection of parasites themselves by microscopic examination is essential for the definitive diagnosis. Thus, the method of in situ LAMP for the parasites was developed.ResultsRed blood cell suspensions, including cultured Plasmodium falciparum, strain 3D7, infected-RBCs, were dispersed on cyclic olefin copolymer (COC) plate surfaces rendered hydrophilic by reactive ion-etching treatment using a SAMCO RIE system (hydrophilic-treated), followed by standing for 10 min to allow the RBCs to settle down on the plate surface. By rinsing the plate with RPMI 1640 medium, monolayers of RBCs formed on almost the entire plate surface. The plate was then dried with a hair drier. The RBCs were fixed with formalin, followed by permeabilization with Triton X-100. Then, amplification of the P. falciparum 18S rRNA gene by the LAMP reaction with digoxigenin (DIG)-labelled dUTP and a specific primer set was performed. Infected RBCs as fluorescence-positive cells with anti-DIG antibodies conjugated with fluorescein using fluorescent microscopy could be detected.ConclusionsThe present work shows that the potential of in situ LAMP for the identification of Plasmodium species at the single cell level on hydrophilic-treated COC palates, allowing highly sensitive and accurate malaria diagnosis. The findings will improve the efficacy of the gold standard method for malaria diagnosis.

Lipopeptide biodiversity in antifungal Bacillus strains isolated from Algeria.

Several Bacillus strains have been well studied for their ability to control soil-borne plant diseases. This property is linked to the production of several families of lipopeptides. Depending of their structure, these compounds show antifungal and/or plant systemic resistance inducing activities. In this work, the biodiversity of lipopeptides produced by different antifungal Bacillus strains isolated from seeds, rhizospheric, and non-rhizospheric soils in Algeria was analyzed. Sixteen active strains were characterized by PCR for their content in genes involved in lipopeptide biosynthesis and by MALDI-ToF for their lipopeptide production, revealing a high biodiversity of products. The difficulty to detect kurstakin genes led us to design two new sets of specific primers. An interesting potential of antifungal activity and the synthesis of two forms of fengycins differing in the eighth amino acid (Gln/Glu) were found from the strain 8. Investigation of its genome led to the finding of an adenylation domain of the fengycin synthetase predicted to activate the glutamate residue instead of the glutamine one. According to the comparison of both the results of MALDI-ToF-MS and genome analysis, it was concluded that this adenylation domain could activate both residues at the same time. This study highlighted that the richness of the Algerian ecosystems in Bacillus strains is able to produce: surfactin, pumilacidin, lichenysin, kurstakin, and different types of fengycins.

Rapid detection of four non-fermenting Gram-negative bacteria directly from cystic fibrosis patient's respiratory samples on the BD MAX™ system

The aim of this study was to develop a multiplex PCR test to detect Achromobacter xylosoxidans (AX), Burkholderia cepacia (BC), Pseudomonas aeruginosa (PSA) and Stenotrophomonas maltophilia (SM) directly from CF patient's respiratory samples using the open mode of the BD MAX™ System. A total of 402 CF respiratory samples were evaluated by culture and PCR. Specific sets of primers and probes for each target were designed in-house. Out of 402 samples tested, 196 were identified as negative and 206 as positive by culture for AX, PSA, BC and SM. Among culture positive samples, PCR detected 21/27 AX, 4/5 BC, 138/140 PSA and 29/34 SM. In addition, PCR assay identified 35 samples as positive that were initially negative by culture for those 4 targets. The CF BDM test proved to be an excellent tool to detect AX, BC, PSA and SM by real-time PCR on an automated platform.

Differential sequences of exosomal NANOG DNA as a potential diagnostic cancer marker.

NANOG has been demonstrated to play an essential role in the maintenance of embryonic stem cells, and its pseudogene, NANOGP8, is suggested to promote the cancer stem cell phenotype. As the roles of these genes are intimately involved with glioblastoma multiforme progression and exosomes are critical in intercellular communication, we conducted a detailed analysis of the association of the NANOG gene family with exosomes to identify diagnostic markers for cancer. Exosomes were precipitated from conditioned culture media from various cell lines, and NANOG gene fragments were directly amplified without DNA isolation using multiple primer sets. The use of the enzymes AlwNI and SmaI with restriction fragment length polymorphism analysis functioned to distinguish NANOGP8 from other NANOG family members. Collectively, results suggest that the NANOG DNA associated with exosomes is not full length and that mixed populations of the NANOG gene family exist. Furthermore, sequence analysis of exosomal DNA amplified with a NANOGP8 specific primer set frequently showed an insertion of a 22 bp sequence into the 3’ UTR. The occurrence rate of this insertion was significantly higher in exosomal DNA clones from cancer cells as compared to normal cells. We have detected mixed populations of NANOG DNA associated with exosomes and have identified preferential modulations in the sequences from cancer samples. Our findings, coupled with the properties of exosomes, may allow for the detection of traditionally inaccessible cancers (i.e. GBM) through minimally invasive techniques. Further analysis of exosomal DNA sequences of NANOG and other embryonic stemness genes (OCT3/4, SOX2, etc.) may establish a robust collection of exosome based diagnostic markers, and further elucidate the mechanisms of cancer formation, progression, and metastasis.

Development of a Simple, Rapid Multiplex PCR Method Using the 16S rRNA Gene to Determine the Country of Origin of Brackish Water Bivalve Corbicula japonica

In this study, a multiplex PCR detection method was developed to identify the country of origin of Corbicula japonica (clams), a commercially important bivalve in Asia. Specific primer sets that have a single nucleotide mismatch at the 3′ terminus were designed after sequencing the mitochondrial 16S rRNA gene of clams identified as C. japonica originating from Korea, China, and Japan. Using this method, each origin was clearly identified based on the PCR products: three bands for Korean C. japonica (100, 283, and 384 bp), one band for Chinese C. japonica (384 bp), and two bands for Japanese C. japonica (384 and 100 bp). These results indicate that the 16S rRNA gene, which is usually used to identify species, can distinguish the country of origin within C. japonica. Our multiplex PCR assay should be a useful tool for the fair trade of the species.