Universal Reverse Primer Research Articles

Comparison of Bacterial Communities in the Throat Swabs from Healthy Subjects and Pharyngitis Patients by Terminal Restriction Fragment Length Polymorphism

Terminal restriction fragment length polymorphism (T-RFLP) analysis was applied to characterize bacterial flora present in the throats of healthy subjects and pharyngitis patients. The 16SrRNA genes of bacteria present in throat metagenome were amplified by PCR with 6-carboxy-fluorescein (6-FAM)-labeled universal forward primer (27F) and a universal reverse primer (1513R). The 16S rDNAs were digested with restriction enzymes with 4-bp recognition sites (MspI or RsaI) and analyzed by using an automated DNA sequencer. T-RFLP patterns were numerically analyzed using computer programs. From analysis of the throat bacterial community, patterns derived from MspI and RsaI digested samples of healthy subjects and pharyngitis patients were grouped into different clusters, though RsaI digested samples showed some uncertainty. Pharyngitis throats generated an average species richness of 9 [±2.1 (SD)] and 10 (±2.9) for MspI and RsaI digests, respectively, whereas healthy throats generated 6.3 (±1.2) and 6.1 (±1.5) in MspI and RsaI digests, respectively. These results suggest that samples from pharyngitis patients contain an unexpected diversity of causative bacteria. The pharyngitis throats were colonized with a rich diversity of bacterial species than that of healthy throats. Using T-RFLP, we are able to detect a model bacterium, Streptococcus pyogenes SF370, and T-RF patterns were consistent with the Streptococcal T-RFLP patterns. Our study indicates that T-RFLP analysis is useful for the assessment of diversity of throat bacterial flora and rapid comparison of the community structure between subjects with and without pharyngitis.

Next Generation Sequencing Spectratyping (NGS-S) Comprehensively Monitors T Cell Receptor Diversity in Children with T Cell Abnormalities

Abstract▪2173▪This icon denotes a clinically relevant abstractThe adaptive arm of the immune system - the T-cell compartment – may become compromised by inherited or acquired defects resulting in cancer, autoimmunity, or increased susceptibility to microbial infectious agents. A normal polyclonal T cell compartment comprises an estimated number of 2.5 × 10E7 individual T cell clones each expressing a unique antigen recognizing T cell receptor (TCR). The functionality of the T cell compartment is thus – at least partly - reflected by TCR sequence diversity. There is a medical need for rapid and robust diagnostic approaches that accurately monitor TCR diversity in patient samples, e.g. after bone marrow transplantation (BMT).Previously, complementarity determining region 3 (CDR3) size spectratyping in TCR β-chain subfamilies (Vβ), an immunoscopic technique, was employed for the analysis of T cell diversity. However, spectratyping is limited to the analysis of CDR3 length polymorphisms only. Underlying diversity of TCR Vβ sequences of equal length remain undetected. Furthermore, spectratyping is time consuming and consequently data can only be interpreted with a delay of weeks.To determine TCR diversity fast and accurately we developed next-generation-sequencing spectratyping (NGS-S), which employs high coverage, massive parallel Roche/454-sequencing of TCR Vβ-chain amplicons.Three different sample groups were analyzed in parallel by spectratyping and NGS-S: T cells from (1) healthy children (n=6), (2) children at diagnosis of severe aplastic anemia (n=7), and (3) children who underwent haploidentical BMT (n=7). In brief, RNA was extracted from bone marrow derived CD8+ cells and transcribed to cDNA. Amplicon libraries were generated by PCR employing two degenerated wobble primers (VP1, VP2), designed to cover most of the known TCR Vβ gene segments and a universal reverse primer (CP1) located in the conserved TCR region (Figure 1). The mean overall coverage of the CDR3 region achieved was 23.133 per patient. [Display omitted] For simultaneous characterization of these individual amplicons we generated the TCR Profiler (Figure 2). This new software tool automatically preprocessed raw sequencing data using a threshold quality value (q=30) to trim the 3’ end of the TCR β-chain sequences. Rearranged germline TCR Vβ and Jβ genes were identified by Smith-Waterman local alignment against each human TCR β-chain germline gene of the IMGT/GENE-DB reference directory. Base call reliability was assured by incorporating phred-like quality values provided by the sequencer into the Smith-Waterman local alignment routine. A quality value, q, was computed for each base as the log-transformation of the probability p of a base being incorrectly called, q = − 10 x log10(p). Transformed into a reliability measure, r = 1- (1/10^(q/10)), q-values indicated the probability for each base to be correctly called. CDR3 regions were delimited using specific flanking amino acid sequence motifs. The 5’ end motif varies dependently on the rearranged TCR Vβ gene and was identified using the IMGT/GENE-DB reference directory sequence set, whereas the 3’ end motif [W/F]GXG (IUPAC code) is conserved in all TCR β-chains. CDR3 length was calculated including all amino acids between the two motifs. Screening for in-frame stop codons was done to exclude non-functional TCR β-chain transcripts. [Display omitted] The TCR profiler identified on average 16165 of the input sequences as unique CDR3 sequences. Of these a mean of 9840 were predicted to be functionally rearranged.Whereas spectratyping gave a rough estimate of CDR3 size and allowed T cell deficient samples to be identified, NGS-S determined the exact length and sequence composition of the CDR3, identified the rearranged TCR Vβ and Jβ genes and the specific recombination (Figure 3A). [Display omitted] Utilization of specific genes, the resulting amino acid composition of the CDR3 region as well as its length and overall diversity were integrated into a novel NGS-S score.This score reliably differentiated not only between normal and T cell deficient samples, but also between the different T cell deficient groups (SAA and BMT) (Figure 3B). We conclude that NGS-S allows rapid and precise determination of TCR diversity in clinical samples. Disclosures:No relevant conflicts of interest to declare.

Culture-independent detection of microorganisms in traditional Slovakian bryndza cheese

In order to investigate the microflora of Slovakian bryndza cheese (a cheese containing unpasteurized or pasteurized ewes' milk component) by a culture-independent method, DNA was extracted directly from 7 bryndza samples and analysed by an innovative method. Using the universal prokaryotic and fungal primers, ribosomal DNA internal transcribed spacer (ITS) regions with variable length were amplified. The standard universal reverse primer L1 aligning to bacterial 23s rDNA was found unsuitable for some lactic acid bacteria and other species based on in silico analysis. Therefore, L1 primer was replaced by a combination of novel primers GplusR and GminusR aligning to the adjacent, more conserved DNA region. The amplification profiles were visualised by both standard electrophoresis and by fluorescent capillary gel electrophoresis. From representative samples, major amplicons were excised from the gel, cloned and sequenced. Sequencing revealed that the samples contained Lactobacillus delbrueckii, Lactobacillus brevis, Streptococcus thermophilus, Lactococcus lactis, Lactococcus raffinolactis, Streptococcus macedonicus, Leuconostoc pseudomesenteroides, Debaromyces hansenii, Mucor fragilis, Yarrowia lipolytica and Galactomyces geotrichum. These results represent an extension of the knowledge on the microflora of Slovakian bryndza cheese. The introduced automated ribosomal DNA intergenic spacer analysis of the bacterial and fungal genomes proved to be very effective in the application of studying microflora of cheese.

Collagen I Induces Discoidin Domain Receptor (DDR) 1 Expression through DDR2 and a JAK2-ERK1/2-mediated Mechanism in Primary Human Lung Fibroblasts

Discoidin domain receptors (DDRs) DDR1 and DDR2 are receptor tyrosine kinases with the unique ability among receptor tyrosine kinases to respond to collagen. Several signaling molecules have been implicated in DDR signaling, including Shp-2, Src, and MAPK pathways, but a detailed understanding of these pathways and their transcriptional targets is still lacking. Similarly, the regulation of the expression of DDRs is poorly characterized with only a few inflammatory mediators, such as lipopolysaccharide and interleukin-1β identified as playing a role in DDR1 expression. DDRs have been reported to induce the expression of various genes including matrix metalloproteinases and bone morphogenetic proteins, but the regulatory mechanisms underlying DDR-induced gene expression remain to be determined. The aim of the present work was to elucidate the molecular mechanisms implicated in the expression of DDRs and to identify DDR-induced signaling pathways and target genes. Our data show that collagen I induces the expression of DDR1 in a dose- and time-dependent manner in primary human lung fibroblasts. Furthermore, activation of DDR2, JAK2, and ERK1/2 MAPK signaling pathways was essential for collagen I-induced DDR1 and matrix metalloproteinase 10 expression. Finally, inhibition of the ERK1/2 pathway abrogated DDR1 expression by blocking the recruitment of the transcription factor polyoma enhancer A-binding protein 3 to the DDR1 promoter. Our data provide new insights into the molecular mechanisms of collagen I-induced DDR1 expression and demonstrate an important role for ERK1/2 activation and the recruitment of polyoma enhancer-A binding protein 3 to the DDR1 promoter.

A diagnostic multiplex polymerase chain reaction method to identify Japanese internal apple-feeding Lepidopteran pests: Grapholita molesta, Grapholita dimorpha (Lepidoptera: Tortricidae), and Carposina sasakii (Lepidoptera: Carposinidae)

We present a multiplex polymerase chain reaction method to differentiate between three Japanese internal apple-feeding pests: the oriental fruit moth, Grapholita molesta (Busck), Grapholita dimorpha Komai, and the peach fruit moth, Carposina sasakii Matsumura. A 1,342-bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was sequenced in each species. The three species showed consistent and diagnostic differences in the region of the COI gene, from which three species-specific forward primers were designed. The forward primers along with one universal reverse primer were used to selectively amplify DNA from specimens of diverse geographic origin for each corresponding target species. This method enabled easy, immediate, and accurate identification of internal feeding Lepidoptera in apples and other fruits.

Sensitive and Specific Universal RT-qPCR Profiling of miRNAs

RATIONALE: MicroRNAs (miRNAs) bind to target mRNAs and regulate their expression in a dose-dependent manner. The amount of specific miRNA in the cell is critical for the regulation process. Their expression pattern can be used as signatures of the developmental stage of cells and tissues, and as a biomarker for diagnosis and prognosis of disease, and miRNAs may be useful as therapeutic agents. Accurate quantitation of miRNAs is essential to their study. However, a highly specific and sensitive but simple method for quantitating miRNAs is still sought. METHODS: The method combines a universal hydrolysis probe with a universal reverse trasncription primer (UPR), which requires only one RT reaction and one hydrolysis probe for detection of all miRNAs. A computer program has been developed to design the probes and primers for the RT-qPCR assay. RESULTS: The method can detect and quantify miRNAs in as little as 1 pg of total RNA, and genomic DNA and mRNAs present in total RNA produce negligible or undetectable signals. Simple extraction methods such as Trizol (Invitrogen, CA, USA) without DNase treatment and small RNA fractionation is sufficient to produce total RNA for this assay. CONCLUSION: This approach affords a highly specific, sensitive, economical and convenient system to profile the expression of all known miRNAs.

Molecular Analysis of Gut Microflora in Captive-Raised Sea Urchins (Lytechinus variegatus)

In the USA, sea urchin aquaculture is in its infancy. One concern with current captive culture involves maintaining roe, or “uni,” quality. Diet of cultured urchins has been shown to affect roe quality. Sea urchins have a unique digestive system and lack certain digestive enzymes, yet they are able to digest high percentages of food biomass consumed. It is hypothesized that their highly efficient digestion is because of the presence of bacteria in the gut. This study determined bacterial profiles in guts of captive-raised sea urchins, Lytechinus variegatus, by analyzing DNA extracted from the urchins. Using a bacteria-specific forward and universal reverse primer, 16s rDNA genes were amplified using L. variegatus gut DNA samples and subsequently cloned. Recombinant colonies containing putative 16S rDNA were randomly selected for sequencing. Sequences generated from these clones were analyzed and compared with published bacterial 16S rDNA sequences available through the National Center for Biotechnology Information database. Sequence data analysis suggested that the captive-raised sea urchins contained a limited number of representative genera. The genera most commonly identified included Pseudomonads, Vibrio, and various epsilon and gamma proteobacterium species.

Molecular Detection of Autosomal-Dominant Feline Polycystic Kidney Disease by Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction

Feline autosomal-dominant polycystic kidney disease (ADPKD), with its characteristic growth of fluid-filled cysts of different sizes, is the most prevalent inherited genetic disease of cats. The point mutation (C-->A transversion) in exon 29 of the PKD1 gene is known to contribute to ADPKD development and can thus serve as a target for the molecular genetic diagnosis of ADPKD. To this end, a simple amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) was designed with 3 primers: 2 forward primers specifically targeting either the mutant or normal allele, and 1 universal reverse primer for amplification of both alleles. The new method was tested on the DNA from 35 feline blood samples, which included 15 mutant cats and 20 wild type cats. As verified by direct DNA sequencing, both sensitivity and specificity of this tri-primer ARMS PCR were 100%. As the multiplex ARMS PCR test can be performed in a single PCR reaction without other post-PCR procedures, it is a simple and accurate method for molecular studies of feline ADPKD.

10.1007/s11171-008-1013-3

A PCR system in the fluorescent amplification-based specific hybridization (FLASH) format was developed for the detection and identification of two important wheat pathogenic fungi Septoria tritici (teleomorph of Mycosphaerella graminicola) and Stagonospora nodorum (teleomorph of Phaeosphaeria nodorum), which cause spots on leaves and glumes, respectively. The pathogen detection system is based on the amplification of a genome fragment in the internal transcribed spacer 1 (ITS1) region and a site encoding the 5.8S ribosomal RNA. The forward primers to ITS1 and a universal reverse primer and a beacon type probe to the 5.8S ribosomal RNA region were chosen to provide the detection of the products in the FLASH format. This system was tested on different isolates of the pathogens, and on infected soil, leaf, and seed samples.

MicroRNA-7 Modulates CD98 Expression during Intestinal Epithelial Cell Differentiation

The transmembrane glycoprotein CD98 regulates multiple cellular functions, including extracellular signaling, epithelial cell adhesion/polarity, amino acid transport, and cell-cell interactions. MicroRNAs post-transcriptionally regulate gene expression, thereby functioning as modulators of numerous cellular processes, such as cell differentiation, proliferation, and apoptosis. Here, we investigated if microRNAs regulate CD98 expression during intestinal epithelial cell differentiation and inflammation. We found that microRNA-7 repressed CD98 expression in Caco2-BBE cells by directly targeting the 3'-untranslated region of human CD98 mRNA. Expression of CD98 was decreased, whereas that of microRNA-7 was increased in well-differentiated Caco2-BBE cells compared with undifferentiated cells. Undifferentiated crypt cells isolated from mouse jejunum showed higher CD98 levels and lower levels of mmu-microRNA-706, a murine original microRNA candidate for CD98, than well-differentiated villus cells. Importantly, microRNA-7 decreased Caco2-BBE cell attachment on laminin-1, and CD98 overexpression recovered this inhibition, suggesting that microRNA-7 modulates epithelial cell adhesion to extracellular matrix, which in turn could affect proliferation and differentiation during the migration of enterocytes across the crypt-villus axis, by regulating CD98 expression. In a pathological context, the pro-inflammatory cytokine interleukin 1-beta increased CD98 expression in Caco2-BBE cells by decreasing microRNA-7 levels. Consistent with the in vitro findings, microRNA-7 levels were decreased in actively inflamed Crohn disease colonic tissues, where CD98 expression was up-regulated, compared with normal tissues. Together, these results reveal a novel mechanism underlying regulation of CD98 expression during patho-physiological states. This study raises microRNAs as a promising target for therapeutic modulations of CD98 expression in intestinal inflammatory disorders.

49 REPROGRAMMING OF MICRO RNA IN BOVINE CLONED ELONGATED EMBRYOS

MicroRNAs (miRNA) are key regulators of gene expression in vertebrate development; however, little information exists about their expression in pre-implantation mammalian embryos. We studied the expression of miRNA in bovine cloned and IVF-elongated embryos using microarrays and quantitative RT-PCR (qRT-PCR). Day-7 IVF or handmade cloned blastocysts from an adult cell line that previously yielded live cloned calves were transferred to synchronized heifers and recovered at Day 17 as elongated embryos. MicroRNAs were purified using miRVANA kit+Flash Page fractionator column (Ambion, Austin, TX, USA) coupled to amine reactive dyes CyDye™ or used in qRT-PCR. Arrays were custom-made from 271 mice, human, and rat and 104 predicted miRNAs. After overnight hybridization, slides were scanned and data analyzed. Only miRNAs showing hybridization in 3 out of 6 features per slide were considered to be expressed. MicroRNAs were simultaneously converted to complementary (c)DNA and elongated using miRNA-specific DNA-oligo with 5′ overhang and primer extension by reverse transcription, followed by SYBR Green qPCR with another miRNA-specific primer and an excess of universal forward and reverse primers. The qPCR were normalized against 5S rRNA. Data were analyzed using Kruskal-Wallis nonparametric test for P-values < 0.05. Cloned (n = 48) and IVF (n = 28) blastocysts were transferred into 10 and 4 surrogate cattle, and 42 elongated embryos were recovered; 13 of these with embryonic disc were used for miRNA extraction and analysis. In experiment 1, miRNA profiling (1 slide) from 4 cloned embryos and donor cells yielded 34 and 22 miRNAs expressed in the embryos and cells, respectively. In experiment 2 (differential expression; 3 replicates), 39 miRNAs were expressed in cloned embryos and 32 in IVF embryos. Fifteen of these miRNAs were differentially expressed: 7 were up-regulated in the cloned elongated embryos and 8 in the elongated IVF embryos. The profiles of miRNA expression of cloned embryos, IVF embryos, and the donor cells were compared and reprogramming patterns analyzed. Reprogramming means similar expression among cloned and IVF embryos, but different from the somatic cells. Overall, 50 miRNAs were subjected to nuclear reprogramming; 31 (62%) were reprogrammed correctly, 10 aberrantly (20%), and 9 were not reprogrammed (18%). Most of the aberrantly reprogrammed miRNAs (90%) were switched on after nucleus transfer. Some of the reprogrammed miRNAs clustered in the same genomic location. Based on microarray data, 3 miRNAs over-expressed in the IVF embryos (let7-b, miR-200c, and miR-24), and 1 equally expressed (miR-16) and 2 over-expressed in the cloned embryos (miR-21 and miR-103) were analyzed by qRT-PCR. Expression of all the assayed miRNAs except miR-200c correlated adequately with microarray data (Pearson coefficient of correlation R2 = 0.9; P = 0.01). Here we present the first description of miRNA expression and reprogramming in bovine elongated cloned embryos. This can have profound implications for the understanding of nuclear reprogramming in somatic cloning.

Multiplex PCR system for identifying the carnivore origins of faeces for an epidemiological study on Echinococcus multilocularis in Hokkaido, Japan

A multiplex PCR system was developed to identify the carnivore origins of faeces collected in Hokkaido, Japan, for epidemiological studies on Echinococcus multilocularis. Primers were designed against the D-loop region of mitochondrial DNA. Two separate primer mixtures (mix 1, specific forward primers to fox, raccoon dog and dog, and a universal reverse primer [prH]; and mix 2, specific forward primers to cat, raccoon and weasels and prH) were used so that the PCR products (160 bp, fox and cat; 240 bp, raccoon dog and raccoon; and 330 bp, dog and weasel) were distinguished by size. The multiplex PCR exhibited no cross-reactivity between carnivore species and did not amplify DNA from rodent prey. When 270 field-collected faeces were examined, 250 showed single PCR products belonging to specific target sizes, suggesting successful carnivore identification for 92.6% of samples. Taeniid eggs were detected in 11.1% of samples and coproantigen in 30.4%; whereas the prevalences of taeniid eggs and coproantigen were 12.9% and 34.0% in fox faeces, and 0% and 26.3% in cat faeces, respectively. These results suggest that the prevalence in different target animals can be evaluated individually and precisely using multiplex PCR system.

NEW PRIMERS FOR AMPLIFICATION OF THE PLANCTOMYCETES 16S rRNA GENE FROM ENVIRONMENTAL SAMPLES

ABSTRACT Although the 16S rDNA primer set Pla‐46‐F/1392‐R is often used to explore the planctomycetes community, the universal reverse primer 1392‐R limits the capability of the forward primer Pla‐46‐F. Here we report two new reverse primers (Pla‐1097‐R and Pla‐1368‐R), which greatly promote the potential ability of the forward primer Pla‐46‐F, both theoretically and experimentally. The tests with field samples showed that PCR induced by the new primer sets both reduced mismatches to non‐planctomycetes and supplemented the diversity of planctomycetes, particularly increasing the numbers of planctomycetes genera in both seawater and sediment samples. Our results strongly suggest that the diversity of planctomycetes revealed by the previously used primer set Pla‐46F/1392‐R has been underestimated. PRACTICAL APPLICATIONSThe planctomycetes are an important bacteria; this paper developed two robust reverse primers which have been verified with filed samples. For natural and artificial ecosystems, the application of the new primers will: 1) reduce mismatches to non‐planctomycetes during PCR amplifying; 2) supplement the diversity of planctomycetes and 3) benefit our understanding of its potential ecological role.

Application of COMPOCHIP Microarray to Investigate the Bacterial Communities of Different Composts

A microarray spotted with 369 different 16S rRNA gene probes specific to microorganisms involved in the degradation process of organic waste during composting was developed. The microarray was tested with pure cultures, and of the 30,258 individual probe-target hybridization reactions performed, there were only 188 false positive (0.62%) and 22 false negative signals (0.07%). Labeled target DNA was prepared by polymerase chain reaction amplification of 16S rRNA genes using a Cy5-labeled universal bacterial forward primer and a universal reverse primer. The COMPOCHIP microarray was applied to three different compost types (green compost, manure mix compost, and anaerobic digestate compost) of different maturity (2, 8, and 16 weeks), and differences in the microorganisms in the three compost types and maturity stages were observed. Multivariate analysis showed that the bacterial composition of the three composts was different at the beginning of the composting process and became more similar upon maturation. Certain probes (targeting Sphingobacterium, Actinomyces, Xylella/Xanthomonas/Stenotrophomonas, Microbacterium, Verrucomicrobia, Planctomycetes, Low G + C and Alphaproteobacteria) were more influential in discriminating between different composts. Results from denaturing gradient gel electrophoresis supported those of microarray analysis. This study showed that the COMPOCHIP array is a suitable tool to study bacterial communities in composts.

Relative quantification of TCR Vbeta-chain families by real time PCR for identification of clonal T-cell populations

BackgroundQuantification of T-cell receptor (TCR) chain families can be utilized for detection of clonal T-cell populations. Besides southern blotting and antibody-based approaches, quantitative real time PCR (qRT PCR) has been more widely applied in this context during the last years. Here, the heterogeneity of sequences within single families is the most challenging problem for exact quantification.MethodVβ-families were quantified using a universal reverse primer and family-specific forward primers with TaqMan technology on a light cycler instrument. Relative concentrations were calculated considering slopes and crossing points of each PCR reaction. Total expression of α/β TCR was assessed by quantification of the constant α-chain as a further control.ResultsThe method was tested by serial dilutions of clonal T-cells in mononuclear cells from healthy volunteers. Calculated percentages were in good correspondence with qRT PCR results demonstrating high reliability. Duplicates showed excellent technical reproducibility. We analyzed blood samples of 20 healthy volunteers for determination of mean and standard deviation for each family. The method was applied both to tissue and blood samples from patients with carcinomas and hematological disorders.ConclusionWe introduce a versatile method for the relative quantification of Vβ-families by real time PCR. The experimental strategy described allows the identification of alterations in the Vβ-family repertoire.

Diagnostics of phytopathogen fungi Septoria tritici and Stagonospora nodorum by fluorescent amplification-based specific hybridization (FLASH) PCR

A PCR system in the fluorescent amplification-based specific hybridization (FLASH) format was developed for the detection and identification of two important wheat pathogenic fungi Septoria tritici (teleomorph of Mycosphaerella graminicola and Stagonospora nodorum (teleomorph of Phaeosphaeria nodorum), which cause spots on leaves and glumes, respectively. The pathogen detection system is based on the amplification of a genome fragment in the internal transcribed spacer 1 (ITS 1) region and a site encoding the 5.8S ribosomal RNA. The forward primers to ITS1 and a universal reverse primer and a Beacon type probe to the 5.8S ribosomal RNA region were chosen to provide the detection of the products in the FLASH format. This system was tested on different isolates of the pathogens, and on infected soil, leaf, and seed samples.

Allele-specific amplification and electrochemiluminescence method for single nucleotide polymorphism analysis

A new approach combined the specificity of allele-specific amplification (ASA) with the sensitivity of electrochemiluminescence (ECL) assay for single nucleotide polymorphism (SNP) analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru(bpy) 3 2+ (TBR)-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K- ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA–ECL assay. The method is useful in SNP analysis due to its sensitivity, safety, and simplicity.

One-Step Rapid Reverse Transcription–PCR Assay for Detecting and Typing Dengue Viruses with GC Tail and Induced Fluorescence Resonance Energy Transfer Techniques for Melting Temperature and Color Multiplexing

Dengue fever is an arthropod-borne infection caused by dengue viruses (DVs; DEN-1 to DEN-4). Early diagnosis is critical to prevent severe disease progression and the spreading of DV because no vaccine or specific treatment is available; therefore, a rapid and specific diagnostic assay capable of detecting and typing all serotypes would be ideal. We amplified RNA samples from all 4 DV serotypes and Japanese encephalitis virus with 4 serotype-specific forward primers and a universal species-specific reverse primer. DEN-1 and DEN-3 forward primers were labeled at their 5' ends with BODIPY 630/650 and Cy5.5, respectively. DEN-1 and DEN-3 amplicons were detected by their characteristic emission generated from induced fluorescence resonance energy transfer. The presence of DEN-2 and DEN-4 amplicons was indicated by SYBR Green I (SGI) signals at specific amplicon melting temperatures (T(m)s). Fluorescence signals with specific emission wavelengths were obtained from DEN-1 and DEN-3. SGI melting profiles showed a T(m) difference between DEN-2 and DEN-4 of 4.7 degrees C, which was sufficient for differentiating these 2 serotypes. The primers did not amplify the Japanese encephalitis virus. The detection limits of DEN-1 to DEN-4 were 1.64 x 10(-4), 1.05 x 10(-3), 8.15 x 10(-4), and 5.80 x 10(-3) plaque-forming units per reaction, respectively. The assay had a dynamic range of 10(3)-10(8) plaque-forming units/L and could be performed in 2 h. A single-tube, 1-step reverse transcription-PCR assay based on T(m) and color multiplexing was developed for detecting and typing all 4 DV serotypes.

Rapid isolation and characteristics analysis of microsatellites from Lutjanus russelli

The (CA)n DNA sequences in Lutjanus russelli were isolated through PCR arrays, and the characteristics of the microsatellite were analyzed. DNA was extracted from a sample of Lutjanus russelli, and digested with Hae+Dra. The fragments were ligated to pUCm-T vector and transferred into DH5alpha to construct a genomic library. The positive clones were isolated with universal M13 reverse and forward primers, M13 forward primer and the simple sequence repeats (SSRs) probes (CA)15, M13 reverse primer and (CA)15. After twice isolation, 121 positive clones, whose PCR products with M13 forward primer and probes (CA)15 or M13 reverse primer and (CA)15 were smaller than those with universal M13 reverse and forward primers probably contained microsatellites, were obtained. Then, 53 (CA)n(n> or =7) microsatellites were obtained by sequencing. The repeat length mainly distributed in 7-15(80.77%). Besides (CA)n repeats, other repeat motifs, such as An, (CAC)n and (AACA)n, were also obtained. Scorable and constant amplification of DNA fragments were observed with 48 pairs of SSR primers designed from the flank sequences. This research makes a positive contribution to explorating genomes of Lutjanus russelli, offers genetic tools to examine the genetic variations and constructs genetic linkage map.

Molecular diversity and anammox activity of novel planctomycete-like bacteria in the wastewater treatment system of a full-scale alcohol manufacturing plant

In this study, the diversity of planctomycete-like bacteria and the presence of anaerobic ammonia-oxidizing bacteria in the aerobic and anaerobic basins of a full-scale alcohol manufacturing wastewater treatment plant were examined by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Two clone libraries were constructed by using a 16S rRNA-targeted universal reverse primer and a forward PCR primer specific for planctomycetes. Phylogenetic analysis of 63 16S rRNA gene sequences defined 52 operational taxonomic units (OTUs). The majority of these sequences (51.6%) clustered in two separated monophyletic groups distantly within the planctomycete tree. 15.9% sequences were related to some uncultured planctomycetes and branched as a monophyletic cluster from the described four genera ( planctomyces, Pirellula, Isosphaera and Gemmata) of Planctomycetales with similarity of 81–89% to the cultured Planctomyces. More interestingly, 17.5% sequences (most retrieved from the anaerobic basin) formed a separated group close to the anammox cluster with similarity of 82–90% to the described anaerobic ammonia-oxidizing species despite of the unsuitable environments for these bacteria in the wastewater treatment plant. However, the following batch experiment results indicated low level of anammox activity (anaerobic ammonia-oxidizing rate of 0.8 mg NH 4 +-N g −1 dry biomass day −1) existing in the anaerobic biomass of alcohol manufacturing wastewater treatment plant.