Overlapping Polymerase Chain Reaction Research Articles

The character of PA3235 virulence factors of Pseudomonas aeruginosa PAO1 – a preliminary study

Introduction: Many virulence factors of Pseudomonas aeruginosa PAO1 are regulated by temperature and host conditions upon infection. Based on microarray data, the PA3235 gene is one of the upregulated genes during cell growth at 37 °C. Until now, no information about its role in PAO1 pathogenicity. Methods: The PAO1∆PA3235 strain was constructed by overlapping polymerase chain reaction (PCR) method and through biparental mating. The deletion was confirmed by PCR and restriction analyses. The virulence factors of both P. aeruginosa PAO1 wild type and ∆PA3235 mutant strains were examined, which consisted of the amount of pyocyanin and pyoverdine, swarming, swimming, and twitching motility, biofilm formation, 3-oxo-dodecanoyl-homoserine lactone concentration, and growth curve profile. Data were analyzed using Student’s t-tests to determine differences between treatments. P-value < 0.05 were considered significant. Results: The ∆PA3235 strain was successfully constructed. At 37 ºC, the mutant produced less pyocyanin (p-value 0.0004), pyoverdine (p-value 0.0009), and swarm area than the wild-type. The dendrites pattern of both strains was similar. The mutant and parental strains showed no differences in swimming and twitching motility when incubated at 22 ºC and 37 ºC. The mutant produced more biofilm compared to the wild-type strain (p-value 0.0013). The AHL was higher in the mutant than in wild type strain (p-value 0.0095) after 24 h incubation. Both the wild type and mutant strains exhibited similar growth patterns in LB broth. The mutant colonies also showed the same morphology as the wild type on the LB plate (not shown here). Conclusion: The deletion of the PA3235 gene from the Pseudomonas aeruginosa genome caused some changes in virulence factors production, as the bacterium grew at body temperature 37°C. We predicted that the PA3235 gene might function to transport molecules involved in the early infection of this bacterium to humans.

Fluorescence Resonance Energy Transfer Based Biosensor from Thermophilic Bacterial Periplasmic Binding Protein to Measure Branched-chain and Aromatic Amino Acids

Aim: Exploring the ligand binding capacity of leucine-isoleucine-valine binding protein from Thermotoga maritima.
 Background: The Fischer ratio, ratio of concentrations between branched-chain and aromatic amino acids, has been considered as an indicator of hepatic disease involving metabolic dysfunctions since its instigation in the 1970's. These amino acids are usually measured by high performance liquid chromatography, gas chromatography and enzymatic spectrophotometry. Considering the future potential of periplasmic binding proteins in clinical diagnosis, leucine-isoleucine-valine binding protein from a thermophilic bacterium Thermotoga maritima was purified and characterized, and evaluated its binding properties with twenty amino acids. The protein's stereo-specificity was also tested due to its importance in astrobiology research. Amino acids and carbohydrates are both known to exist in extraterrestrial environments, and stereo-chemistry is the key aspect for distinguishing between abiotic and biotic origin.
 Results: Single amino acid substitutions were generated by overlapping polymerase chain reaction mediated mutagenesis using mutagenic primers to construct two mutants of leucine-isoleucine-valine binding protein (LIVBP). The first mutant of LIVBP in which phenylalanine (F) at 118 position was replaced with cysteine (C) was termed as LIVBP-F118C and the resultant plasmid was named as pKM242. Another mutant of LIVBP in which aspartic acid (D) at 221 position was replaced with cysteine was termed as LIVBP-D221C and the resultant plasmid was named as pKM244. Since the LIVBP-F118C showed higher labeling and fluorescence resonance energy transfer efficiency compared to that of D221C, the LIVBP-F118C was used for further study.
 Conclusion: Fluorescence resonance energy transfer technology was applied to measure dissociation constant (Kd) where chimeric isoleucine-valine binding protein (LIVBP) was engineered to conjugate a donor fluorophore at amino-terminal and an acceptor fluorophore at the incorporated cysteine residue. Ligand binding studies revealed that LIVBP from Thermotoga maritima was able to bind with fourteen amino acids out of twenty including branched-chain amino acids (BCAAs) and aromatic amino acids (AAAs) with Kd values ranging from 10-6 to 10-9 M. The Kd values of BCAAs, AAAs, methionine and cysteine were observed at nM level. Highest Kd value was observed for L-leucine (37.2 nM) and L-phenylalanine (44.6 nM). These results indicated that LIVBP from Thermotoga maritima has a broader substrate specificity than previously reported for LIVBP from other organisms, which might be helpful for the development of a miniaturized and noninvasive biosensor to measure BCAAs and AAAs.

Heterologous fusion gene expression and characterization of a novel carbohydrate binding module (Cbm36) to laccase (Lcc2)

Carbohydrate binding modules (CBMs) are commonly known to have the potential to increase binding of carbohydrate-active enzymes to target polysaccharides, thus the catalytic module can easily contact the substrate via CBM. Laccase is one of the most important enzymes used for delignification step in paper industry. This enzyme improves the product's quality, industrial process, and environmentally sound technologies. In this study, we have constructed the fusion gene encoding a novel CBM36 from β-Xylosidase B (GbtXyl43B) of Geobacillus thermoleovorans IT-08 and laccase (LCC2) of Pleurotus salmoneostramineus using overlapping polymerase chain reaction (PCR), and inserted into expression vectors pET-32a and pYHM1 for Escherichia coli BL21 codon plus and Saccharomyces cerevisiae BJ1824, respectively. The fusion protein, Cbm36-Lcc2, was successfully expressed both in E. coli and S. ceerevisiae and optimum pH and temperature were determined to be 5.0 and 60 °C, and 6.0 and 60 °C, respectively, with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate; ABTS) as the substrate. The thermostability and pH stability indicated that Cbm36-Lcc2 expressed in S. cerevisiae BJ1824 was more stable than in E.coli BL21 codon plus. The laccase activity for Cbm36-Lcc2 was two to three times higher than that for Lcc2 itself. This study developed the new insight that fusion of novel Cbm36 into Lcc2 could improve the thermostability of Lcc2 in its chimeric laccase form of Cbm36-Lcc2 compared to Lcc2 itself.

Biodegradation of Fumonisins by the Consecutive Action of a Fusion Enzyme.

Fumonisins (FBs) are toxic mycotoxins that commonly exist in food and feed. FBs can induce many aspects of toxicity, leading to adverse effects on human and animal health; therefore, investigating methods to reduce fumonisin contamination is necessary. In our study, we generated a recombinant fusion enzyme called FUMDI by linking the carboxylesterase gene (fumD) and the aminotransferase gene (fumI) by overlapping polymerase chain reaction (PCR). The fusion enzyme FUMDI was successfully, secretively expressed in the host Pichia pastoris (P. pastoris) GS115, and its expression was optimized. Our results demonstrated that the fusion enzyme FUMDI had high biodegradation activity of fumonisin B1 (FB1) and other common FBs, such as fumonisin B2 (FB2) and fumonisin B3 (FB3), and almost completely degraded 5 μg/mL of each toxin within 24 h. We also found that FUMDI enzyme and its reaction products had no negative effect on cell viability and did not induce cell apoptosis, oxidative stress, or endoplasmic reticulum (ER) stress in a human gastric epithelial cell line (GES-1). The results indicated that these FBs degradation products cannot have adverse effects in a cell model. In conclusion, a safe and efficient fumonisin-degrading enzyme was discovered, which could be a new a technical method for hazard control of FBs in the future.

GENETIC ENGEENERING OF ESCHERICHIA COLI STRAIN PRODUCING KERATINASE OF BACILLUS LICHENIFORMIS

The method of continuous overlapping polymerase chain reaction was applied to clone gene encoding keratinase of Bacillus licheniformis within vector pET42a(+). The resulting genetic construction was used to transform Escherichia coli BL21 (DE3) cells. The strain with maximum level of protein synthesis was selected. Specific activity of purified enzyme solution (purity grade ~95 %) in the reaction with azocasein substrate equaled 73.18 units/mg protein. The recombinant E. coli strain could serve as a source of enzyme capable to process keratinous wastes.

Recombinant pseudorabies virus with gI/gE deletion generated by overlapping polymerase chain reaction and homologous recombination technology induces protection against the PRV variant PRV-GD2013

BackgroundSince 2011, numerous highly virulent and antigenic variant viral strains have been reported in pigs that were vaccinated against the swine pseudorabies virus. These infections have led to substantial economic losses in the Chinese swine industry.ResultsThis study, constructed a novel recombinant vaccine strain with gI/gE deletion (PRV-GD2013-ΔgI/gE) by overlapping PCR and homologous recombination technology. The growth curves and plaque morphology of the recombinant virus were similar to those of the parental strain. However, PRV-GD2013-ΔgI/gE infection was significantly attenuated in mice compared with that of PRV-GD2013. Two-week-old piglets had normal rectal temperatures and displayed no clinical symptoms after being inoculated with 105 TCID50 PRV-GD2013-ΔgI/gE, indicating that the recombinant virus was avirulent in piglets. Piglets were immunized with different doses of PRV-GD2013-ΔgI/gE, or a single dose of Bartha-K61 or DMEM, and infected with PRV-GD2013 at 14 days post-vaccination. Piglets given high doses of PRV-GD2013-ΔgI/gE showed no obvious clinical symptoms, and their antibody levels were higher than those of other groups, indicating that the piglets were completely protected from PRV-GD2013.ConclusionsThe PRV-GD2013-ΔgI/gE vaccine strain could be effective for immunizing Chinese swine herds against the pseudorabies virus (PRV) strain.

Evaluation of the immunogenicity of an omp A and staphylococcal target of RNAIII activating fusion protein displayed on the surface of Escherichia coli

The purpose of this investigation was to construct a recombinant Escherichia coli strain displaying the Staphylococcus aureus target of RNAIII activating protein (TRAP) on its surface, and to investigate the strain for its immunogenicity. The lpp'ompA and lpp'ompA-TRAP genes were fused by the overlap polymerase chain reaction and then ligated into expression plasmid pQE30 producing pLO and pLO-TRAP. These two recombinant plasmids were transformed into E. coli XL1-Blue, resulting in XL1-Blue/pLO and XL1-Blue/pLO-TRAP, which were induced to express protein. The expressed TRAP protein was displayed on the surface of XL1-Blue as judged by whole cell ELISA, flow cytometric analysis, and laser scanning confocal microscopy using the lpp'ompA surface display system. ICR mice were intramuscularly immunized with recombinant strains XL1-Blue/pLO and XL1-Blue/pLO-TRAP as well as recombinant protein TRAP. Immunized mice were assessed for anti-TRAP antibody and lymphocytes for secreted IL-4 and IFN-γ by ELISPOT and secreted IL-17A by indirect ELISA. Immunized mice were challenged with S. aureus Newman and HLJ23-1 strains. The results showed both XL1-Blue/pLO-TRAP and TRAP protein immunized mice to produce better cellular and humoral immunity than XL1-Blue/pLO and PBS injected mice.

Molecular epidemiology of chicken anaemia virus in sick chickens in China from 2014 to 2015.

Chicken anaemia virus (CAV), a member of the genus Gyrovirus, is the etiological agent of chicken infectious anaemia. CAV infects bone marrow-derived cells, resulting in severe anaemia and immunosuppression in young chickens and a compromised immune response in older birds. We investigated the molecular epidemiology of CAV in sick chickens in China from 2014 to 2015 and showed that the CAV-positive rate was 13.30%, in which mixed infection (55.56%) was the main type of infection. We isolated and identified 15 new CAV strains using different methods including indirect immunofluorescence assay and Western Blotting. We used overlapping polymerase chain reaction to map the whole genome of the strains. Phylogenetic analyses of the obtained sequences and related sequences available in GenBank generated four distinct groups (A–D). We built phylogenetic trees using predicted viral protein (VP) sequences. Unlike CAV VP2s and VP3s that were well conserved, the diversity of VP1s indicated that the new strains were virulent. Our epidemiological study provided new insights into the prevalence of CAV in clinical settings in recent years in China.

Rational engineering of Cel5E from Clostridium thermocellum to improve its thermal stability and catalytic activity.

The celH gene from Clostridium thermocellum encodes a protein containing 900 residues and three components, including Cel5E, Lic26a, and carbohydrate-binding domains. Cel5E is a member of the glycoside hydrolase-5 family and is a bifunctional xylanase/cellulase enzyme. We targeted a semi-hydrophobic pocket near the Cel5E active site and theoretically screened mutated variants for enhanced levels of thermal stability. Cel5E mutations were inserted into celH by overlapping polymerase chain reaction, followed by expression of wild-type and mutant enzymes in Escherichia coli BL21 (DE3) and purification by affinity chromatography. Thermal-stabilizing mutations were subjected to molecular dynamics simulation, and measurement of the in vacuo potential energy, van der Waals forces, electrostatic interactions, and net nonbonded potential energies obtained an overall binding affinity of - 64.964KJ/mol for wild-type Cel5E and - 176.148, - 200.921, and - 120.038KJ/mol for the N94F, N94W, and E133F mutants, respectively. Additionally, the N94W, N94F, E133F, and N94A variants exhibited 1.92-, 1.29-, 1.1-, and 1.15-fold better carboxymethyl cellulase (CMCase) and 1.46-, 1.29-, 1.11-, and 1.12-fold better β-glucanase activity on barley β-glucan relative to the wild-type enzyme. Interestingly, the optimal temperature for CMCase activity by the N94W variant was shifted 2 °C higher than that for the wild-type enzyme. Mutated variants showed improved CMCase and β-glucanase activity and shifted toward higher temperature of maximum activity.

Development and evaluation of armored RNA-based standards for quantification of BCR-ABL1p210/p190 fusion gene transcripts.

Standards play an important role in detection of the BCR-ABL1 fusion gene (FG) transcript. However, the standards widely used in laboratories are mainly based on plasmids or cDNA, which cannot accurately reflect the process of RNA extraction and cDNA synthesis. Therefore, we aimed to develop armored RNA-based standards for p210 and p190 BCR-ABL1FG transcripts' quantification. Using overlapping polymerase chain reaction (PCR) technology, we first linked a segment of the p210 or p190 BCR-ABL1FG transcript with four control genes (CGs; ABL1, BCR, GUSB, and B2M) to form p210FG-CG and p190FG-CG. Subsequently, using armored RNA technology, we prepared p210FG-CG- and p190FG-CG-armored RNAs and the p210FG-CG and p190FG-CG standards, the values of which were assigned by digital PCR (dPCR). The p210FG-CG and p190FG-CG standards were stable and homogeneous, and were significantly linear with r2 >0.98. A field trial including 52 laboratories across China showed that the coefficient of variation (CV%) of BCR-ABL1 values among samples was in the range of 58.6%-129.6% for p210 samples and 73.2%-194.0% for p190 samples when using local standards. By contrast, when using the p210FG-CG and p190FG-CG standards, the CV% of BCR-ABL1 values was decreased to 35.6%-124.9% and 36.6%-170.6% for p210 and p190 samples, respectively. In addition, 33.3% (3/9) of the p210 and p190 samples had CV% values <50.0%, whereas 44.4% (4/9) and 77.8% (7/9) of the samples had lower CV% values when using the p210FG-CG and p190FG-CG standards. The overall variability of detection of BCR-ABL1 transcripts decreased significantly when using the p210FG-CG or p190FG-CG standards, especially the p190FG-CG standard.

The mitochondrial genome of the Kentish Plover Charadrius alexandrinus (Charadriiformes: Charadriidae) and phylogenetic analysis of Charadrii.

The suborder Charadrii (Aves: Charadriiformes), one of the most species-rich radiations within shorebirds, which contains good source for studies of ecology, behaviour and evolution. The resources of mitogenome have rapidly accumulated in recent years due to the advanced genomic sequencing, while suborder Charadrii's mitogenome has not been well studied. The primary objective of this study was to determine the complete mitogenome sequence of Charadrius alexandrinus, and investigated the evolutionary relationship within Charadrii. The mitogenome of C. alexandrinus were generated by amplification of overlapping Polymerase Chain Reaction (PCR) fragments. In this study, we determined the complete mitogenome sequence of the Kentish Plover Charadrius alexandrinus, and comparative analysed 11 species to illustrate mitogenomes structure and investigated their evolutionary relationship within Charadrii. The Charadrii mitogenomes displayed moderate size variation, the mean size was 16,944bp (SD = 182, n = 11), and most of the size variation due to mutations in the control region (CR). Nucleotide composition was consistently biased towards AT rich, and the A+T content also varies for each protein-coding genes. The variation in ATP8 and COIII was the highest and lowest respectively. The GC skew was always negative, with the ATP8 had higher value than other regions. The average uncorrected pairwise distances revealed heterogeneity of evolutionary rate for each gene, the COIII, COI and COII have slow evolutionary rate, whereas the gene of ATP8 has the relative fast rate. The highest value of Ks and Ka were ND1 and ATP8, and the ratios of Ka/Ks are lower than 0.27, indicating that they were under purifying selection. Phylogenomic analysis based on the complete mitochondrial genomes strongly supported the monophyly of the suborder Charadrii. This study improves our understanding of mitogenome structure and evolution, and providing further insights into phylogeny and taxonomy in Charadrii. In future, sequencing more mitogenomes from various taxonomic levels will significantly improve our understanding of phylogenetic relationships within Charadrii.

Construction of srtA-deletion mutant of Streptococcus mutans by an in-frame deletion system

To construct srtA-gene deletion mutant of Streptococcus mutans (S. mutans) UA159 with IFDC2 cassette through overlapping polymerase chain reaction (PCR) and allelic homologous recombination. First, the upstream and downstream fragments surrounding the srtA and IFDC2 cassette were PCR amplified and ligated through overlapping PCR. The resulting amplicon was transformed into UA159, and positive transformants were selected on BHI plates containing erythromycin. Second, upstream and downstream fragments of srtA with overlap regions were generated by PCR and were overlapped to create upΔ-down amplicon. Then, the upΔ-down amplicon was transformed into the aforementioned positive transformants and selected on BHI plates containing p-Cl-Phe. The PCR analysis and DNA sequencing results indicated that the coding region of the srtA was completely deleted, and the upstream and downstream regions flanking the srtA were ligated seamlessly. The markerless srtA-deletion mutant of S. mutans was constructed successfully, which laid a foundation for further study of its biological function and influence on the biofilm formation of S. mutans.

Optimization of a Polymerase Chain Reaction System for Whole Genome Amplification of Human Immunodeficiency Virus Type 2 (HIV-2)

BackgroundIn this study, attempts were made to amplify the whole genome of HIV-2 using polymerase chain reaction (PCR) from viral RNA and proviral DNA extracted from archived human plasma and whole blood, respectively. The aim this study is to develop a PCR system that can be used to amplify the entire genome of all known subtypes and recombinant forms of HIV-2MethodsProviral DNA and viral RNA were extracted from archived human whole blood and plasma using Zymo Research Viral DNA kit and Zymo Research Viral nucleic acids kit, respectively. Primers that target the conserved sites of the 3’ and 5’ long terminal repeat were selected based on in silico analysis using Snapgene® tool version 2.1.0. Eight overlapping Primers for PCR whole genome amplification of HIV-2 were also selected and used for overlap PCR amplification of whole genome of HIV-2. Long range and overlapping PCR of whole genome of HIV-2 were carried out using long range Kit (New England Biolabs Inc., Ipswich, MA, USA) and cDNA synthesized using NEB ProtoScript II for proviral DNA and viral RNA templates. Amplified whole genome of HIV-2 was gel purified and PCR confirmed using two sets of primers ENVF/ENVG and EB2/EB5 and gel electrophoresis.ResultsProviral DNA and viral RNA were successfully extracted from archived whole blood and plasma. Six primers were selected out of the 68 primer sequences retrieved using in silico analysis for long range single PCR amplification of HIV-2 whole genome. Primers P1/P8 and HIV2upA/HIV2lowA were successfully used in the whole genome amplification of HIV-2. Overlapping primers P1/P4, P6/P8, PolF/EnvG and Pol4F/EnvG covering the entire genome of HIV-2 were also successfully used in the whole genome amplification of HIV-2. The amplified whole genome fragment was confirmed to be HIV-2 by PCR using primers EB2/EB5 and EnvF/EnvG.ConclusionThe techniques of long-range and overlapping amplification of HIV-2 whole genome may be useful in HIV-2 genotyping using viral RNA and proviral DNA. This study has led to the selection and shown novel primer combinations of already existing primers which can be used to amplify the entire genome of HIV-2.Disclosures All authors: No reported disclosures.

STITCHER 2.0: primer design for overlapping PCR applications

Overlapping polymerase chain reaction (PCR) is a common technique used by researchers in very diverse fields that enables the user to ‘stitch’ individual pieces of DNA together. Previously, we have reported a web based tool called STITCHER that provides a platform for researchers to automate the design of primers for overlapping PCR applications. Here we present STITCHER 2.0, which represents a substantial update to STITCHER. STITCHER 2.0 is a newly designed web tool that automates the design of primers for overlapping PCR. Unlike STITCHER, STITCHER 2.0 considers diverse algorithmic parameters, and returns multiple result files that include a facility for the user to draw their own primers as well as comprehensive visual guides to the user’s input, output, and designed primers. These result files provide greater control and insight during experimental design and troubleshooting. STITCHER 2.0 is freely available to all users without signup or login requirements and can be accessed at the following webpage: www.ohalloranlab.net/STITCHER2.html.

High-level expression of two thermophilic β-mannanases in Yarrowialipolytica

Two thermophilic β-mannanases (ManA and ManB)were successfully expressed in Yarrowialipolytica using vector pINA1296I. The sequences of manA from Aspergillus niger CBS 513.88 and manB from Bacillus subtilis BCC41051 were optimized based on codon-usage bias in Y.lipolytica and synthesized by overlapping polymerase chain reaction (PCR). We utilized the pINA1296I vector, which allows inserting and expression of multiple copies of an expression cassette, to engineer recombinant strains containing multiple copies of manA or manB. Following verification of target-gene expression by quantitative PCR, fermentation experiments indicated that recombinant protein levels and enzyme activity increased along with increasing manA/manB copy number.After production in a 10 l fermenter, we obtained maximum enzyme activity from strains YLA6 and YLB6 of3024 U/mL and 1024 U/mL, respectively. Additionally, purification and characterization results revealed that the optimum pH and temperature for manA activity were pH∼5 and ∼70 °C, and for manB activity were pH∼7 and 60 °C, respectively. These results indicated that the thermo stabilities of these two enzymes were higher than most other mannanases, making them potentially useful for industrial applications.

High-Frequency Targeted Mutagenesis in Pseudomonas stutzeri Using a Vector-Free Allele-Exchange Protocol.

The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes. Several protocols have been developed to inactivate genes from the genus Pseudomonas. Those protocols are complicated and time-consuming and mostly do not enable easy construction of multiple knock-ins/outs. The current study describes a single and double crossover-recombination system using an optimized vector-free allele-exchange protocol for gene disruption and gene replacement in a single species of the family Pseudomonadaceae. The protocol is based on self-ligation (circularization) for the DNA cassette which has been obtained by overlapping polymerase chain reaction (Fusion-PCR), and carries an antibiotic resistance cassette flanked by homologous internal regions of the target locus. To establish the reproducibility of the approach, three different chromosomal genes (ncRNA31, rpoN, rpoS) were knocked-out from the root-associative bacterium Pseudomonas stutzeri A1501. The results showed that the P. stutzeri A1501 mutants, which are free of any plasmid backbone, could be obtained via a single or double crossover recombination. In order to optimize this protocol, three key factors that were found to have great effect on the efficiency of the homologous recombination were further investigated. Moreover, the modified protocol does not require further cloning steps, and it enables the construction of multiple gene knock-in/out mutants sequentially. This work provides a simple and rapid mutagenesis strategy for genome editing in P. stutzeri, which may also be applicable for other gram-negative bacteria.

Artificial Synthesis of Conserved Segment S Gene Fragment of Rift Valley Fever Virus and Preliminary Study of Its Reverse Transcription Loop-Mediated Isothermal Amplification Detection Method

Purpose: To develop a rapid detection method for Rift Valley fever virus (RVFV) diagnosis.Methods: According to the reference sequences of RVFV published in GenBank, nine overlapping polymerase chain reaction (PCR) primers and four specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) primers were designed using DNAStar and LAMP primer design software, respectively. Based on the synthesis of a conserved part of the RVFV S segment gene sequence using overlapping PCR, RT-LAMP assay was first established and evaluated after a series of tests, including, optimization of reaction conditions, and sensitivity and specificity tests.Result: A target RVFV S segment gene fragment of 288 bp was synthesised. The optimal reaction conditions for RT-LAMP assay were 63 °C for 45 min: the assay has a specific ladder-like pattern of amplification bands from about 120 bp. The lowest target gene copy number of RT-LAMP for RVFV detection was 70 copies. The assay showed good specificity as only the synthesised target RVFV gene was amplified with no amplification for the detection of Peste des petits ruminants virus, Epidemic encephalitis B virus, E. coli, Pasteurella multocida, or Salmonella.Conclusion: This study provides a rapid, sensitive, specific RT-LAMP method for RVFV detection.Keywords: Rift valley fever virus, Overlapping polymerase chain reaction, Reverse transcription loopmediated isothermal amplification, Rapid diagnosis test

Development of a dot blot assay with antibodies to recombinant "core" 14-3-3 protein: Evaluation of its usefulness in diagnosis of Creutzfeldt-Jakob disease.

Background and Purpose:Definitive diagnosis of Creutzfeldt–Jakob disease (CJD) requires demonstration of infective prion protein (PrPSc) in brain tissues by immunohistochemistry or immunoblot, making antemortem diagnosis of CJD difficult. The World Health Organization (WHO) recommends detection of 14-3-3 protein in cerebrospinal fluid (CSF) in cases of dementia, with clinical correlation, as a useful diagnostic marker for CJD, obviating the need for brain biopsy. This facility is currently available in only a few specialized centers in the West and no commercial kit is available for clinical diagnostic use in India. Hence the objective of this study was to develop an in-house sensitive assay for quantitation of 14-3-3 protein and to evaluate its diagnostic potential to detect 14-3-3 proteins in CSF as a biomarker in suspected cases of CJD.Materials and Methods:A minigene expressing the “core” 14-3-3 protein was synthesized by overlapping polymerase chain reaction (PCR) and the recombinant protein was produced by employing a bacterial expression system. Polyclonal antibodies raised in rabbit against the purified recombinant protein were used for developing a dot blot assay with avidin-biotin technology for signal amplification and quantitation of 14-3-3 protein in CSF.Results:The results in the present study suggest the diagnostic potential of the dot blot method with about 10-fold difference (P< 0.001) in the CSF levels of 14-3-3 protein between the CJD cases (N= 50) and disease controls (N= 70). The receiver operating characteristic (ROC) analysis of the results suggested an optimal cutoff value of 2 ng/mL.Conclusions:We have developed an indigenous, economical, and sensitive dot blot method for the quantitation of 14-3-3 protein in CSF.

An efficient method for multiple site-directed mutagenesis using type IIs restriction enzymes

Site-directed mutagenesis (SDM) methods are very important in modern molecular biology, biochemistry, and protein engineering. Here, we present a novel SDM method that can be used for multiple mutation generation using type IIs restriction enzymes. This approach is faster and more convenient than the overlap polymerase chain reaction (PCR) method due to its having fewer reaction steps and being cheaper than, but as convenient as, enzymatic assembly. We illustrate the usefulness of our method by introducing three mutations into the bacterial Streptococcus thermophilus Cas9 (bStCas9) gene, converting the humanized S. thermophilus Cas9 (hStCas9) gene into nuclease dead or H847A nickase mutants and generating sunnyTALEN mutagenesis from a wild-type TALEN backbone.

Comparison of vanA gene mRNA levels between vancomycin-resistant Enterococci presenting the VanA or VanB phenotype with identical Tn1546-like elements.

During the routine screening of vancomycin-resistant Enterococci faecium (VREfm) we found that VanA phenotype-vanA genotype and VanB phenotype-vanA genotype isolates had an identical Tn1546-like element structure. This study aimed to evaluate the genetic background and vanA gene expression to identify the mechanisms of the development of the VanB phenotype-vanA genotype VREfm. Twelve VREfm isolates were collected from a 1500-bed tertiary-care teaching hospital in Beijing. Genetic variations of the Tn1546-like element were determined by an overlapping polymerase chain reaction assay and sequencing. The genetic background wasdetermined by pulsed-field gel electrophoresis and mutilocus sequence typing. vanA gene expression was evaluated using a TaqMan quantitative real-time polymerase chain reaction. For the 12 isolates, six isolates with the VanA phenotype-vanA genotype and six with the VanB phenotype-vanA genotype were identified. According to the structure analysis of the Tn1546-like elements, our isolates were divided into two types. In the four isolates of type A, IS1542 and IS1216V were inserted into the orf2-vanR and vanX-vanY regions, respectively. In the eight isolates of type D, a similar insertion as type A occurred, except for an ISEfa4 insertion into IS1542. A significant difference in vanA gene expression was observed between the VanA and VanB phenotype isolates in type A, but not in type D. Mutilocus sequence typing and pulsed-field gel electrophoresis analysis showed that these isolates have a different genetic background. Our results indicated that the occurrence of the VanB phenotype-vanA genotype might not completely depend on the structure of Tn1546-like elements and vanA gene expression.