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Rapid DNA detection is a long-pursuing goal in molecular detection, especially in combating infectious diseases. Loop-mediated isothermal amplification (LAMP) is a robust and prevailing DNA detection method in pathogen detection, which has been drawing broad interest in improving its performance. Herein, we reported a new strategy and developed a new LAMP variant named TLAMP with a superior amplification rate. In this strategy, the turn-back loop primers (TLPs) were devised by ingeniously extending the 5' end of the original loop primer, which conferred the new role of being the inner primer for TLPs while retaining its original function as the loop primer. In theory, based on the bifunctional TLPs, a total of eight basic dumbbell-like structures and four cyclic amplification pathways were produced to significantly enhance the amplification efficiency of TLAMP. With the enhancing effect of TLPs, TLAMP exhibited a significantly reduced amplification-to-result time compared to the conventional six-primer LAMP (typically 1 h), enabling rapid DNA detection within 20 min. Furthermore, TLAMP proved to be about 10 min faster than the fast LAMP variants reported so far, while still presenting comparable sensitivity and higher repeatability. Finally, TLAMP successfully achieved an ultrafast diagnosis of Monkeypox virus (MPXV), capable of detecting as few as 10 copies (0.67copies/μL) of pseudovirus within 20 min using real-time fluorescence assay or within 30 min using a colorimetric assay, suggesting that the proposed TLAMP offers a sensitive, specific, reliable, and, most importantly, ultrafast DNA detection method when facing the challenges posed by infectious diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

PCR-based DNA walking is of efficacy for capturing unknown flanking genomic sequences. Here, an uracil base PCR (UB-PCR) with satisfying specificity has been devised for DNA walking. Primary UB-PCR replaces thymine base with uracil base, resulting in a primary PCR product composed of U-DNAs. A single-primer (primary nested sequence-specific primer) single-cycle amplification, using the four normal bases (adenine, thymine, cytosine, and guanine) as substrate, is then performed on the primary PCR product. Clearly, only those U-DNAs, ended by the primary nested sequence-specific primer at least at one side, will produce the corresponding normal single strands. Next, the single-cycle product undergoes uracil-DNA glycosylase treatment to destroy the U-DNAs, while the normal single strands are unaffected. Afterward, secondary even tertiary PCR is performed to exclusively enrich the target product. The feasibility of UB-PCR has been checked by obtaining unknown sequences bordering the three selected genetic sites., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ribosomal RNA gene as a high-copy number nucleo-biomarker is extremely conserved among bacteria which limits its application to the discriminative detection approaches. We have developed a colorimetric isothermal amplification method called "single specific primer-LAMP (SSP-LAMP)" requiring only one specific primer for the amplification of the target and applied to the identification of the 16S rRNA gene in the Shigella genus. A region with high sequence homology in the genus and low homology with other bacteria was considered as the most appropriate. In that regard, a 23 bp sequence in the 16S rRNA gene of the genus was targeted based on the alignment of the gene with fifty-three closely related bacterial species, and a single specific primer along with five degenerate primers were designed. Using hydroxy-naphthol blue (HNB) as an indicator and gel electrophoresis, the proposed approach of SSP-LAMP was able to detect S. boydii, S. sonnei, S. flexneri and S. dysenteriae specifically while other species remained unidentified. The SSP-LAMP method could provide a rapid one-pot point-of-care method for molecular diagnostics of pathogens in many circumstances mainly samples with high genetic homogeneity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Intramolecular secondary structures within templates have been shown to lower PCR performance. Whereas many approaches have been developed to mitigate such impairment on PCR, their effects can vary greatly depending on template sequences. Here we present a novel, universally effective approach to improve PCR performance involving specifically designed oligonucleotides called disruptors. A disruptor contained three functional components, an anchor designed to initiate template binding, an effector to disrupt intramolecular secondary structure, and a 3' blocker to prevent its elongation by DNA polymerase. A functional mechanism for a disruptor to improve PCR efficiency was proposed where anchor first binds to template followed by effector-mediated strand displacement to unwind intramolecular secondary structure. Such a mechanism was consistent with the observation that anchor played a more critical role for disruptor function. As an example of potential disruptor applications, inverted terminal repeat sequences of recombinant adeno-associated virus vectors were successfully amplified in the presence of disruptors despite their well-known reputation as some of the most difficult templates for PCR amplification and Sanger sequencing due to their ultra-stable T-shaped hairpin structures. In stark contrast, both DMSO and betaine, two PCR additives routinely used to facilitate PCR amplification and Sanger sequencing of GC-rich templates, did not demonstrate any improving effect., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Technological advancements have revolutionized ancient and degraded DNA analysis, moving the field to the Next Generation Sequencing era. One of the advancements, the ancient DNA-oriented high-throughput library preparation methods, enabled the sequencing of more endogenous molecules. Although fairly optimized, both single- and double-stranded library preparation methods hold the potential for further improvement. Here, we test a series of modifications made at different steps of both single- and double-stranded library preparation methods. Given all the modifications tested, we found that two of them provide further benefits, including the use of Endonuclease VIII as a pre-treatment step before preparing single-stranded libraries and the use of a modified second adapter of the single stranded-libraries as an alternative option to enable sequencing of single stranded-libraries with the standard Illumina sequencing primer instead of the custom designed as described in the single stranded library preparation method. Furthermore, we propose uracil-DNA-glycosylase (UDG) could also be considered for both single- and double-stranded library preparation methods, although additional parameters should be taken into account depending on the sequencing strategy and the sample characteristics. Further modifications were also tested and although they were not advantageous, they could be considered as equivalent to the published options., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: Current polymerase chain reaction (PCR) methods for the diagnosis of infections are time consuming and require large sample volume and skilled technicians. We developed a novel, easy-to-use, and rapid (processing time, 1 minute; total time, 33 minutes) multiplex real-time PCR test (Direct Strip PCR) that did not require DNA extraction to detect 9 pathogens that could cause uveitis in 20-μl samples., Design: Multicenter prospective evaluation of a diagnostic PCR test., Participants: A total of 511 participants (patients with infectious uveitis and controls) were examined at 18 institutes worldwide., Methods: After validation, intraocular fluid samples were subjected to etiologic or exclusive diagnosis, including intraoperative rapid diagnosis., Main Outcome Measures: The concordance and correlations between Direct Strip PCR and quantitative PCR (qPCR) results., Results: Direct Strip PCR exhibited rapid detection, good repeatability and specificity, long storage stability, and detection ability equal to that of qPCR. It also showed low interinstitutional variability compared with qPCR, even when PCR beginners used various real-time PCR machines. The Direct Strip PCR for 9 pathogens exhibited high concordance against the qPCR (positive concordance rate, 98.8%-100%; negative concordance rate, 99.8%-100%; κ coefficient, 0.969-1.000; P < 0.001-0.031). Additionally, results obtained using Direct Strip PCR and qPCR were highly correlated (ρ = 0.748; P < 0.001). This assay was used for rapid intraoperative diagnosis., Conclusions: The Direct Strip PCR test may improve the prognosis of various infectious diseases because it facilitates rapid etiologic evaluation at the first hospital visit and can be used for intraoperative diagnosis., (Copyright © 2020 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Novel coronavirus disease (COVID-19) caused by SARS-CoV-2 is an ongoing global pandemic associated with high rates of morbidity and mortality. RT-qPCR has become the diagnostic standard for the testing of SARS-CoV-2 in most countries. COVID-19 diagnosis generally relies upon RT-qPCR-mediated identification of SARS-CoV-2 viral RNA, which is costly, labor-extensive, and requires specialized training and equipment. Herein, we established a novel one-tube rapid diagnostic approach based upon formamide and colorimetric RT-LAMP (One-Pot RT-LAMP) that can be used to diagnose COVID-19 without the extraction of specific viral RNA. The technique could visually detect SARS-CoV-2 within 45 min with a limit of detection of 5 copies per reaction in extracted RNA, and about 7.66 virus copies per μL in viral transport medium. The One-Pot RT-LAMP test showed a high specificity without cross-reactivity with 12 viruses including SARS-CoV, MERS-CoV, and human infectious influenza virus (H1N1/H3N2 of influenza A and B virus, ect. We validated this One-Pot RT-LAMP approach by its successful use for the analysis of 45 clinical nasopharyngeal swab samples, yielding results identical to those of traditional RT-qPCR analyses, while achieving good selectivity and sensitivity relative to a commercial RT-qPCR approach. As such, this One-Pot RT-LAMP technology may be a valid means of conducting high-sensitivity, low-cost and rapid SARS-CoV-2 identification without the extraction of viral RNA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Denaturation bubble-mediated strand exchange amplification (SEA) is a novel, rapid isothermal nucleic acid amplification has been applied for point-of-care molecular diagnostic in food safety, meat adulteration, forest disease and animal disease. Nevertheless, the absence of specialized strategy for SEA primers design led to long-time of primer screening progress before SEA reaction execution, which would largely increase the time consuming when SEA is utilized for detecting other new targets. In this present work, we investigated the impact of the following primers' attributes on SEA efficiency, including Tm value, 3' end G/C content, self-complementary and 3' complementary, according to which we demonstrated that optimal Tm value and reaction temperature were all 61 °C, while 3'-terminal nucleotide should be G/C, as the SEA reaction induced by the primers possessing these attributes exhibited significantly lower threshold time (Tt) value. Moreover, self-complementary and 3' complementary of primers should be avoided. Besides, we also discussed the consideration priority order of these factors, which was self-complementary and 3' complementary, Tm value and 3' end G/C content in turn. Because the SEA primer design strategy is first presented, our work will greatly promote the application of SEA in point-of-care test., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Isothermal DNA amplification only using a Bacillus stearothermophilus (Bst) DNA polymerase such as loop-mediated isothermal amplification typically entails multiple target sites for primer design and is thereby not suited for the amplification of short gene sequences, for example, the sequences with size below 200 nucleotides (nt). Here we present SLIMP, a novel single enzyme-based isothermal amplification of short gene sequence mediated by both stem-loop and linear primers. In SLIMP, a pair of stem-loop primers and a pair of linear primers are specifically designed to recognize only two target sites. Linear primers in SLIMP are similar as conventional PCR primers, but stem-loop primers are the modified linear primers through attaching a stem-loop structure at their 5'-ends. Attributed to this unique primer design, three basic reaction modes including linear-priming, single stem-loop-priming, and double stem-loop-priming amplifications co-mediate the SLIMP process under the function of Bst DNA polymerase. As a proof-of-concept assay, a synthetic 80 nt sequence from hepatitis B virus S gene was used as the template to develop SLIMP. On performance, SLIMP detection possesses high sensitivity and specificity, good selectivity, and the potential for analysing real sample. Therefore, SLIMP is expected as a novel alternative to amplify short gene sequences using a single enzyme., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Recombinant antibodies serve as therapeutic molecules for a broad range of applications. High affinity antibodies are typically isolated following an active and effective immunization. Human-like antibodies may be obtained from immunized nonhuman primates (NHP), such as rhesus macaque, when immunized human origin is not available. For the isolation of such antibodies, strategies like phage and yeast display, are employed. These strategies are primarily based on the amplification of the rearranged variable (V) regions coded by mRNA, obtained from lymphatic source of immunized animals. To amplify these genomic sequences, designated set of primers are required, ideally covering the immune animal V-gene repertoire. Such primer sets are commonly designed based on the germline repertoire of specific animals according to immunoglobulin databases. However, In case of rhesus macaque, however, the known immunoglobulin germline V-gene database is still limited. The emergence and continuous improvements in high-throughput sequencing (HTS) technologies now enable the profiling of an immune repertoire for both basic and applicative studies, among which is the identification and expression of novel alleles. We report here on the profiling of non-immunized rhesus macaque (Macaca mulatta) expressed antibody repertoire, using HTS and advanced tailored bioinformatics tools. This analysis resulted in 32,480 and 73,354 complete heavy and light variable gene (V H and V L ) sequences, respectively. Further analysis of these sequences, using the IgDiscover tool, resulted in the identification of 102, 214 and 48 inferred V H , V κ and V λ germline sequences, respectively, of which over 50% are novel alleles. This dataset, together with other recently published datasets, enabled the design of a comprehensive primer set (v2018), which demonstrated the broadest coverage of rhesus macaque germline genes identified up to date. The newly designed primer set was confirmed for its extent of coverage of the V-genes in various datasets of rhesus macaque germlines as well as the expressed repertoire mapped in this study. Among other things, an improvement of 28% and 50% in the coverage of the VH and VL expressed repertoire was demonstrated in comparison to a primer set we have previously designed. This primer set can be further used for various applications that require the complete coverage of the NHP V-gene repertoire., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Although various methods have been developed to suffice the oligonucleotide demand of molecular biology laboratories, in vitro production of high-purity ssDNAs remains to be a challenging task. We hypothesized that complementing the asymmetric PCR with 3' phosphate blocked limiting primer decreases the mispriming thus reduces polymerisation of DNA by-products. The presented results attest our assumption that the primer blocked asymmetric PCR (PBA-PCR) selectively produces ssDNA of interest and is even suitable for effective amplification of DNA libraries of large sequence space. The high-throughput sequence analysis demonstrated that PBA-PCR also alleviates the PCR bias obstacle since it does not distort the sequence space. The practicability of the novel method was verified by monitoring the process of SELEX and screening of aptamer candidates using PBA-PCR produced ssDNAs in Amplified Luminescent Proximity Homogeneous Assay. In summary, we have developed a generally applicable method for straightforward, cost-effective production of ssDNA with on demand labelling., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Loop-mediated isothermal amplification is a promising method in the area of nucleic acid detection. However, it suffers from a high rate of false-positive amplifications that largely restrict its application. In this study, we observed gold nanoparticles (AuNP) absorbing single-stranded DNA primers and interacting with Bst DNA polymerase via electrostatic adsorption. As a result of these interactions, the presence of the gold nanoparticles exerted a hot-start effect on the loop-mediated isothermal amplification system. Based on these results, we developed a novel AuNP-mediated nucleic acid isothermal amplification assay. This assay displays significantly enhanced specificity-the proportion of false positive decreased from 76% to 0% and from 100% to 0% for the detection of rotavirus and the β-actin gene, respectively, with the hot-start temperature of 48 °C. Moreover, our AuNP-based assay maintained good sensitivity and a satisfactory detection limit (1 × 10 3 copies/μL) compared with the conventional assay. This approach has the potential to solve the nonspecificity problem of loop-mediated isothermal amplification, thereby promoting its real-world application, particularly, in clinical settings., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Advanced methods for developing and applying responsive DNA nanodevices are of great interest. Herein, we report a stretchable DNAzyme that allows simple, multiplexed and sensitive fluorescent detection of antibodies. We find that rigid antibody can tightly stretch soft, antigen-labelled DNAzyme strand and disrupt the hybridization between DNAzyme and its substrate. Based on this finding, we develop a novel strategy to detect antibodies. Due to the robustness and high activity of DNAzyme, this assay can easily detect target as low as 1 ± 0.25 pM and achieve multiplexed detection by using a cocktail of DNAzymes. The proposed assay not only provides a new approach to readily measure antibody, but broadens the application of DNAzyme that is usually employed to detect metal ions or indirectly analyze biomolecules without the cumbersome design., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: β-thalassaemia is a group of inherited single-gene disorders worldwide. Each ethnic population has its own common mutations. Heterogeneity of β-thalassaemia mutations in multi-ethnic population of Surat, makes molecular diagnosis expensive and time consuming., Methods: Specific primers were used to differentiate four common mutations, IVS I-5 (G→C), Codon 41/42 (- TCTT), 619-bp deletion and FS 8/9 (+G), by a simple PCR involving a multiplex amplification refractory mutation system., Results: Several high prevalence β-Thalassemia trait groups constituted by Muslims, Patels, Sindhis, ModhBanias, and Mahayavanshi. Four most common mutations detected in them are IVS I-5 (G→C), Codon 41/42 (- TCTT), 619-bp deletion and FS 8/9 (+G). We identified each of these β-thalassemia mutations in multiplexed ARMS from positive control samples. Our multiplex-ARMS-PCR system was first standardized on positive DNA samples with above known four most common β-thalassemia mutations, and these positive samples had been diagnosed with β-thalassemia and also all these samples belonged to Surat ethnic groups. The system was subsequently tested on 110 blood samples from different ethnic backgrounds with unknown β-thalassemia mutations which were in all specimens., Conclusion: The ARMS multiplex system was found reliable, cost effective, fast and most applicable for mutation screening of Thalassemia in Surat populations., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Betaine was used as a common additive to isothermal nucleic acid amplification reactions because of lowering the melting temperature (Tm) of DNA. Herein, we reported a novel finding that betaine was inhibiting the reaction efficiency of isothermal amplification reactions. In this work, we have verified this finding by classical loop-mediated isothermal amplification that the addition of 0.8 M betaine inhibited the efficiency of reaction dropping to approximately 1%. Additionally, we clarified the mechanism of betaine hindering isothermal amplification reactions with a molecular barrier to lower associate rate constant K1 for intermolecular hybridization. This finding would be very significant for studies on the interaction between small molecule substance and DNA, and the development of point-of-care testing because of simplifying reaction system and increasing reaction efficiency., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The chemokine receptor CXCR4 plays a key role in the bone marrow microenvironment maintenance and in the hematopoietic stem and progenitor cells migration. In addition, CXCR4 is expressed in a broad spectrum of solid tumors where its methylation state has been recently proposed as a biomarker for cancer prognosis. To evaluate methylation status of CXCR4 promoter we developed a sensitive, accurate, specific and cost-effective two-step PCR method that does not require any specific equipment other than a conventional real-time PCR instrument. The principle of the technique relies on a novel normalization strategy which allows the detection and quantification of small methylation differences among pre-amplified DNA samples deriving from low amount of starting material. In addition, the analysis of melting curve profiles of PCR products provides additional information about the methylation status of CpG sites in between the primers. Finally, the principle of this technique can potentially be adapted for the investigation of the methylation status of any other DNA region., (Copyright © 2016 Elsevier Inc. All rights reserved.)

DNA analysis of biological specimens containing degraded nucleic acids such as mortal remains, archaeological artefacts, forensic samples etc. has gained more attention in recent years. DNA extracted from these samples is often inapplicable for conventional polymerase chain reaction (PCR), so for its amplification the nearby primers are commonly used. Here we report the data that clarify the features of PCR with nearby and abutting primers. We have shown that the proximity of primers leads to significant reduction of the reaction time and ensures the successful performance of DNA amplification even in the presence of PCR inhibitors. The PCR with abutting primers is usually characterized by the absence of nonspecific amplification products that causes extreme sensitivity with limit of detection on single copy level. The feasibility of PCR with abutting primers was demonstrated on species identification of 100 years old rotten wood., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL). VL is the most severe form of leishmaniasis and can be fatal if it is not properly treated. Although several PCR works are intended to detect L. infantum, in silico analysis of available primers and/or primer-probes reveals potential cross species amplification. Here, a TaqMan-based quantitative real time PCR (qPCR) assay was developed for specific detection and quantitation of L. infantum in tissue samples from experimentally or naturally infected animals, mice or dogs, respectively. For this assay, primers and probes were designed for the kinetoplast minicircle DNA of L. infantum. The qPCR assay achieved a detection limit of 0.01pg of parasite DNA, and allowed specific amplification of L. infantum in both asymptomatic and symptomatic naturally infected dogs with inter-assay variation coefficients between 0.05-0.11. There was no cross amplification with dog DNA or with L. braziliensis, L. donovani, L. major, L. tropica or Trypanosoma cruzi. In addition, our assay detected a significantly higher parasite load in symptomatic than in the asymptomatic animals (p<0.0001). We believe this approach will be a valuable tool for the specific detection of L. infantum in regions of sympatric transmission of VL-causing parasites., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) provides a powerful tool for precise gene expression analysis. The accuracy of the results highly depends on careful selection of a reference gene for data normalization. HPRT1 (hypoxanthine phosphoribosyl transferase 1) is a frequently used housekeeping gene for normalizing relative expression values. However, the existence of processed pseudogenes for HPRT1 might interfere with reliable results obtained in qRT-PCR due to amplification of unintended products. Here, we designed a primer pair for pseudogene-free amplification of HPRT1 in qRT-PCR. We demonstrate that this primer pair specifically amplified HPRT1 messenger RNA (mRNA) sequence while avoiding coamplification of the pseudogenes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Pseudomonas aeruginosa is a nosocomial pathogen, which, due to its inherent and acquired resistance to a wide range of antibiotics, causes high mortality rates. Therefore, rapid detection of the bacterium with high specificity and sensitivity plays a critical role in the control of the pathogenic bacterium. The aim of this study was to evaluate the accuracy and specificity of a prompt detection of the bacterium based on a triplex polymerase chain reaction that amplifies the lasI, lasR and gyrB genes. For this purpose, 30 clinical isolates of P. aeruginosa and 30 wound biopsy samples were retrieved from clinical diagnostic laboratories. After the extraction of the chromosomal DNA, the desired genes were amplified using uniplex and triplex PCR with appropriate primers. The specificity of the primers was evaluated by a comparison of the PCR results for P. aeruginosa clinical samples and non-Pseudomonas species control samples. The sensitivity of the primers was determined using a serial dilution of the genomic DNA template (100 ng to 100 fg) and by a comparison of the PCR and bacterial culture results. The results showed that the triplex PCR assay was positive for all of the samples (100%), while the PCR identifications were negative for non-Pseudomonas species. Additionally, at 10(-4) and 10(-5) diluted genomic DNA from P. aeruginosa (10 pg and 1 pg), the triplex PCR test was positive for the Las and gyrB genes in all of the samples, respectively. Based on these results, the designed primers can be used for the rapid, specific and sensitive diagnosis of P. aeruginosa in a triplex PCR assay., (Copyright © 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.)

Background: MicroRNA-429 (miR-429), a short noncoding RNA belonging to the miR-200 superfamily, plays a crucial role in tumorigenesis and tumor progression. It also acts as a modulator of epithelial-to-mesenchymal transition, a cell development regulating process that affects tumor development and metastasis. The aim of this study was to investigate the potential role of miR-429 in regulating growth and metastasis of renal cell carcinoma., Methods: miR-429 expression was stably up-regulated or down-regulated in the renal cell carcinoma ACHN and A498 cell lines, and cell proliferation and metastasis were assessed., Results: miR-429 overexpression inhibited cell proliferation, colony formation, migration, and invasion. Suppression of endogenous miR-429 promoted cell growth and metastasis. miR-429 was shown to directly target the 3' untranslated regions of B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) and E2F transcription factor 3 (E2F3) transcripts, regulating their expression, as well as that of the downstream epithelial-to-mesenchymal transition markers E-cadherin, N-cadherin, vimentin, p14, and p16., Conclusions: These results revealed a tumor suppressive role for miR-429 in renal cell carcinoma through directly targeting BMI1 and E2F3., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Inhibition of EGFR is a strategy for treating metastatic colorectal cancer (CRC) patients. KRAS sequencing is mandatory for selecting wild-type tumor patients who might benefit from this treatment. DNA from formalin-fixed paraffin-embedded (FFPE) tissues is commonly used for routine clinical detection of mutations, and its amplification succeeds only when all preanalytical histological processes have been controlled. In cases that are not properly processed, the DNA results can be poor, with low peak pyrosequencing findings. We designed and tested a pair of forward and reverse primers for a nested PCR method, followed by pyrosequencing, in a single Latin American institution series of 422 unselected CRC patients, correlating KRAS mutations with pathological and clinical data., Materials and Methods: Patient DNA samples from tumors were obtained by scraping or laser microdissection of cells from FFPE tissue and extracted using a commercial kit. DNA was first amplified by PCR using 2 primers that we designed; then, nested PCR was performed with the amplicon from the preamplification PCR using the KRAS PyroMark™ Q96 V2.0 kit (Qiagen). Pathological data were retrieved from pathology reports., Results: KRAS mutation was observed in 33% of 421 cases. Codon 12 was mutated in 76% of cases versus codon 13 in 24%. Right-sided CRCs harbored more KRAS mutations than left-sided tumors, as did tumors that presented with perineural invasion., Conclusion: Our findings in this Latin American population are consistent with the literature regarding the frequency of KRAS mutations in CRC, their distribution between codons 12 and 13, and type of nucleotide substitution. By combining nested PCR and pyrosequencing, we achieved a high rate of conclusive results in testing KRAS mutations in CRC samples - a method that can be used as an ancillary test for failed assays by conventional PCR., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Amplification of fluorescently labeled products is one of the most popular methods for genotyping genetic variations. Two-step amplification using fluorescent universal primers simultaneously produces multiple targeted fragments labeled with fluorescent dyes, and this strategy is applicable to large-scale, cost-effective genotyping. In this study, we developed a fast PCR-based, multiple short tandem repeat (STR) genotyping method using fluorescent universal primers containing locked nucleic acids (LNAs). Four amplification reactions, each assaying six or seven markers and using 0.5-1.0 ng of genomic DNA, produced obvious Fam-labeled peaks in all 26 loci tested (25 autosomal STRs and amelogenin). The overall amplification time was 37 min. Moreover, fluorescent signals for the 25 STRs obtained from LNA-containing primers were 1.5-9.0 fold higher compared to those from non-LNA primers. Using genomic DNA from 120 Japanese individuals, 16 out of the 25 STRs had observed heterozygosity greater than 0.7. Some of these 25 STRs also had high discriminatory power, similar to that of the 13 core STRs in the Combined DNA Index System dataset. The probability of incorrectly assigning a match based on the accumulated matching probability for these 25 STRs is 1.2 × 10(-22), and their combined use can provide robust information for Japanese forensics., (Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Objective: To describe the changes in dark-adapted (DA) retinal electrophysiological function after prolonged dark adaptation in a cohort of patients with late-onset retinal degeneration (L-ORD)., Design: Prospective case series., Participants: Nine patients with either stage 2 or 3 L-ORD., Methods: International Society for Clinical Electrophysiology of Vision standard DA electroretinograms (ERGs) were performed before and after a period of extended dark adaptation (16 hours) in a cohort of patients heterozygous for the Ser163Arg mutation in C1QTNF5., Results: Rod function was abnormal in 8 of 9 patients after standard (20 min) of dark adaptation. After extended dark adaptation, rod function normalized in 4 patients and there was a mean improvement in the DA 0.01 rod-specific ERG b-wave amplitude of 310% (p = 0.004). A significant improvement in DA 3.0 a-wave ERG amplitude localized the improvement in rod function at the level of the photoreceptor., Conclusions: This study demonstrates that a significant proportion of rod dysfunction in L-ORD can be reversed by extended dark adaptation and suggests that an abnormality of the visual cycle contributes to the pathogenesis of the disease. These findings would suggest that some retinal function could be restored, even in advanced cases of the disease if a suitable treatment is found., (Copyright © 2015 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.)

Febrile neutropenia is the main treatment-related cause of mortality in cancer patients. During June 2012 to April 2014, 97 blood culture samples were collected from patients receiving chemotherapy for hematological malignancy and cancer with febrile neutropenia episodes (FNEs). The samples were examined for the presence of bacteria and fungi using real-time PCR amplification and sequencing of 16S and 18S rRNA genes. Bacteria were identified in 20 of 97 samples (20.6%) by the real-time PCR assay and in 10 of 97 (10.3%) samples by blood culture. In 6 blood culture-positive samples, the real-time PCR assay detected the same type of bacteria. No fungi were detected by the real-time PCR assay or blood culture. During antibiotic therapy, all samples were negative by blood culture, but the real-time PCR assay yielded a positive result in 2 cases of 2 (100%). The bacterial DNA copy number was not well correlated with the serum C-reactive protein titer of patients with FNEs. We conclude that a real-time PCR assay could provide better detection of causative microbes' in a shorter time, and with a smaller blood sample than blood culture. Using a real-time PCR assay in combination with blood culture could improve microbiological documentation of FNEs., (Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 °C, and no cross-reactivity was observed for other types of β-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory., (Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

A highly sensitive fluorescence method of quantitative detection for specific DNA sequence is developed based on molecular beacon (MB) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. It is demonstrated by an oligonucleotide sequence of wild-type HBV (target DNA) as a model system. In this strategy, the fluorophore of MB is designed to be 6-carboxyfluorescein group (FAM), and the maximum excitation wavelength and maximum emission wavelength are both very close to that of SYBR Green I. In the presence of targets DNA, the MBs hybridize with the targets DNA and form double-strand DNA (dsDNA), the fluorophore FAM is separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. At the same time, SYBR Green I binds to dsDNA, the fluorescence intensity of SYBR Green I is significantly enhanced. When targets DNA are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for DNA can be realized. Under the optimum conditions, the total fluorescence intensity of FAM and SYBR Green I exhibits good linear dependence on concentration of targets DNA in the range from 2×10(-11) to 2.5×10(-9)M. The detection limit of target DNA is estimated to be 9×10(-12)M (3σ). Compared with previously reported methods of detection DNA with MB, the proposed method can significantly enhance the detection sensitivity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Extension rates of a thermostable, deletion-mutant polymerase were measured from 50°C to 90°C using a fluorescence activity assay adapted for real-time PCR instruments. Substrates with a common hairpin (6-base loop and a 14-bp stem) were synthesized with different 10-base homopolymer tails. Rates for A, C, G, T, and 7-deaza-G incorporation at 75°C were 81, 150, 214, 46, and 120 seconds(-1). Rates for U were half as fast as T and did not increase with increasing concentration. Hairpin substrates with 25-base tails from 0% to 100% GC content had maximal extension rates near 60% GC and were predicted from the template sequence and mononucleotide incorporation rates to within 30% for most sequences. Addition of dimethyl sulfoxide at 7.5% increased rates to within 1% to 17% of prediction for templates with 40% to 90% GC. When secondary structure was designed into the template region, extension rates decreased. Oligonucleotide probes reduced extension rates by 65% (5'-3' exo-) and 70% (5'-3' exo+). When using a separate primer and a linear template to form a polymerase substrate, rates were dependent on both the primer melting temperature (Tm) and the annealing/extension temperature. Maximum rates were observed from Tm to Tm - 5°C with little extension by Tm + 5°C. Defining the influence of sequence and temperature on polymerase extension will enable more rapid and efficient PCR., (Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Sex identification of birds is of great interest in ecological studies, however this can be very difficult in many species because their external features are almost monomorphic between the sexes. Molecular methodology has simplified this process but limitations still occur with widely accepted methods using polymerase chain reaction and gel electrophoresis, especially when applied to degraded DNA. Real-time polymerase chain reaction assays are emerging as a more efficient, sensitive, and higher throughput means of identification, but there are very few techniques validated using fecal samples and small target sizes. We present a real-time melt curve analysis assay targeting a small region of the CHD-1 gene allowing for high-throughput, sensitive, specific, and easy-to-interpret sexing results for a variety of Southern Ocean seabirds using fecal and tissue samples., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

This study has developed a new method, near infrared fluorescent bridge polymerase chain reaction (NIRF-bPCR), for analyzing transcription factor (TF) activity. This method was first used to detect the activity of purified nuclear factor kappa B (NF-κB) p50. The results demonstrated that this method could quantitatively detect the activity of p50 protein at less than 115ng (∼ 2320fmol), and the detection limit reached as little as 6.94ng (∼ 140fmol) of p50 protein. This method was then used to detect TF activity in cell extracts. The results revealed that this method could specifically detect NF-κB activity in HeLa cell nuclear extracts. Finally, this method was used to detect the activities of multiple TFs in a protein sample. The results showed that this method could detect the activities of six TFs-NF-κB, AP-1, TFIID, CREB, NF-E2, and p53-in the TNFα-induced and -uninduced HeLa cell nuclear extracts. Calculation of the fold induction of six TFs revealed that NF-κB, CREB, and AP1 were activated by TNFα induction in HeLa cells, in agreement with the detection results of other methods. Therefore, this study provides a new tool for analyzing TF activity. This study also revealed that NIRF-bPCR may be used as a new method for detecting DNA molecules., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The increasing need to multiplex nucleic acid reactions presses test designers to the limits of amplification specificity in PCR. Although more than a dozen hot starts have been developed for PCR to reduce primer-dimer formation, none can stop the propagation of primer-dimers once formed. Even a small number of primer-dimers can result in false-negatives and/or false-positives. Herein, we demonstrate a new class of primer technology that greatly reduces primer-dimer propagation, showing successful amplification of 60 template copies with no signal dampening in a background of 150,000,000 primer-dimers. In contrast, normal primers, with or without a hot start, experienced signal dampening with as few as 60 primer-dimers and false-negatives with only 600 primer-dimers. This represents more than a 2.5 million-fold improvement in reduction of nonspecific amplification. We also show how a probe can be incorporated into the cooperative primer, with 2.5 times more signal than conventional fluorescent probes., (Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Many individuals infected with the human immunodeficiency virus (HIV), especially children in African countries, die of co-infections with Mycobacterium tuberculosis (MTB) (coinfection rate: 50%) or Pneumocystis carinii pneumonia (PCP) (coinfection rate: 81%). The present proposal describes a rapid, portable, low-cost, multiplexed point-of-care diagnostic technique for simultaneously detecting HIV, MTB, and PCP. This technique incorporates a creative micro-device (hardware) and a loop-mediated isothermal amplification strategy (software)., (© 2013 Elsevier B.V. All rights reserved.)

Epizootic ulcerative syndrome (EUS) is a devastating fish disease caused by the fungus, Aphanomyces invadans. Rapid diagnosis of EUS is needed to control and treat this highly invasive disease. The current diagnostic methods for EUS are labor intensive. We have developed a highly sensitive and specific electrochemical genosensor towards the 18S rRNA and internal transcribed spacer regions of A. invadans. Multiple layers of latex were synthesized with the help of polyelectrolytes, and labeled with gold nanoparticles to enhance sensitivity. The gold-latex spheres were functionalized with specific DNA probes. We describe here the novel application of this improved platform for detection of PCR product from real sample of A. invadans using a premix sandwich hybridization assay. The premix assay was easier, more specific and gave higher sensitivity of one log unit when compared to the conventional method of step-by-step hybridization. The limit of detection was 0.5 fM (4.99 zmol) of linear target DNA and 1 fM (10 amol) of PCR product. The binding positions of the probes to the PCR amplicons were optimized for efficient hybridization. Probes that hybridized close to the 5' or 3' terminus of the PCR amplicons gave the highest signal due to minimal steric hindrance for hybridization. The genosensor is highly suitable as a surveillance and diagnostic tool for EUS in the aquaculture industry., (© 2013 Elsevier B.V. All rights reserved.)

The tailing genome walking strategies are simple and efficient. However, they sometimes can be restricted due to the low stringency of homo-oligomeric primers. Here we modified their conventional tailing step by adding polythymidine and polyguanine to the target single-stranded DNA (ssDNA). The tailed ssDNA was then amplified exponentially with a specific primer in the known region and a primer comprising 5' polycytosine and 3' polyadenosine. The successful application of this novel method for identifying integration sites mediated by φC31 integrase in goat genome indicates that the method is more suitable for genomes with high complexity and local GC content., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Hemolytic uremic syndrome (HUS) is a disease of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. About 90% of cases are secondary to infections by Escherichia coli strains producing Shiga-like toxins (STEC-HUS), while 10% are associated with mutations in genes encoding proteins of complement system (aHUS). We describe two patients with a clinical history of STEC-HUS, who developed end-stage renal disease (ESRD) soon after disease onset. They received a kidney transplant but lost the graft for HUS recurrence, a complication more commonly observed in aHUS. Before planning a second renal transplantation, the two patients underwent genetic screening for aHUS-associated mutations that revealed the presence of a heterozygous CFI mutation in patient #1 and a heterozygous MCP mutation in patient #2, and also in her mother who donated the kidney. This finding argues that the two cases originally diagnosed as STEC-HUS had indeed aHUS triggered by STEC infection on a genetic background of impaired complement regulation. Complement gene sequencing should be performed before kidney transplantation in patients who developed ESRD following STEC-HUS since they may be undiagnosed cases of aHUS, at risk of posttransplant recurrence. Furthermore, genetic analysis of donors is mandatory before living-related transplantation to exclude carriers of HUS-predisposing mutations., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

We report the development of a fluorescently labeled oligonucleotide primer that can be used to monitor real-time PCR. The primer has two parts, the 3'-end of the primer is complimentary to the target and a universal 17-mer stem loop at the 5'-end forms a hairpin structure. A fluorescent dye is attached to 5'-end of either the forward or reverse primer. The presence of guanosine residues at the first and second position of the 3' dangling end effectively quenches the fluorescence due to the photo electron transfer (PET) mechanism. During the synthesis of nucleic acid, the hairpin structure is linearized and the fluorescence of the incorporated primer increases several-fold due to release of the fluorescently labeled tail and the absence of guanosine quenching. As amplicons are synthesized during nucleic acid amplification, the fluorescence increase in the reaction mixture can be measured with commercially available real-time PCR instruments. In addition, a melting procedure can be performed to denature the double-stranded amplicons, thereby generating fluorescence peaks that can differentiate primer dimers and other non-specific amplicons if formed during the reaction. We demonstrated the application of PET-PCR for the rapid detection and quantification of Cryptosporidium parvum DNA. Comparison with a previously published TaqMan® assay demonstrated that the two real-time PCR assays exhibited similar sensitivity for a dynamic range of detection of 6000-0.6 oocysts per reaction. PET PCR primers are simple to design and less-expensive than dual-labeled probe PCR methods, and should be of interest for use by laboratories operating in resource-limited environments., (Published by Elsevier Inc.)

Rapid amplification of cDNA 5' ends (5'-RACE) is routinely used for the sequence analysis of the upstream noncoding regions of cellular mRNAs; however, it represents a tedious and cost-intensive procedure. By employing 5' phosphorylated gene-specific primers for first-strand cDNA synthesis, we cut short the previously established reverse ligation and amplification protocol of Mandl and coworkers (BioTechniques, 1991, vol. 10, pp. 484-486) to a streamlined three-step procedure that no longer depends on enzymatic mRNA decapping or linker ligation. The novel three-step protocol has been validated by mapping the transcriptional start sites of heterologously expressed yellow fever virus genomic RNAs from cultured mammalian cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Viral infection is a leading cause of disease and death. Although vaccines are the most effective method of controlling viral infections, antiviral drugs are also important. Here, we established an in vitro transcription system by using fluorescein isothiocyanate-conjugated primers for RNA polymerases of viruses that are important disease-causing human pathogens (influenza, hepatitis C, Japanese encephalitis viruses, and enterovirus 71). This technology will allow us to analyze RNA polymerase activity without using radioisotopes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

T7 endonuclease I is a dimeric nuclease that is selective for four-way DNA junctions. Previous crystallographic studies have found that the N-terminal 16 amino acids are not visible, neither in the presence nor in the absence of DNA. We have now investigated the effect of deleting the N-terminus completely or partially. N-terminal deleted enzyme binds more tightly to DNA junctions but cleaves them more slowly. While deletion of the N-terminus does not measurably affect the global structure of the complex, the presence of the peptide is required to generate a local opening at the center of the DNA junction that is observed by 2-aminopurine fluorescence. Complete deletion of the peptide leads to a cleavage rate that is 3 orders of magnitude slower and an activation enthalpy that is 3-fold higher, suggesting that the most important interaction of the peptide is with the reaction transition state. Taken together, these data point to an important role of the N-terminus in generating a central opening of the junction that is required for the cleavage reaction to proceed properly. In the absence of this, we find that a cruciform junction is no longer subject to bilateral cleavage, but instead, just one strand is cleaved. Thus, the N-terminus is required for a productive resolution of the junction., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: 17β-Estradiol (E2) has been thought to produce cardioprotective effects by mediating bone marrow-derived endothelial progenitor cells (EPC) for cardiac repair in the setting of acute myocardial infarction (AMI). However, the underlying mechanism of action of E2 on EPC remains unclear. CXCR4 is a critical modulator in homing of EPC. Accordingly, we hypothesized that E2 exerts beneficial effects through enhancing EPC homing to infarcted myocardium via mediating CXCR4 pathway., Methods and Results: Migratory capacity and CXCR4 expression of EPC from ovariectomized BALB/C mice were detected after being incubated with various E2 concentrations for various incubation times. For in vivo studies, EPC were labeled with superparamagnetic ion oxide (SPIO) for tracing, and ovariectomized mice were grouped (n=11) after inducing AMI to receive saline without cells or with 3 × 10(6) non-preconditioned EPC, 100 nmol/L E2 preconditioned EPC, CXCR4 inhibitor AMD3100 (5 μg/mL) preconditioned EPC, or EPC pretreated with E2 plus AMD3100. The number of homing EPC in infarcted myocardium and left ventricular (LV) function, dimensions and fibrosis were measured. In vitro data showed that E2 increased migratory activity and functional CXCR4 expression of EPC. However, these effects were completely blocked by AMD3100. In vivo data in E2 group displayed a greater number of homing EPC, decreased fibrosis of LV, and significant improvement in cardiac function. Nevertheless, effects of E2 preconditioning were abrogated by AMD3100., Conclusions: We conclude that E2 enhances the recruitment of EPC into infarcted myocardium by up-regulating functional CXCR4 expression, resulting in improving recovery after myocardial infarction., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Emerging evidence has indicated that the endothelial-to-mesenchymal transition (EndMT) is a crucial event during early stages of cardiac fibrosis. In the present study, we first investigated the influence of Irbesartan (Irb) on myocardial EndMT in diabetic rats., Methods: Diabetic rats were divided into two groups: the diabetic group (DM) and the Irb-treated group (DM+Irb). Wistar-Kyoto rats served as controls. The pathological changes were investigated by microscopy. Immunofluorescence was performed to evaluate the co-expression of CD31 and fibroblast-specific protein 1 (FSP1). FSP1 and α-SMA expressions were detected by RT-PCR and Western blot analysis. EndMT was also studied in human aortic endothelial cells (HAECs) that had been exposed to high glucose (HG) levels., Results: Increased interstitial fibrosis was detected in the DM group. Double labeling revealed CD31 expression in FSP1-positive cells in the DM group, and this expression was diminished by Irb treatment (P<0.05). In vitro, we found that HG stimulated angiotensin II synthesis in HAECs. When HAECs were exposed to HG, some of the cells acquired a spindle-shaped morphology and demonstrated a loss of CD31 labeling, which was attenuated by Irb treatment. FSP1 and α-SMA mRNA and protein expression levels were markedly upregulated in diabetic rats compared to controls, and their expressions were inhibited by Irb treatment (P<0.05)., Conclusion: The results provide the novel insight that an angiotensin II receptor blocker might prevent diabetic cardiomyopathy by abrogating EndMT in diabetic rats., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background and Aims: We designed this study to evaluate the ability of a plasmid carrying an RU486 regulatory system to induce expression of interleukin-2 (IL-2) gene and to examine the antitumour efficacy of the induced IL-2 gene., Methods: The plasmid pRS-mIL-2,which contains an RU486 inducible system and IL-2 gene was injected into mice. Sera and tissues from liver, spleen, lungs and kidneys were taken to test the properties of the plasmid. To examine the antitumour efficacy of pRS-mIL-2, tumours were established in the liver by direct inoculation of H22 hepatoma cells., Results: The IL-2 levels in serum correlated with the dose of plasmid and RU486. High and sustained IL-2 levels could be achieved by administration of RU486 every day. The mRNA of transgene IL-2 was found only in the liver. Treatment of mice with pRS-mIL-2 plus RU486 resulted in the significant reduction in tumour volume compared with control groups., Conclusions: Tight temporal and spatial control of transgene IL-2 expression can be achieved by a plasmid containing an RU486 inducible system driven by liver specific promoter. pRS-mIL-2 exhibited strong antitumour efficacy following consecutive induction with RU486., (Copyright © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: Colon carcinogenesis is associated with increased expression levels of Toll-like receptor 2 and Toll-like receptor 4., Aim: To determine in a Caucasian population the role of Toll-like receptor 2 and Toll-like receptor 4 polymorphisms in colorectal cancer development., Methods: Hospital based multicentre case control study involving 193 colorectal cancer patients and 278 healthy individuals. DNA samples were extracted from blood cells and genotyping of TLR2+597T>C, TLR2-4760T>C, TLR4-3745A>G, TLR2Arg753Gln, TLR4Asp299Gly was performed. Functionality of risk polymorphisms was evaluated through production of TNF-α in cell culture and Toll-like receptors levels quantified by real-time RT-PCR., Results: TLR2+597CC homozygous had 5-fold decreased risk (odds ratio (OR)=0.21, 95% CI: 0.09-0.50, p<0.001) and TLR4 299Gly homozygous 3-fold increased risk of colorectal cancer (OR=3.30, 95% CI: 1.18-9.28, p=0.015). In stratified analysis, TLR2+597CC genotype protective effect was even higher in overweight individuals (OR=0.17, 95% CI: 0.06-0.53, p<0.001) and in never smokers (OR=0.11, 95% CI: 0.02-0.51, p=0.001). Also, the increased risk effect for TLR4 299Gly homozygous genotype was higher in overweight individuals (OR=8.67, 95% CI: 1.11-87.85, p=0.011). TLR2+597T>C polymorphism conferred 41% less (p=0.03) and TLR4Asp299Gly 65% more TNF-α production (p=0.02) with no differences in Toll-like receptors levels., Conclusion: Functional Toll-like receptor 2 and Toll-like receptor 4 polymorphisms significantly alter the risk to have colorectal cancer. Obesity and smoking may influence the risk for colorectal cancer in individuals presenting these genetic profiles., (Copyright © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: Recent data suggest that the serine protease inhibitor Kazal type 1 (SPINK1) gene mutation is associated with idiopathic chronic pancreatitis. However, few studies have focused on the serine protease inhibitor Kazal type 1 c.194+2T>C mutation. Therefore, our goal was to study the prevalence and impact of serine protease inhibitor Kazal type 1 mutations on the clinical profile of idiopathic chronic pancreatitis patients in China., Methods: A retrospective-cohort study of 118 Chinese patients with idiopathic chronic pancreatitis was performed, and genetic tests were carried out to detect SPINK1 mutations. Subjects without pancreatitis were used as controls. In total, 118 idiopathic chronic pancreatitis patients and 100 control subjects were evaluated., Results: The serine protease inhibitor Kazal type 1 c.194+2T>C variant was present in 44.9% of patients with idiopathic chronic pancreatitis. The frequency of diabetes in idiopathic chronic pancreatitis patients with the serine protease inhibitor Kazal type 1 c.194+2T>C mutation (39.6%) was higher than that of patients without the mutation (9.2%). The time to occurrence of diabetes mellitus after idiopathic chronic pancreatitis symptom onset is significantly influenced by the c.194+2T>C mutation (p<0.001). In addition, the mean age of diabetes onset in patients with the serine protease inhibitor Kazal type 1 c.194+2T>C mutation (38.33 ± 9.50) was significantly younger than that of patients without this mutation (49.67 ± 6.74)., Conclusions: The presence of the serine protease inhibitor Kazal type 1 c.194+2T>C mutation seems to be associated with idiopathic chronic pancreatitis and could predispose individuals to pancreatic diabetes onset at an earlier age., (Copyright © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

MusPV, a novel papillomavirus (PV) that naturally infects laboratory mice, was isolated and characterized from a colony of NMRI-Foxn1(nu)/Foxn1(nu) (nude) mice in India. Because MusPV may have been missed during routine pathogen screening of mice in colonies worldwide, a variety of detection methods are described to detect MusPV. The clinical and histologic lesions of productive MusPV infections fit PV-associated features, including papillomas, koilocytes within the stratum granulosum of the hyperplastic/acanthotic papillomatous epithelium, and the presence of intranuclear virus particles in koilocytotic cells visualized by electron microscopy. Antiserum against disrupted PV virions, isolated from another species (canine), identified conserved viral antigens in productively infected cells by immunohistochemistry. A rolling circle technique was used to amplify viral circular DNAs followed by endonuclease restriction enzyme digestion to determine the correct size of PV DNA. Consensus PV degenerative primers, My09/11, commonly used to detect many different types of PVs by polymerase chain reaction (PCR), particularly mucosotropic HPVs, also identified MusPV and all rodent PVs tested. Since there was one nucleotide mismatch between the My09/11 primer set and the MusPV template, a new primer set, MusPV-My09/11, was designed to specifically detect MusPV in latent infections and spontaneous MusPV-induced papillomas. Southern blot analysis verified the presence of full size PV DNA in infected tissues. Virus-like particles (VLPs), generated from MusPV L1 genes, provided a substrate for serological testing of naturally and experimentally infected mice. In summary, a series of diagnostic assays were developed and validated to detect MusPV infection in skin tumors and serological response in laboratory mice., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background and Study Aim: Several genes of Helicobacter pylori, such as vacA, cagA, iceA and babA, have been reported to significantly increase the risk of gastrointestinal diseases. The aim of this study was to study the relationship between H. pylori virulence factors and clinical outcomes and identify the independent markers of peptic ulcer disease in Iraq., Patients and Methods: DNA was extracted from specimens taken from 154 unselected H. Pylori positive Iraqi patients. Genotyping was performed by the polymerase chain reaction (PCR), using specific primers for cagA, vacA (s, m), iceA and babA2 genes., Results: A total of 56 (82%) peptic ulcer disease (PUD) patients carried cagA+ strains, significantly more than the 56 (65%) non-ulcer disease (NUD) patients (p=0.017). The difference in the prevalence of babA2 positivity was significant between patients with NUD (33.7%) and PUD (58.8%) (p=0.002). In addition, babA2 was associated as an independent factor, with PUD (p=0.005; odds ratio (OR)=0.4; confidence interval (CI)=0.18-0.68) followed by cagA (p=0.05; OR=0.4; CI=0.18-0.85). Forty-five isolates (29%) were typed as 'triple positive' strains, and their presence was significantly associated with PUD (p=0.001)., Conclusion: The cagA and babA2 genotypes might be considered as useful markers for PUD patients. However, iceA1 and iceA2 seem not to be good markers for the disease. The presence of H. pylori strains with triple-positive status is of high clinical relevance to H. pylori-associated diseases., (Copyright © 2012 Arab Journal of Gastroenterology. Published by Elsevier Ltd. All rights reserved.)

Medical practitioners in nine countries submitted samples of Gardasil (Merck & Co.) to be tested for the presence of human papillomavirus (HPV) DNA because they suspected that residual recombinant HPV DNA left in the vaccine might have been a contributing factor leading to some of the unexplained post-vaccination side effects. A total of 16 packages of Gardasil were received from Australia, Bulgaria, France, India, New Zealand, Poland, Russia, Spain and the United States. A nested polymerase chain reaction (PCR) method using the MY09/MY11 degenerate primers for initial amplification and the GP5/GP6-based nested PCR primers for the second amplification were used to prepare the template for direct automated cycle DNA sequencing of a hypervariable segment of the HPV L1 gene which is used for manufacturing of the HPV L1 capsid protein by a DNA recombinant technology in vaccine production. Detection of HPV DNA and HPV genotyping of all positive samples were finally validated by BLAST (Basic Local Alignment Search Tool) analysis of a 45-60 bases sequence of the computer-generated electropherogram. The results showed that all 16 Gardasil samples, each with a different lot number, contained fragments of HPV-11 DNA, or HPV-18 DNA, or a DNA fragment mixture from both genotypes. The detected HPV DNA was found to be firmly bound to the insoluble, proteinase-resistant fraction, presumably of amorphous aluminum hydroxyphosphate sulfate (AAHS) nanoparticles used as adjuvant. The clinical significance of these residual HPV DNA fragments bound to a particulate mineral-based adjuvant is uncertain after intramuscular injection, and requires further investigation for vaccination safety., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A simple, rapid, and efficient site-directed mutagenesis method using TA strategy with synthetic mutagenic oligonucleotides is described. Briefly, a 3' A-overhang vector was prepared by polymerase chain reaction (PCR) using a classical Taq polymerase with terminal transferase activity, a reverse vector primer starting the complement nucleotide prior to the 5' end adenosine of the target, and a forward vector primer starting the nucleotide posterior to the 3' end thymidine. The 3' T-overhang mutagenic double-strand oligonucleotide was synthesized and cloned directly into the PCR-amplified 3' A-overhang vector. Thus, direct ligation of synthetic mutagenic oligonucleotides and PCR-amplified vector via TA sticky ends provides us with simple, rapid, and efficient site-directed mutagenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)