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BACKGROUND: Despite the decline of the COVID-19 pandemic, there continues to be a persistent requirement for reliable testing methods that can be adapted to future outbreaks and areas with limited resources. While the standard approach of using reverse transcription-polymerase chain reaction (RT-PCR) with Taq polymerase is effective, it faces challenges such as limited access to high-quality enzymes and the presence of bacterial DNA contamination in commercial kits, which can impact the accuracy of test results. METHODS: This study investigates the production of recombinant Taq polymerase in yeast cells and assesses its crude lysate in a multiplex RT-PCR assay for detecting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) and N genes, with human Ribonuclease P serving as an internal control. RESULTS: The unpurified yeast Taq polymerase demonstrates sensitivity comparable to commercially purified bacterial Taq polymerase and unpurified bacterial counterparts in detecting the RdRP and N genes. It exhibits the highest specificity, with 100% accuracy, for the N gene. The specificity for the RdRP gene closely aligns with that of commercially purified bacterial Taq polymerase and unpurified bacterial Taq polymerase. CONCLUSIONS: The use of unpurified recombinant yeast Taq polymerase shows promise as a cost-effective approach for conducting in-house COVID-19 RT-PCR testing. By eliminating the need for chromatography purification steps, the production of RT-PCR kits can be streamlined, potentially improving accessibility and scalability, especially in resource-limited settings and future pandemics.

BACKGROUND: Despite the decline of the COVID-19 pandemic, there continues to be a persistent requirement for reliable testing methods that can be adapted to future outbreaks and areas with limited resources. While the standard approach of using reverse transcription-polymerase chain reaction (RT-PCR) with Taq polymerase is effective, it faces challenges such as limited access to high-quality enzymes and the presence of bacterial DNA contamination in commercial kits, which can impact the accuracy of test results. METHODS: This study investigates the production of recombinant Taq polymerase in yeast cells and assesses its crude lysate in a multiplex RT-PCR assay for detecting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) and N genes, with human Ribonuclease P serving as an internal control. RESULTS: The unpurified yeast Taq polymerase demonstrates sensitivity comparable to commercially purified bacterial Taq polymerase and unpurified bacterial counterparts in detecting the RdRP and N genes. It exhibits the highest specificity, with 100% accuracy, for the N gene. The specificity for the RdRP gene closely aligns with that of commercially purified bacterial Taq polymerase and unpurified bacterial Taq polymerase. CONCLUSIONS: The use of unpurified recombinant yeast Taq polymerase shows promise as a cost-effective approach for conducting in-house COVID-19 RT-PCR testing. By eliminating the need for chromatography purification steps, the production of RT-PCR kits can be streamlined, potentially improving accessibility and scalability, especially in resource-limited settings and future pandemics. [ABSTRACT FROM AUTHOR]

The basic principles of ultrafast plasmonic PCR have been promulgated in the scientific and technological literature for over a decade. Yet, its everyday diagnostic utility remains unvalidated in pre-clinical and clinical settings. Although the impressive speed of plasmonic PCR reaction is well-documented, implementing this process into a device form compatible with routine diagnostic tasks has been challenging. Here, we show that combining careful system engineering and process control with innovative and specific PCR biochemistry makes it possible to routinely achieve a sensitive and robust "10 min" PCR assay in a compact and lightweight system. The critical analytical parameters of PCR reactions are discussed in the current instrument setting. [ABSTRACT FROM AUTHOR]

Detecting mutations related to tumors holds immense clinical significance for cancer diagnosis and treatment. However, the presence of highly redundant wild DNA poses a challenge for the advancement of low-copy mutant ctDNA genotyping in cancer cases. To address this, a Taqman qPCR strategy to identify rare mutations at low variant allele fractions (VAFs) has been developed. This strategy combines mutant-specific primers with wild-specific blockers. Diverging from other blocker-mediated PCRs, which rely on primer-induced strand displacement or the use of modified oligos resistant to Taq polymerase, our innovation is built upon the cleavage of specific blockers by Taq polymerase. Given its unique design, which does not hinge on strand displacement or base modification, we refer to this novel method as unmodified-blocker cleavage PCR (UBC-PCR). Multiple experiments consistently confirmed that variant distinction was improved significantly by introduction of 5′ unmatched blockers into the reaction. Moreover, UBC-PCR successfully detected mutant DNA at VAFs as low as 0.01% across six different variant contexts. Multiplex UBC-PCR was also performed to identify a reference target and three mutations with a sensitivity of 0.01% VAFs in one single tube. In profiling the gene status from 12 lung cancer ctDNA samples and 22 thyroid cancer FNA DNA samples, UBC-PCR exhibited a 100% concordance rate with ddPCR and a commercial ARMS kit, respectively. Our work demonstrates that UBC-PCR can identify low-abundance variants with high sensitivity in multiplex reactions, independent of strand displacement and base modification. This strategy holds the potential to significantly impact clinical practice and precision medicine. [ABSTRACT FROM AUTHOR]

Real-time qPCR and digital PCR are powerful techniques for detecting nucleic acids with high specificity and sensitivity, relying on sequence-specific labeled probes. However, each target requires the design and synthesis of specific probes, incurring significant costs. Therefore, the development of a specific and universal detection technique is both urgent and beneficial. Previously, a universal detection method was developed using a modified mediator probe and a universal hairpin reporter. However, our experiments uncovered that the mediator primers, generated by Taq polymerase cleaving mediator probes, deviate from the expected sequence, indicating a severe compromise in detection ability. Additionally, the high cost of the modified mediator probe and hairpin reporter impedes the practicality of their method. To overcome these challenges, we have developed a novel universal detection method (MP-UP) using Taqman probes, unmodified mediator probes and helper target, enabling the utilization of all mediator primers to produce abundant fluorescence. Enhanced MP-UP qPCR with an increased signal-to-noise ratio, MP-UP ddPCR, and multiplex MP-UP were established, exhibiting remarkable sensitivity, specificity, and precision comparable to specific probe assays or commercial testing kit. The mediator probe and helper target cost only one-tenth of a specific Taqman probe, rendering it feasible to replace the latter in specific nucleic acid detection. • Mediator primers are tailed with one or two template-specific nucleotides at the 3'3′ end. • Taqman probes and unmodified MP, more accessible and cheaper, were used in MP-UP. • Improve signal-to-noise ratio of MP-UP by adding heated step or using hairpin helper target. • MP-UP ddPCR was established by utilizing double-quenched probes. [ABSTRACT FROM AUTHOR]

A DNA polymerase from Thermus aquaticus remains the most popular among DNA polymerases. It was widely applied in various fields involving the application of polymerase chain reaction (PCR), implying the high commercial value of this enzyme. For this reason, an attempt to obtain a high yield of Taq DNA polymerase is continuously conducted. In this study, the l -rhamnose-inducible promoter rhaBAD was utilized due to its ability to produce recombinant protein under tight control in E. coli expression system. Instead of full-length Taq polymerase, an N-terminal deletion of Taq polymerase was selected. To obtain a high-level expression, we attempted to optimize the codon by reducing the rare codon and GC content, and in a second attempt, we optimized the culture conditions for protein expression. The production of Taq polymerase using the optimum culture condition improved the level of expression by up to 3-fold. This approach further proved that a high level of recombinant protein expression could be achieved by yielding a purified Taq polymerase of about 8.5 mg/L of culture. This is the first research publication on the production of Taq polymerase with N-terminal deletion in E. coli with the control of the rhaBAD promoter system. [Display omitted] • Synthesis of a TaqPol using rhaBAD was successfully demonstrated. • Optimization of codon and culture condition were employed to improve protein yield. • Expression of TaqPol increased by up to 3-fold with a yield of about 7.7 mg/L. • TaqPol was produced in its active form based on quantitative and qualitative assay. [ABSTRACT FROM AUTHOR]

Due to its advantages of high precision and low cost, automated Sanger sequencing technology remains useful for many applications despite the emergence of next-generation sequencing (NGS) platforms. Few studies revealed the sequence errors in sanger sequencing because the instrumentation and chemistry currently used for Sanger sequencing is highly reliable and reproducible. Unexpectedly, in our study, the sequencing artifacts were identified in sequencing the PCR product of rAAV-ITR (recombination Adeno-Associated Virusinverted terminal repeat) which formed highly stable T-shaped hairpin and played critical role in virus production. The artifactual sequences were characterized by the 3’ end palindromic sequence and nucleotide substitutions. We confirmed that the presence of stable hairpin in template results in sequencing artifacts by employing disruptor to eliminate the structure, sequencing the PCR product and the synthezized strands containing stable or weak hairpin respectively for comparation. Furthermore, we proposed that endonuclease activity of Taq polymerase cleaves the templates containing stable structure to generate annealed 3’end which acts as primer to synthesize the artifactual palindrome. [ABSTRACT FROM AUTHOR]

The methylation status of the MGMT promoter represents the valuable prognostic and predictive marker in glioblastoma (GBM) patients undergoing treatment with alkylating agents such as Temozolomide. Although Formalin-Fixed and Paraffin-Embedded Tissue (FFPE) signifies the most commonly used source for tissue-based molecular testing, its use in Methylation-Specific Polymerase Chain Reaction (MSP) analysis manifests certain limitations due to low DNA integrity. Our study aimed to identify the optimal MGMT promoter MSP reaction conditions concerning the utilization of bisulfite-converted FFPE-derived template DNA. Several optimizing reactions were conducted and subjected to ImageJ software analysis. As a result, 4U of HotStarTaq and 125 ng of template DNA were specified as necessary for successful MSP reactions. The confirmation of optimization success was obtained through comparison of semi-quantitative values of DNA methylation levels between reference Fresh Frozen tissue and corresponding FFPE sample obtained from the same GBM patient. [ABSTRACT FROM AUTHOR]

[Display omitted] • Pioneering use of Taq polymerase-mediated hairpin amplicon cleavage to discriminate variants. • HAC-PCR allows for the effective identification of rare variants without strand displacement, base modification, or additional reagents. • Our method consistently achieved a remarkable sensitivity of 0.01% Variant Allele Fraction (VAF). Highly sensitive detection of gene variants holds immense significance for cancer diagnosis and treatment. However, the presence of highly redundant wild DNA poses a substantial challenge when identifying low-frequency mutant circulating tumor DNA (ctDNA) in cancer cases. Here, a novel technique called hairpin amplicon cleavage PCR (HAC-PCR) for detecting low-level DNA mutations with exceptional sensitivity was presented. What sets HAC-PCR apart is its ability to achieve high sensitivity independently of strand-displacement, base modification, or the use of additional reagents. The discrimination between wild-type targets (WT) and mutant targets (MT) is significantly improved by introducing uniquely designed hairpin structures. These hairpins hinder the amplification of WT and are cleaved during the elongation of MT. For the first time, the unique mechanism of hairpin amplicon cleavage by Taq polymerase was exploited for mutation identification. The optimized HAC-PCR consistently demonstrates the capability to discriminate mutant DNA at levels as low as 0.01% variant allele fraction (VAF) for five mutation types. Furthermore, this impressive sensitivity of 0.01% VAF has also been achieved in a multiplex assay. When applied to the testing of tissue DNA from 24 patients with thyroid cancers and plasma ctDNA from 12 patients with non-small cell lung cancers (NSCLCs), HAC-PCR exhibited a 100% concordance rate with the existing commercial kit and droplet digital PCR (ddPCR). Therefore, HAC-PCR stands as a cost-effective and highly effective tool for variant identification in clinical testing. [ABSTRACT FROM AUTHOR]

Abstract In this study a one-tube real-time RT-qPCR assay was developed using the TaqMan chemistry for the universal detection and quantification of PPV, one of the most important pathogens affecting stone fruit trees. In order to design appropriate primers and probe, nucleotide sequences from different PPV isolates originating from all known strains were recovered from the databases. Various genomic regions were screened and finally primers were selected from a conserved region of the 3′- terminal part of the CP gene amplifying a 146 bp DNA fragment while the probe was designed to bind within the amplicon. Ten-fold serial dilutions of in vitro synthesized RNA transcripts were applied for the construction of standard curve. The amplification efficiency of the assay was 93.8% and the linear range of quantification was from 40 up to 4 × 108 RNA copies. The real time RT-PCR was successfully tested with a collection of genetically diverse isolates with different geographical origin belonging to seven PPV strains. The present method is proposed as a useful tool for various basic or applied research studies of PPV as well as for routine testing of plant material during phytosanitary control or in certification schemes of Prunus species. [ABSTRACT FROM AUTHOR]

Background: Increasing evidences support the role of genetic factors in susceptibility to chronic periodontitis. The aim of the present pilot study was to explore the association of two potential single nucleotide polymorphisms (SNPs): Interleukin (IL)-1β + 3954 (rs1143634, C > T) and vitamin D receptor (VDR) TaqI (rs731236, T > C) with chronic periodontitis in a North Indian population. Materials and Methods: Twenty-eight chronic periodontitis subjects and 47 periodontally healthy controls were recruited. Individual samples of venous blood were obtained from each subject. Genotyping was done by polymerase chain reaction, followed by restriction fragment length polymorphism (PCR-RFLP). Logistic regression and chi square test were used for genetic association analysis and a P value less than 0.05 taken as statistical significance. Statistical Analysis Used: Chi square test and odds ratio (OR) was used. Results: Genotypes and alleles of SNP IL-1β + 3954 did not show a significant association (P > 0.05) with chronic periodontitis. Genotype CC and allele C of VDR TaqI were significantly associated with a higher risk for chronic periodontitis as compared to subjects with TT genotype (CC/TT OR = 4.615; 95% confidence interval [CI]: 1.17 to 18.078 P = 0.028) and allele T (C/T OR = 2.423; 95% CI: 1.179 to 4.980). Conclusion: In North Indian population, genotype CC and allele C of VDR TaqI were associated with risk of chronic periodontitis. No significant correlation was found for IL-1β + 3954 polymorphism and chronic periodontitis.

Aim. To assess the inhibitory activity of ellagitannin complex of the purified extract from the fruits of white alder and black alder in non-eukaryotic polymerase systems and to analyze the effects of these substances on the reverse transcriptase of murine leukemia virus and HIV reproduction in vitro. Methods. Chronically infected MT4/BIII culture of human leukemic cells was used as the HIV source. Anti-HIV activity of the ellagitannin-containing extract was estimated by the reduction of HIV infectious titer in the MT-4 cells. The inhibitory effects of the extract on retroviral reverse transcriptase, T7 RNA-polymerase and Taq polymerase were also studied in cell-free systems. Results. The ellagitannin extract inhibited activities of the reverse transcriptase of murine leukemia retrovirus, T7 RNA-polymerase, and Taq polymerase. It also reduced the HIV infectious titer in MT-4 cells with IC50 of 1.5 μg/ml and selectivity index of 87. Conclusions. The ellagitannin complex possesses a significant anti-HIV activity. Its in vitro antiviral effect is achieved with a relatively low toxicity. [ABSTRACT FROM AUTHOR]

Ligictaluridus michaelalicea n. sp. (Monogenea: Dactylogyridae, Ancyrocephalinae) is described from the gills of Pylodictis olivaris (Siluriformes: Ictaluridae) from Wisconsin and Iowa portions of the upper Mississippi River. Diagnostic features include a relatively large, strongly curved tubular cirrus with minor terminal flare; an accessory piece with a prominent basal lobe and a simple, thick terminal limb featuring a thin lateral flange and blunt distal tip devoid of recurved hooks. The sinistral vagina, two prostatic reservoirs, and a terminal seminal vesicle of the vas deferens are prominent. The anchors, ventral and dorsal bars, and hooks are similar in overall form to those of other members of the genus. The description includes sequence data for the 18S rRNA gene, which aligned most closely with species of ancyrocephaline monogeneans, with the highest similarity being with Ligictaluridus pricei (Mueller, 1936). Other monogenean species identified from the flathead catfish examined included L. pricei and Ligictaluridus mirabilis (Mueller, 1937). L. michaelalicea n. sp. is the fourth species to be described from P. olivaris within its natural range in central and eastern North America. Implications resulting from taxonomic name changes, including species of Ligictaluridus, and United States Food and Drug Administration treatment regulations are discussed. An updated key to species of the genus Ligictaluridus is presented. [ABSTRACT FROM AUTHOR]

Standard Illumina libraries are biased toward sequences of intermediate GC-content. This results in an underrepresentation of GC-rich regions in sequencing projects of genomes with heterogeneous base composition, such as mammals and birds. We developed a simple, cost-effective protocol to enrich sheared genomic DNA in its GC-rich fraction by subtracting AT-rich DNA. This was achieved by heating DNA up to 90 °C before applying Illumina library preparation. We tested the new approach on chicken DNA and found that heated DNA increased average coverage in the GC-richest chromosomes by a factor up to six. Using a Taq polymerase supposedly appropriate for PCR amplification of GC-rich sequences had a much weaker effect. Our protocol should greatly facilitate sequencing and resequencing of the GC-richest regions of heterogeneous genomes, in combination with standard short-read and long-read technologies. [ABSTRACT FROM AUTHOR]

Background The association between vitamin D receptor (VDR) gene polymorphisms and multiple sclerosis (MS) has been extensively studied, but results were controversial. Methods This meta-analysis aimed to confirm whether VDR gene polymorphisms were associated with MS. Meta-analysis on the association between MS and VDR Apa I, Bsm I, Taq I and Fok I polymorphisms were conducted using allelic contrast, recessive, homozygotes and dominant models. Data were extracted by standardized forms and odds ratios (OR) with 95% confidence intervals (CI) were calculated using the random effects model if the results were heterogeneous. Stratification analysis by the selected study characteristics were performed to detect potential source of heterogeneity. Results A total of 21 relevant studies involving 3593 MS patients and 3917 controls were included in the analysis. The association between Taq I polymorphism and MS risk was significant in the homozygous model (p = 0.006) indicated a significant protective effect of TT Taq I genotype. High latitude (40.1–50°N) was also found markedly influenced Taq I polymorphism and MS risk in the recessive and homozygous models (p = 0.045 and p = 0.015, respectively). Additionally, Asian or low latitude (20.1–30°N) people with Apa I homozygous genotype, ‘> 2013’ publication year people in the allele contrast and dominant models of Fok I, ‘> 40 years’ age people with Bsm I recessive model also indirectly significantly affected the association between VDR gene polymorphisms and MS risk. Conclusion Taq I polymorphism is a significant protective factor for MS. However, the associations between Apa I, Fok I and Bsm I polymorphisms and MS were found only by study characteristics. [ABSTRACT FROM AUTHOR]

Seven ferrocenyl naphthalene diimide (FND) ligands were synthesized. Each had a higher affinity for tetraplex DNA than for either single- or double-stranded DNA. The FND binding affinities were > 10 5 M − 1 in 0.10 M AcOH-AcONa or AcOH-AcOK (pH 5.5) containing 0.10 M NaCl or KCl. The FNDs with the highest binding affinities for tetraplex DNA showed 23- or 8-times higher preference for tetraplex DNA than for single- or double-stranded DNA, respectively. The current signals generated from the seven FNDs bound to the tetraplex DNA immobilized on the electrode were found to correlate with the binding affinities of these ligands for the tetraplex DNA. Furthermore, using the telomerase repeat amplification protocol assay, the FND ligands could be categorized into three groups: (a) inhibiting both telomerase and Taq polymerase, (b) inhibiting telomerase alone, and (c) inhibiting neither telomerase nor Taq polymerase. [ABSTRACT FROM AUTHOR]

Next-generation sequencing technology has enabled the detection of rare genetic or somatic mutations and contributed to our understanding of disease progression and evolution. However, many next-generation sequencing technologies first rely on DNA amplification, via the Polymerase Chain Reaction (PCR), as part of sample preparation workflows. Mistakes made during PCR appear in sequencing data and contribute to false mutations that can ultimately confound genetic analysis. In this report, a single-molecule sequencing assay was used to comprehensively catalog the different types of errors introduced during PCR, including polymerase misincorporation, structure-induced template-switching, PCR-mediated recombination and DNA damage. In addition to well-characterized polymerase base substitution errors, other sources of error were found to be equally prevalent. PCR-mediated recombination by Taq polymerase was observed at the single-molecule level, and surprisingly found to occur as frequently as polymerase base substitution errors, suggesting it may be an underappreciated source of error for multiplex amplification reactions. Inverted repeat structural elements in lacZ caused polymerase template-switching between the top and bottom strands during replication and the frequency of these events were measured for different polymerases. For very accurate polymerases, DNA damage introduced during temperature cycling, and not polymerase base substitution errors, appeared to be the major contributor toward mutations occurring in amplification products. In total, we analyzed PCR products at the single-molecule level and present here a more complete picture of the types of mistakes that occur during DNA amplification. [ABSTRACT FROM AUTHOR]

The use of molecular methods to investigate the community structure and diversity of microalgae has largely replaced the previous morphological methods that were routinely carried out by microscopy. Different DNA polymerases can lead to bias in PCR amplification and affect the downstream community and diversity analysis. In this study, two clone libraries were constructed with two different DNA polymerases, Q5 high-fidelity DNA polymerase and exTaq polymerase, to compare the differences in their capability to accurately reflect the cyanobacterial community structure and diversity in a subtropical deep freshwater reservoir, Dongzhen reservoir. The results indicated that the two cyanobacterial clone libraries constructed by using Q5 high-fidelity DNA polymerase and exTaq DNA polymerase did not show significant differences, although a slightly higher community diversity was revealed by using Q5 high-fidelity DNA polymerase. It is noteworthy that so far Q5 high-fidelity DNA polymerase was the first time to be employed in the genetic analysis of cyanobacterial community. And it is for the first time that the cyanobacterial community structure in Dongzhen reservoir was analyzed using molecular methods. Phylogenetic analysis revealed that most of the clones belonged to Cyanophyta and chloroplast, among which Cyanobium sp. Suigetsu-CR5 made up the largest fraction of cyanobacteria in winter. [ABSTRACT FROM AUTHOR]

Background: Taq DNA polymerase is a very important enzyme for molecular biological studies such as DNA amplification and DNA sequencing by the PCR. It is a standard enzyme that is used in 90% of molecular biology labs today. The aim of this study was to produce Taq DNA polymerase enzyme in E. coli by a reliable, practical, simple and low cost method.Materials and Methods: In this study, the Taq gene was amplified from the genomic DNA of Thermus aquaticus and cloned into pTrc99A vector. Recombinant plasmid is expressed in E. coli strain TOP10. Product protein is extracted and purified. Expression of gene was analyzed by SDS-PAGE and gene amplification.Results: SDS-PAGE showed an approximately 94 KDa protein. The density of protein bands in agarose gel electrophoresis indicated that the purified enzyme is more active than the non purified one.Conclusion: The protocols described in this paper lead to the production of pure and active enzyme that can be applied in both teaching and research laboratories.

The polymerase chain reaction (PCR) method is widely used for the reproduction and amplification of specific DNA segments, and a novel PCR method using nanomaterials such as gold nanoparticles has recently been reported. This paper reports on the effects of superparamagnetic nanoparticles on PCR amplification without an external magnetic field, and clarifies the mechanism behind the effects of superparamagnetic particle clusters on PCR efficiency by estimating the structures of such clusters in PCR. It was found that superparamagnetic nanoparticles tend to inhibit PCR amplification depending on the structure of the magnetic nanoparticle clusters. The paper also clarifies that Taq polymerase is captured in the spaces formed among magnetic nanoparticle clusters, and that it is captured more efficiently as a result of their motion from heat treatment in PCR thermal cycles. Consequently, Taq polymerase that should be used in PCR is reduced in the PCR solution. These outcomes will be applied to novel PCR techniques using magnetic particles in an external magnetic field.

We present a new, general concept termed Hydrodynamic Thermal Confinement (HTC), and its implementation for the creation of microscale dynamic thermo-chemical microenvironments on biological surfaces. HTC is based on a scanning probe and operates under physiological conditions. The temperature can be regulated between 30° and 80 °C with ±0.2 °C precision and temperature ramps of 5 °C s−1 over a footprint of ∼50 μm × 80 μm in a volume of ∼50 × 80 × 15 μm3 (∼50 pl). [ABSTRACT FROM AUTHOR]

Here we develop a new method for the sensitive and selective detection of cell-surface proteins with an aptamer probe designed for binding-responsive catalysis of Taq DNA polymerase. Taking the biotin receptor as a model, the method allows the detection of target protein on surfaces of different types of cancer cells. [ABSTRACT FROM AUTHOR]

Microcystin-RR (MC-RR) is a highly acute hepatotoxin produced by cyanobacteria. It is harmful to both humans and the environment. A novel aptamer was identified by the systemic evolution of ligands by exponential enrichment (SELEX) method as a recognition element for determination of MC-RR in aquatic products. The graphene oxide (GO) SELEX strategy was adopted to generate aptamers with high affinity and specificity. Of the 50 aptamer candidates tested, sequence RR-33 was found to display high affinity and selectivity, with a dissociation constant of 45.7 ± 6.8 nM. Aptamer RR-33 therefore was used as the recognition element in a fluorometric assay that proceeds as follows: (1) Biotinylated aptamer RR-33 is immobilized on the streptavidinylated wells of a microtiterplate, and carboxyfluorescein (FAM) labelled complementary DNA is then allowed to hybridize. (2) After removal of excess (unbound) cDNA, sample containing MC-RR is added and incubated at 37 °C for 2 h. (3) Displaced free cDNA is washed away and fluorescence intensity measured at excitation/emission wavelengths of 490/515 nm. The calibration plot is linear in the 0.20 to 2.5 ng·mL concentration range, and the limit of detection is 80 pg·mL. The results indicate that the GO-SELEX technology is appropriate for the screening of aptamers against small-molecule toxins. The detection scheme was applied to the determination of MC-RR in (spiked) water, mussel and fish and gave recoveries between 91 and 98 %. The method compares favorably to a known ELISA. Conceivably, this kind of assay is applicable to other toxins for which appropriate aptamers are available. [ABSTRACT FROM AUTHOR]

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Aims: Sickle-cell anemia and β-thalassemia are two of the most common autosomal recessive disorders in the developing world. The severity of the problem and the pressure it exerts on the health services in the Kingdom of Saudi Arabia forced the introduction of a national premarital screening program to lessen its impact on the society. Furthermore, a significant effort has been exerted in the elucidation of the genetic causes of such diseases to facilitate diagnosis and detection of carriers. Methods: We have designed and validated the use of custom TaqMan® genotyping assays for the rapid detection of IVS-I-1 (G>A), IVS-I-5 (G>C), codon 39 (C>T), and IVS-I-110 (G>A) mutations in transfusion-dependent β-thalassemia patients' cohort. Results: We demonstrated that IVS-I-5 (rs33915217) is the most common single-nucleotide variant in our cohort, with the variant allele constituting 26% of the total alleles investigated. However, this variant was not found in 352 alleles screened from buccal swab DNA obtained from healthy volunteers. Conclusion: The TaqMan single nucleotide polymorphism (SNP) genotyping assays are a rapid, accurate, and cost-effective method for the initial screening of β-thalassemia cases, which will minimize the need for direct sequencing of the HBB gene, thus reducing detection costs and increasing throughput. [ABSTRACT FROM AUTHOR]

We propose a facile fluorometric system for detection of gene mutations using graphene oxide (GO). A fluorescent probe DNA anneals to a specific mutant gene and is degraded by the 5′→ 3′ exonuclease activity of Taq polymerase during PCR, and the released fluorophore retains fluorescence after addition of GO without quenching. [ABSTRACT FROM AUTHOR]

Background: The Cobas TaqMan MTB assay is used for rapid detection of the Mycobacterium tuberculosis complex (MTC) in clinical samples. It is only validated for respiratory samples, but is often requested by physicians for non-respiratory specimens. The aim of this study was therefore to evaluate the performance of this assay in clinical praxis in a country with low prevalence of tuberculosis (TB). Methods: The results from 2388 respiratory and 1005 non-respiratory human specimens were analyzed by this real-time PCR technique. Using culture results as the gold standard, the sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV) of the PCR results were calculated. Results: For smear-positive respiratory specimens all the four values investigated were 100%. The number of smear-positive nonrespiratory specimens was only eight, and all of these eight specimens reacted positively. Sensitivity was 51% for smearnegative respiratory specimens, and 47% for smear-negative non-respiratory specimens. When all non-respiratory specimens were analyzed together the sensitivity was 51%. Specimens from 16 patients were PCR-positive but culture-negative and these cases are discussed. In one case, TB DNA was still present in sputum 2 years after a successful treatment. Conclusion: The study shows that the Cobas TaqMan MTB assay performs well in smear-positive samples, while the sensitivities are unsatisfactory for both respiratory and non-respiratory smear-negative specimens. Furthermore, the analyses emphasize that this assay cannot be used to evaluate treatment or contagiousness, or to detect relapses or screen for TB. [ABSTRACT FROM AUTHOR]

CYP2C19 is a polymorphic enzyme that metabolizes a wide variety of therapeutic drugs that has been associated with altered enzymatic activity and adverse drug reactions. Differences in allele frequencies of the CYP2C19 gene have been detected in populations worldwide. Thus, we analysed the alleles CYP2C19*2, CYP2C19*3, CYP2C19*4 and CYP2C19*5 related to the poor metabolizer (PM) phenotype in a Mexican population sample (n = 238), as well as CYP2C19*17, unique allele related to ultrarapid metabolizer phenotype (UMs). Genotypes were determined using SNaPshot and TaqManqPCR assays. In addition to the wild-type CYP2C19*1 allele (77.1%), we only found CYP2C19*17 (14.3%) and CYP2C19*2 (8.6%). Comparison with previous population reports demonstrated that these two SNPs are homogeneously distributed in Latin America (P > 0.05). Based on comparison with a previous pharmacokinetic study that determined the frequency of CYP2C19 phenotypes in the same population (western Mexican), we obtained the following findings: (i) based on the difference between the frequency of genotypes CYP2C19*2/*2 (presumably PM) versus the observed prevalence of PM phenotypes (0.4 versus 6.3%; X² = 9.58, P = 0.00196), we inferred the plausible presence of novel CYP2C19 alleles related to the PM phenotype; (ii) the prevalence of UMs was in disagreement with the dominant inheritance pattern suggested for CYP2C19*17 (23.1 versus 4%; P < 0.00001); (iii) the apparent recessive inheritance pattern of CYP2C19*17, based on the agreement between homozygous CYP2C19*17/*17 (presumably UMs) and the observed prevalence of UMs (2.1 versus 4%; (X² = 1.048; P = 0.306). [ABSTRACT FROM AUTHOR]

Background: Increasing evidences support the role of genetic factors in susceptibility to chronic periodontitis. The aim of the present pilot study was to explore the association of two potential single nucleotide polymorphisms (SNPs): Interleukin (IL)-1 β + 3954 (rs1143634, C > T) and vitamin D receptor (VDR) TaqI (rs731236, T > C) with chronic periodontitis in a North Indian population. Materials and Methods: Twentyeight chronic periodontitis subjects and 47 periodontally healthy controls were recruited. Individual samples of venous blood were obtained from each subject. Genotyping was done by polymerase chain reaction, followed by restriction fragment length polymorphism (PCR-RFLP). Logistic regression and chi square test were used for genetic association analysis and a P value less than 0.05 taken as statistical significance. Statistical Analysis Used: Chi square test and odds ratio (OR) was used. Results: Genotypes and alleles of SNP IL-1 β + 3954 did not show a significant association (P > 0.05) with chronic periodontitis. Genotype CC and allele C of VDR TaqI were significantly associated with a higher risk for chronic periodontitis as compared to subjects with TT genotype (CC/TT OR = 4.615; 95% confidence interval [CI]: 1.17 to 18.078 P = 0.028) and allele T (C/T OR = 2.423; 95% CI: 1.179 to 4.980). Conclusion: In North Indian population, genotype CC and allele C of VDR TaqI were associated with risk of chronic periodontitis. No significant correlation was found for IL-1 β + 3954 polymorphism and chronic periodontitis. [ABSTRACT FROM AUTHOR]

Introduction: The component of commercial available master mix most sensitive to unfavorable conditions is a polymerase. Available commercial polymerase chain reaction (PCR) master mixes are generally recommended for storage at 208C, otherwise they may lose their activity. Aim: The aim of the experiment was to verify if storing mixes in adverse and extreme conditions may influence the quality of a PCR product. In the second phase of the research, it was to indicate if inactive PCR reagents that have lost their activity, may recover their enzymatic properties. Material and methods: Five different commercially available master mixes were incubated in unfavorable conditions. After the PCR, an electrophoresis was carried out and the obtained product was an evidence of a proper PCR reaction. Results and discussion: Total degradation of mixes was caused by their incubation at room temperature for 28 days and incubation at 1008C for 60 minutes. Addition of polymerases to the degraded mixes (incubation at room temperature for 28 days) resulted in a regeneration of all of five mixes. In the case of polymerases incubated at 1008C for 60 minutes, regeneration was effective only in two of the five mixes. Conclusions: Our research confirms that PCR master mix is characterized by high resistance to varied conditions as well as in some cases can be repaired after degradation. [ABSTRACT FROM AUTHOR]

DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, DNA labeling, mutagenesis, and other experiments. Thermostable DNA polymerases are especially useful and became quite valuable after the development of PCR technology. A DNA polymerase from Thermus aquaticus (Taq polymerase) is the most famous DNA polymerase as a PCR enzyme, and has been widely used all over the world. In this study, the gene fragments of the family A DNA polymerases were amplified by PCR from the DNAs from microorganisms within environmental soil samples, using a primer set for the two conserved regions. The corresponding region of the pol gene for Taq polymerase was substituted with the amplified gene fragments, and various chimeric DNA polymerases were prepared. Based on the properties of these chimeric enzymes and their sequences, two residues, E742 and A743, in Taq polymerase were found to be critical for its elongation ability. Taq polymerases with mutations at 742 and 743 actually showed higher DNA affinity and faster primer extension ability. These factors also affected the PCR performance of the DNA polymerase, and improved PCR results were observed with the mutant Taq polymerase. [ABSTRACT FROM AUTHOR]

The thermophilic bacterium Thermus aquaticus is a well-known source of Taq polymerase. Here, we studied the structure and dynamics of the T. aquaticus cold-shock protein ( Ta- Csp) to better understand its thermostability using NMR spectroscopy. We found that Ta- Csp has a five-stranded β-barrel structure with five salt bridges which are important for more rigid structure and a higher melting temperature (76 °C) of Ta- Csp compared to mesophilic and psychrophilic Csps. Microsecond to millisecond time scale exchange processes occur only at the β1–β2 surface region of the nucleic acid binding site with an average conformational exchange rate constant of 674 s −1 . The results imply that thermophilic Ta- Csp has a more rigid structure and may not need high structural flexibility to accommodate nucleic acids upon cold shock compared to its mesophile and psychrophile counterparts. [ABSTRACT FROM AUTHOR]

A simple and inexpensive method is reported for the long-term stabilization of enzymes and other unstable reagents in premeasured quantities in water-soluble tablets (cast, not compressed) made with pullulan, a nonionic polysaccharide that forms an oxygen impermeable solid upon drying. The pullulan tablets dissolve in aqueous solutions in seconds, thereby facilitating the easy execution of bioassays at remote sites with no need for special reagent handling and liquid pipetting. This approach is modular in nature, thus allowing the creation of individual tablets for enzymes and their substrates. Proof-of-principle demonstrations include a Taq polymerase tablet for DNA amplification through PCR and a pesticide assay kit consisting of separate tablets for acetylcholinesterase and its chromogenic substrate, indoxyl acetate, both of which are highly unstable. The encapsulated reagents remain stable at room temperature for months, thus enabling the room-temperature shipping and storage of bioassay components. [ABSTRACT FROM AUTHOR]

ABSTRACT The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. A comparative thermodynamic characterization of the large fragment domains of Taq (Klentaq) and E. coli (Klenow) DNA polymerases has been performed by obtaining full Gibbs-Helmholtz stability curves of the free energy of folding (Δ G) versus temperature. This analysis provides the temperature dependencies of the folding enthalpy and entropy (Δ H and Δ S), and the heat capacity (Δ Cp) of folding. If increased or enhanced non-covalent bonding in the native state is responsible for enhanced thermal stabilization of a protein, as is often proposed, then an enhanced favourable folding enthalpy should, in general, be observed for thermophilic proteins. However, for the Klenow -Klentaq homologous pair, the folding enthalpy (Δ Hfold) of Klentaq is considerably less favorable than that of Klenow at all temperatures. In contrast, it is found that Klentaq's extreme free energy of folding (Δ Gfold) originates from a significantly reduced entropic penalty of folding (Δ Sfold). Furthermore, the heat capacity changes upon folding are similar for Klenow and Klentaq. Along with this new data, comparable extended analysis of available thermodynamic data for 17 other mesophilic-thermophilic protein pairs (where enough applicable thermodynamic data exists) shows a similar pattern in seven of the 18 total systems. When analyzed with this approach, the more familiar 'reduced Δ Cp mechanism' for protein thermal stabilization (observed in a different six of the 18 systems) frequently manifests as a temperature dependent shift from enthalpy driven stabilization to a reduced-entropic-penalty model. Proteins 2014; 82:785-793. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: The identification of rapid methods for the surveillance of recreational waters and aquatic environments is necessary to provide adequate levels of health safety and quality standards. Molecular techniques have been proposed in recent years as a valuable alternative to traditional microbiological cultural methods, having numerous advantages (speed, specificity and sensibility). We have previously described a method for the detection of Staphylococcus aureus in recreational waters by a molecular enrichment approach, based on a two steps PCR performed on 23S rDNA. The approach has been now expanded to other microbiological indicators, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli. This methodological extension to other prokaryotic genomes has required a partial primer re-design and protocol optimization, in order to improve the assay. The usage of native not recombinant Taq-polymerases for detection of E. coli eliminated the background probably due to traces of the bacterial genome in many commercially available reagents. The obtained results clearly underline the feasibility of a fast and integrated molecular approach to quantify the standard biological indicators for assessment of safety and quality in recreational waters. [Copyright &y& Elsevier]

Directed evolution allows us to explore protein functionalities not required in the natural environment. It mimics natural genetic processes and selective pressures. This approach is used when the molecular basis is not completely understood and rational design is a difficult task. This approach consists of serial cycles of consecutive diversification and selection which eventually lead to the accumulation of beneficial mutations. Here are presented two cases where directed evolution is used to modify two different proteins: Taq polymerase, enzyme used for DNA extension in PCR, and the LacI repressor protein which regulates gene expression on E.coli.

This study examines the relationship between the DNA binding thermodynamics and the enzymatic activity of the Klenow and Klentaq Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Both polymerases bind DNA with nanomolar affinity at temperatures down to at least 5°C, but have lower than 1% enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding (ΔHbinding) crosses zero (TH) and becomes favorable (negative). This TH/activity upshift temperature is 15°C for Klenow and 30°C for Klentaq. The results indicate that a negative free energy of DNA binding alone is not sufficient to proceed to catalysis, but that the enthalpic versus entropic balance of binding may be a modulator of the temperature dependence of enzymatic function. Analysis of the temperature dependence of the catalytic activity of Klentaq polymerase using expanded Eyring theory yields thermodynamic patterns for ΔG‡, ΔH‡, and TΔS‡ that are highly analogous to those commonly observed for direct DNA binding. Eyring analysis also finds a significant ΔCp‡ of formation of the activated complex, which in turn indicates that the temperature of maximal activity, after which incorporation rate slows with increasing temperature, will correspond with the temperature where the activation enthalpy (ΔH‡) switches from positive to negative. [ABSTRACT FROM AUTHOR]

Several 7-(hydroxy, amino, methylureido, and guanidino)alkynyl-substituted 8-aza-7-deaza- hypoxanthine analogues were investigated as potential universal nucleobases. 7-Aminobutynyl-8-aza-7-deazahypoxanthine was found to be the most promising quasi-universal nucleobase with improved hybridization and polymerase chain reaction (PCR) enhancing properties as compared to commonly used hypoxanthine (the nucleobase of inosine). It demonstrated improved ambiguity for pairing with A, T, and C bases and its base pairing properties can be summarized as follows: X:C∼X:A∼X:T > X:G. The improvement in PCR performance directly correlated with primer's Tm. Primers containing multiple 7-aminobutynyl-8-aza-7-deazahypoxanthines were successfully used without noticeable inhibition of Taq polymerase activity provided the modifications are positioned more than two bases away from the 3′ end. [ABSTRACT FROM PUBLISHER]

It has been suggested that Wigner's clock inequalities can be applied to biological systems. Pešsić was the first to suggest this by applying these inequalities to the reproduction time of a mycoplasma (1993 Eur. J. Phys. 14 90). More recently, Goel applied Wigner's clock inequalities to the system formed by a Taq DNA polymerase reading a phage lambda DNA strand, concluding that the behaviour of polymerases is governed by quantum mechanical processes. By means of a counterexample, using a Pfu DNA polymerase, I show that current experimental evidence does not allow us to conclude that Wigner's inequalities govern the behaviour of polymerases. Furthermore, both works are based on an incorrect interpretation of the concept of position uncertainty. [ABSTRACT FROM AUTHOR]

Abstract: A TaqMan-based real-time PCR assay was developed for specific detection of strains of X. fastidiosa causing oleander leaf scorch. The assay uses primers WG-OLS-F1 and WG-OLS-R1 and the fluorescent probe WG-OLS-P1, designed based on unique sequences found only in the genome of oleander strain Ann1. The assay is specific, allowing detection of only oleander-infecting strains, not other strains of X. fastidiosa nor other plant-associated bacteria tested. The assay is also sensitive, with a detection limit of 10.4fg DNA of X. fastidiosa per reaction in vitro and in planta. The assay can also be applied to detect low numbers of X. fastidiosa in insect samples, or further developed into a multiplex real-time PCR assay to simultaneously detect and distinguish diverse strains of X. fastidiosa that may occupy the same hosts or insect vectors. Specific and sensitive detection and quantification of oleander strains of X. fastidiosa should be useful for disease diagnosis, epidemiological studies, management of oleander leaf scorch disease, and resistance screening for oleander shrubs. [Copyright &y& Elsevier]

SERS primers have been used to directly detect specific PCR products utilizing the difference in adsorption of single-stranded and double-stranded DNA onto nanoparticle surfaces. Herein, seven parameters important for improved positive SERS assays for real applications were investigated. First, we applied a model system for optimization experiments, followed by a PCR assay to detect pathogen DNA, and then the introduction of a new assay that utilizes the 5' → 3' exonuclease activity of Taq DNA polymerase to partly digest the SERS probe, generating dye-labeled single-stranded DNA increasing the SERS signals for detection of pathogen DNA. Applying the model system, it was found that uni-molecular SERS primers perform better than bi-molecular SERS primers. However, within the PCR assays, it was found that uni- and bi-molecular SERS primers performed very similarly, and the most reproducible results were obtained using the 5' → 3' exonuclease digestion assay. These SERS-based assays offer new routes over conventional fluorescence-based techniques without compromising sensitivity or selectivity. [ABSTRACT FROM AUTHOR]

Abstract: The multiplex system allows the detection of 19 autosomal short tandem repeat (STR) loci [including all Combined DNA Index System (CODIS) STR loci as well as D2S1338, D6S1043, D12S391, D19S433, Penta D and Penta E] plus the sex-determining locus Amelogenin in a single reaction, comprising all STR loci in various commercial kits used in the China national DNA database (NDNAD). Primers are designed so that the amplicons are distributed ranging from 90base pairs (bp) to 450bp within a five-dye fluorescent design with the fifth dye reserved for the internal size standard. With 30 cycles, 125pg to 2ng DNA template showed optimal profiling result, while robust profiles could also be achieved by adjusting the cycle numbers for the DNA template beyond that optimal DNA input range. Mixture studies showed that 83% and 87% of minor alleles were detected at 9:1 and 1:9 ratios, respectively. When 4ng of degraded DNA was digested by 2-min DNase and 1ng undegraded DNA was added to 400μM haematin, the complete profiles were still observed. Polymerase chain reaction (PCR)-based procedures were examined and optimized including the concentrations of primer set, magnesium and the Taq polymerase as well as volume, cycle number and annealing temperature. In addition, the system has been validated by 3000 bloodstain samples and 35 common case samples in line with the Chinese National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The total probability of identity (TPI) can reach to 8×10−24, where DNA database can be improved at the level of 10 million DNA profiles or more because the number of expected match is far from one person (4×10−10) and can be negligible. Further, our system also demonstrates its good performance in case samples and it will be an ideal tool for forensic DNA typing and databasing with potential application. [Copyright &y& Elsevier]

Abstract: The increasing use of low temperature co-fired ceramic (LTCC) for the fabrication of biological microfluidic devices necessitates further research on LTCC biocompatibility. In this study we explore the inhibitory effect of DuPont''s 951 LTCC on Polymerase Chain Reaction (PCR), and demonstrate a novel mechanism to increase biocompatibility between LTCC and PCR with the addition of a common passivation substance, bovine serum albumin (BSA). We show that DuPont''s 951 LTCC binds negatively charged proteins including BSA and ovalbumin (OVA). This is a significant discovery as proteins (enzymes) are an essential component of most biological reactions, and a frequent addition to microfluidic devices. A proposed model for LTCC inhibition of PCR by enzyme adsorption is presented. [Copyright &y& Elsevier]

Abstract: Introduction: Polymorphisms of the dopamine receptor D2 (DRD2) gene have been associated with obesity phenotypes. Our aim was to examine if the genotype of TaqIA Restriction Fragment Length Polymorphism (RFPL) was related to an attenuated weight loss response or to changes in energy expenditure (EE) and food preference before and after weight loss. Methods: Obese post-menopausal women (age=57.1±4.6yr, weight=85.4±15.4kg and BMI=32.8±4.5kg/m2) were genotyped for TaqIA (n =127) by using PCR–RFLP analysis and categorized as possessing at least one copy of the A1 allele (A1+) or no copy (A1−). Women were randomized into two groups, caloric restriction (CR) and caloric restriction+resistance training (CRRT) and in this study were further classified as follows: A1+CR, A1+CRRT, A1-−CR and −A1−CRRT. Body composition, total daily EE, physical activity EE, Resting EE (REE), and energy intake were obtained at baseline and post-intervention using DXA, doubly-labeled water, indirect calorimetry, and 3-day dietary records, respectively. Results: Overall, all of the anthropometric variables and REE significantly decreased post-intervention (p <0.001). Women in the CRRT group lost significantly more fat mass (FM) than the CR women (p <0.05). There were significant time by group by allele interactions for attenuated body weight (BW), BMI, and FM loss for A1+ (vs. A1−) in CRRT (p <0.05) and for increased % carbohydrate intake (p <0.01). Conclusion: TaqIA genotype was associated with body weight loss post-intervention; more specifically, carriers of the A1 allele lost significantly less BW and FM than the A1− and had increased carbohydrate intake in the CRRT group. [Copyright &y& Elsevier]

Polymerase chain reaction (PCR) is an in vitro technique to produce million fold copies of a particular segment of DNA. PCR should optimally yield a unique product unless designed otherwise. Artefacts appear many times, which affect the success of downstream applications. Prevention of non specific amplification using formamide and manipulation of cycling time is discussed in this article. [ABSTRACT FROM AUTHOR]

Gap repair is a fast and efficient method for assembling recombinant DNA molecules in Saccharomyces cerevisiae. This method produces a circular DNA molecule by homologous recombination between two or more linear DNA fragments, one of which is typically a vector carrying replicative sequences and a selective marker. This technique avoids laborious and costly in vitro purification and ligation of DNA. The DNA repair machinery can also close and ligate the linear vector by mechanisms other than homologous recombination, resulting in an empty vector. The frequency of these unwanted events can be lowered by removing the 5′-phosphate groups using phosphatase, which is the standard method used for in vitro ligation. However, phosphatase treatment is less effective for gap repair cloning than for in vitro ligation, presumably due to the ability of the S. cerevisiae DNA repair machinery to efficiently repair the missing phosphate group to allow religation. We have developed a more efficient method to prevent vector religation, based on treatment of the vector fragment with Taq DNA polymerase and dATP. This procedure prevents vector recircularization almost completely, facilitating the screening for true recombinant clones. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: White spot syndrome virus (WSSV) is an important pathogen in shrimp aquaculture. The susceptibility of crayfish (Procambarus clarkii) was assessed by means of serial dilutions of a solution containing WSSV. A TaqMan real-time PCR was used to quantify the WSSV challenge dose in P. clarkii. The results showed that WSSV copies could be detected at concentrations from 1.365×104 to 1.129×109 copies/μl. The viral infectivity (LD50), measured as the mortality of infected crayfish, indicated 60% mortality in the 105 dilution group (1.524×105 copies/μl). TaqMan real-time PCR represents a novel standard method, based on the by quantitation of WSSV copies, for determining the appropriate concentration of WSSV for use in infection experiments. [Copyright &y& Elsevier]

Abstract: To determine the burden of rabies in developing countries, a reliable and accurate diagnostic test for the examination of the brains of animals is needed. Recently, the number of samples and carcasses submitted to rabies diagnostic units has been declining. Methods for obtaining tissues from different regions of the brain are even more difficult, and direct florescent antibody examination may fail if the samples decomposed. The spread of rabies virus to peripheral non-nervous tissues starts early during the pre-clinical phase. It has been shown that saliva and skin biopsies taken at the neck and containing hair follicles can be used in the ante-mortem diagnosis of rabies in humans. Obtaining oral swab samples, whisker or hair follicles from the heads of canines is easy and practical and can be performed without special equipment. The objective of this study was to determine whether these non-neural specimens can be used for the detection of rabies viral RNA. The RNAs extracted from these specimens were tested using a real-time reverse transcriptase polymerase chain reaction (RT-PCR). The sensitivity of the TaqMan real-time RT-PCR analysis using samples from dogs confirmed to be infected with rabies virus was 84.6% (55/65), 81.8% (54/66) and 66.7% (44/66) when using oral swab samples, extracted whisker follicles and extracted hair follicles; the specificity of all specimen types was 100%. The negative predictive values were 77.8%, 74.4% and 61.4%, respectively. Although the rate of positivity when combining the three non-neural specimen types was increased to 86.4%, this level of sensitivity was not sufficient to help physicians whether to administer post exposure prophylaxis. However, these oral swab and whisker specimens may serve to enhance epidemiological surveillance; such data will contribute in the planning of rabies control programs. [Copyright &y& Elsevier]

Abstract: Real-time polymerase chain reaction (real-time PCR) is an accurate, rapid and reliable method that can be used for the detection and also for the quantitation of specific DNA molecules. It can be non-specific, with intercalating dyes (SYBR Green I dye) able to bind to any dsDNA, or specific with a probe (TaqMan), whereby the probe is designed to bind within the amplified PCR fragment. A new real-time reverse transcription and polymerase chain reaction (real time RT-PCR) assay with TaqMan probe for specific detection of acute bee paralysis virus was designed. The assay was optimized to be highly sensitive and analytically specific and tested with a selection of genetically diverse ABPV strains originating from Slovenia, the United Kingdom (UK), Hungary and Germany. The detection limit of the assay and sensitivity comparisons with conventional RT-PCR were analyzed and this assay can detect a minimum of 44 copies of ABPV/reaction and is 230 times more sensitive than conventional RT-PCR. In addition, the assay is highly reproducible, with an average slope of standard curve made of ten-fold dilutions of standard copies/reaction −3.479±0.19 and an average slope of standard curve made of ten-fold dilutions of RNA −3.409±0.18. [Copyright &y& Elsevier]

Background: The aim of this project was to isolate and purify the highly active recombinant Taq DNA polymerase from the strain of Escherichia coli BL21. This enzyme, with a molecular weight of about 94 kDa, is widely used in polymerase chain reaction (PCR). In PCR, the activity and fidelity of Taq polymerase can significantly influence the results. One of the active regions of Taq polymerase has been suggested to be the O-helix region. In previous studies, an expression vector containing mutated Asn 666 Glu Taq polymerase gene was designed. In order to investigate the effects of this mutation on the function of the enzyme, Taq polymerase needs to be purified first. Methods: In this study, after transformation of competent cells, enzyme expression was induced by isopropyl â D thiogalactopyranoside (IPTG) method. Modified Desai protocol with DNase and RNase, nickel-nitrilotriacetic acid (Ni-NTA) resin, trichloroacetic acid (TCA), and refolding protocols were subsequently used for purification of this enzyme. These protocols were finally compared. Findings: Using Desai protocol resulted in the production of a sharp band in the expected region (94 kDa) and several other visible bands. After further modification of Desai protocol, only the desirable band was observed. In protocols using TCA and Ni-NTA resin, the expected bands were weak. Refolding protocol caused a band in an undesirable region (66 kDa). Conclusion: From the different purification techniques that were used in this study, the modified method of Desai containing RNase and DNase worked best. Addition of TCA can precipitate proteins that had not been affected by heat. Using Ni-NTA resin resulted in elimination of unwanted bands. However, the amount of Taq polymerase was also decreased. The extra band that was observed in refolding protocol was probably due to the presence of proteases that were isolated with inclusion body and could digest Taq polymerase. [ABSTRACT FROM AUTHOR]