A high-throughput assay for a human telomerase protein-human telomerase RNA interaction

The rapid rate at which cancer cells divide necessitates a mechanism for telomere maintenance, and in approximately 90% of all cancer types the enzyme telomerase is used to maintain the length of telomeric DNA. Telomerase is a multi-subunit enzyme that minimally contains a catalytic protein subunit, hTERT, and an RNA subunit, hTR. Proper assembly of telomerase is critical for its enzymatic activity and therefore is a requirement for the proliferation of most cancer cells. We have developed the first high-throughput screen capable of identifying small molecules that specifically perturb human telomerase assemblage. The screen uses a scintillation proximity assay to identify compounds that prevent a specific and required interaction between hTR and hTERT. Rather than attempting to disrupt all of the individual hTR-hTERT interactions, we focused the screen on the interaction of the CR4-CR5 domain of hTR with hTERT. The screen employs a biotin-labeled derivative of the CR4-CR5 domain of hTR that independently binds [(35)S]hTERT in a functionally relevant manner. The complex between hTERT and biotin-labeled RNA can be captured on streptavidin-coated scintillation proximity beads. Use of 96-well filter plates and a vacuum manifold enables rapid purification of the beads. After optimization, statistical evaluation of the screen generated a Z' factor of 0.6, demonstrating the high precision of the assay.     

A spectrophotometric, enzymatic assay for D-3-hydroxybutyrate that is not dependent on hydrazine

Because of the potential carcinogenic properties of hydrazine and because of other health hazards associated with its use in the laboratory, an enzymatic assay has been developed for D-3-hydroxybutyrate that is not dependent on hydrazine to drive the reaction toward completion. The use of a high concentration of NAD+ and a buffer at pH 9.5 resulted in a favorable conversion of D-3-hydroxybutyrate to acetoacetate by D-3-hydroxybutyrate dehydrogenase even though the reaction favors D-3-hydroxybutyrate formation under physiological conditions. The assay was also completed faster than previous assays using hydrazine so that the amount of enzyme used for the assay could be reduced. The recovery of D-3-hydroxybutyrate added to liver samples was 98 +/- 1% (mean +/- SEM, n = 6). The assay was found to be suitable for the measurement of D-3-hydroxybutyrate in samples such as perchloric acid extracts of isolated hepatocytes even when the acetoacetate to D-3-hydroxybutyrate ratio was 4 to 1. This assay presents a reliable alternative to the use of hydrazine and may be used for the assay of D-3-hydroxybutyrate in a variety of physiological and experimental samples.     

A quantitative real-time polymerase chain reaction assay for the seagrass pathogen Labyrinthula zosterae

The protist Labyrinthula zosterae (Phylum Bigyra, sensu Tsui et al. 2009) has been identified as a causative agent of wasting disease in eelgrass (Zostera marina), of which the most intense outbreak led to the destruction of 90% of eelgrass beds in eastern North America and western Europe in the 1930s. Outbreaks still occur today, albeit at a smaller scale. Traditionally, L. zosterae has been quantified by measuring the necrotic area of Z. marina leaf tissue. This indirect method can however only lead to a very rough estimate of pathogen load. Here, we present a quantitative real-time polymerase chain reaction (qPCR) approach to directly detect and quantify L. zosterae in eelgrass tissue. Based on the internal transcribed spacer (ITS) sequences of rRNA genes, species-specific primers were designed. Using our qPCR, we were able to quantify accurately and specifically L. zosterae load both from culture and eelgrass leaves using material from Europe and North America. Our detection limit was less than one L. zosterae cell. Our results demonstrate the potential of this qPCR assay to provide rapid, accurate and sensitive molecular identification and quantification of L. zosterae. In view of declining seagrass populations worldwide, this method will provide a valuable tool for seagrass ecologists and conservation projects.     

A novel assay for allelic discrimination that combines the fluorogenic 5' nuclease polymerase chain reaction (TaqMan) and mismatch amplification mutation assay

Recently much attention has been focused on single nucleotide polymorphisms (SNPs) within fundamentally important genes, such as those involved in metabolism, cell growth regulation, and other disease-associated genes. Methodologies for discriminating different alleles need to be specific (robust detection of an altered sequence in the presence of wild-type DNA) and preferably, amenable to high throughput screening. We have combined the fluorogenic 5' nuclease polymerase chain reaction (TaqMan) and the mismatch amplification mutation assay (MAMA) to form a novel assay, TaqMAMA, that can quickly and specifically detect single base changes in genomic DNA. TaqMan chemistry utilizes fluorescence detection during PCR to precisely measure the starting template concentration, while the MAMA assay exploits mismatched bases between the PCR primers and the wild-type template to selectively amplify specific mutant or polymorphic sequences. By combining these assays, the amplification of the mutant DNA can be readily detected by fluorescence in a single PCR reaction in 2 hours. Using the human TK6 cell line and specific HPRT-mutant clones as a model system, we have optimized the TaqMAMA technique to discriminate between mutant and wild-type DNA. Here we demonstrate that appropriately designed MAMA primer pairs preferentially amplify mutant genomic DNA even in the presence of a 1,000-fold excess of wild-type DNA. The ability to selectively amplify DNAs with single nucleotide changes, or the specific amplification of a low copy number mutant DNA in a 1,000-fold excess of wild-type DNA, is certain to be a valuable technique for applications such as allelic discrimination, detection of single nucleotide polymorphisms or gene isoforms, and for assessing hotspot mutations in tumor-associated genes from biopsies contaminated with normal tissue.     

A novel pyrazolone, 4,4-dichloro-1-(2,4-dichlorophenyl)-3-methyl-5-pyrazolone, as a potent catalytic inhibitor of human telomerase

A new derivative of 1-phenyl-3-methyl-5-pyrazolone, 4,4-dichloro-1-(2,4-dichlorophenyl)-3-methyl-5-pyrazolone, named TELIN, was chemically synthesized and identified as a potent inhibitor of human telomerase in the cell-free telomeric repeat amplification protocol. TELIN inhibited telomerase activity at submicromolar level with IC50 of approximately 0.3 microM. Kinetic studies revealed that TELIN does not bind to DNA but to telomerase protein, and the mode of inhibition by this substance was competitive-noncompetitive mixed-type with respect to the TS primer, whereas it was uncompetitive or noncompetitive-uncompetitive mixed-type with respect to the three deoxyribonucleosides. These results demonstrate that TELIN is a specific potent catalytic blocker of telomerase,and is considered to be a valuable substance for medical treatment of cancer and related diseases.     

The use of a quantitative real-time polymerase chain reaction assay for identification and enumeration of Lactobacillus buchneri in silage

Aims:  To detect and quantify Lactobacillus buchneri in plant samples with the aid of polymerase chain reaction (PCR) methods.
Methods and Results:  DNA from silage samples spiked with different amounts of L. buchneri cells was isolated using a lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform extraction method. The DNA served as a template for PCR amplification with primers specific for the bacterium. The primers were developed by comparison of 16S rDNA sequences from different lactic acid bacteria (LAB) and testing for specificity with 11 different strains of LAB. As few as 100 L. buchneri colony-forming units per gram of silage could be detected. Additionally, the technique was successfully applied to quantify the population of L. buchneri in two cultivars of corn with or without inoculation.
Conclusions:  The PCR assay provided a specific and rapid tool for identifying and enumerating L. buchneri in silage samples.
Significance and Impact of the Study:  The use of microbial inoculants for silage production is a safe and environment friendly practice, but the full potential of such additives can only be achieved with a better understanding of the fate and activity of the microbes involved. The current study describes a methodology to detect and enumerate L. buchneri , a micro-organism used as an inoculant.     

A quantitative polymerase chain reaction assay for detecting and identifying fungal contamination in human allograft tissue

To complement donor selection and tissue processing, rapid and reliable detection, discrimination, and quantification of fungal pathogens are extremely important for tissues destined to be implanted into humans. The current detection method for fungal pathogens, in particular, is difficult and time-consuming. Quantitative polymerase chain reaction (qPCR) technology is considered one of the most sensitive methods to detect low levels of DNA. Here a qPCR method is described that can detect clinically relevant, pathogenic fungal organisms. The assay allowed the quantification of fungal organisms within a tissue implant and provides a means to identify the contaminating species. The primers for the qPCR assay were designed to amplify a conserved region of the L2 region of the large ribosomal subunit (LSU) gene. This set of primers was able to detect fewer than 10 colony forming units from Aspergillus and Candida species in spiked samples. Clinical samples were also evaluated using this method and the data compared positively to the existing accepted 28-day fungal culture method for fungal detection. The qPCR method described herein significantly reduced the time required to identify fungal contamination in tissue implants.     

A real-time polymerase chain reaction assay for quantification of allele ratios and correction of amplification bias

Allele-specific epigenetic modifications are crucial for several important biological functions, including genomic imprinting and X-inactivation in mammals. Consequently, an ever increasing number of investigations requires accurate quantification of the relative abundance of parental alleles of a specific sequence in a DNA sample. Here, combining the use of polymorphic restriction sites with real-time polymerase chain reaction (PCR) amplification, we describe a simple and quantitative assay to measure allele ratios. The efficiency of the assay was assessed on genomic DNA for several polymorphic restriction sites located in the mouse Igf2/H19 imprinted locus. The assay was also successfully applied to quantify allele ratio in cDNA samples. In addition, we provide an experimental procedure for detection and correction of potential PCR amplification bias which significantly extends the range of application of the assay.     

Commercial enzyme-linked immunosorbent assay versus polymerase chain reaction for the diagnosis of chronic Chagas disease: a systematic review and meta-analysis

Chronic Chagas disease diagnosis relies on laboratory tests due to its clinicalcharacteristics. The aim of this research was to review commercial enzyme-linkedimmunosorbent assay (ELISA) and polymerase chain reaction (PCR) diagnostic testperformance. Performance of commercial ELISA or PCR for the diagnosis of chronicChagas disease were systematically searched in PubMed, Scopus, Embase, ISI Web, andLILACS through the bibliography from 1980-2014 and by contact with the manufacturers.The risk of bias was assessed with QUADAS-2. Heterogeneity was estimated with theI² statistic. Accuracies provided by the manufacturers usuallyoverestimate the accuracy provided by academia. The risk of bias is high in mosttests and in most QUADAS dimensions. Heterogeneity is high in either sensitivity,specificity, or both. The evidence regarding commercial ELISA and ELISA-recsensitivity and specificity indicates that there is overestimation. The currentrecommendation to use two simultaneous serological tests can be supported by the riskof bias analysis and the amount of heterogeneity but not by the observed accuracies.The usefulness of PCR tests are debatable and health care providers should not orderthem on a routine basis. PCR may be used in selected cases due to its potential todetect seronegative subjects.     

Gregariousness does not vary with geography,developmental stage,or group relatedness in feeding redheaded pine sawfly larvae

Aggregations are widespread across the animal kingdom, yet the underlying proximate and ultimate causes are still largely unknown. An ideal system to investigate this simple, social behavior is the pine sawfly genus Neodiprion, which is experimentally tractable and exhibits interspecific variation in larval gregariousness. To assess intraspecific variation in this trait, we characterized aggregative tendency within a single widespread species, the redheaded pine sawfly (N. lecontei). To do so, we developed a quantitative assay in which we measured interindividual distances over a 90‐min video. This assay revealed minimal behavioral differences: (1) between early‐feeding and late‐feeding larval instars, (2) among larvae derived from different latitudes, and (3) between groups composed of kin and those composed of nonkin. Together, these results suggest that, during the larval feeding period, the benefits individuals derive from aggregating outweigh the costs and that this cost‐to‐benefit ratio does not vary dramatically across space (geography) or ontogeny (developmental stage). In contrast to the feeding larvae, our assay revealed a striking reduction in gregariousness following the final larval molt in N. lecontei. We also found some intriguing interspecific variation: While N. lecontei and N. maurus feeding larvae exhibit significant aggregative tendencies, feeding N. compar larvae do not aggregate at all. These results set the stage for future work investigating the proximate and ultimate mechanisms underlying developmental and interspecific variation in larval gregariousness across Neodiprion.     

A novel nested polymerase chain reaction assay targeting Plasmodium mitochondrial DNA in field‐collected Anopheles mosquitoes

Sensitive techniques for the detection of Plasmodium (Aconoidasida: Plasmodiidae) sporozoites in field‐collected malaria vectors are essential for the correct assessment of risk for malaria transmission. A real‐time polymerase chain reaction (RT‐PCR) protocol targeting Plasmodium mtDNA proved to be much more sensitive in detecting sporozoites in mosquitoes than the widely used enzyme‐linked immunosorbent assay targeting Plasmodium circumsporozoite protein (CSP‐ELISA). However, because of the relatively high costs associated with equipment and reagents, RT‐PCRs are mostly used to assess the outcomes of experimental infections in the frame of research experiments, rather than in routine monitoring of mosquito infection in the field. The present authors developed a novel mtDNA‐based nested PCR protocol, modified from a loop‐mediated isothermal amplification (LAMP) assay for Plasmodium recognition in human blood samples, and compared its performance with that of routinely used CSP‐ELISAs in field‐collected Anopheles coluzzii (Diptera: Culicidae) samples. The nested PCR showed 1.4‐fold higher sensitivity than the CSP‐ELISA. However, nested PCR results obtained in two laboratories and in different replicates within the same laboratory were not 100% consistent, probably because the copy number of amplifiable Plasmodium mtDNA was close in some specimens to the threshold of nested PCR sensitivity. This implies that Plasmodium‐positive specimens should be confirmed by a second nested PCR to avoid false positives. Overall, the results emphasize the need to use molecular approaches to obtain accurate estimates of the actual level of Plasmodium circulation within malaria vector populations.     

A simple polymerase chain reaction/restriction fragment length polymorphism assay capable of identifying medically relevant filamentous fungi

Because of the accumulating evidence that suggests that numerous unhealthy conditions in the indoor environment are the result of abnormal growth of the filamentous fungi (mold) in and on building surfaces, it is necessary to accurately reflect the organisms responsible for these maladies and to identify them in precise and timely manner. To this end, we have developed a method that is cost effective, easy to perform, and accurate. We performed a simple polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis on multiple members of species known to negatively influence the indoor environment. The genera analyzed were Stachybotrys, Penicillium, Aspergillus, and Cladosporium. Each organism underwent PCR with universal primers that amplified ribosomal sequences generating products from 550 to 600 bp followed by enzymatic digestion with EcoRI, HaeIII, MspI, and HinfI. Our results show that using this combination of restriction enzymes enables the identification of these fungal organisms at the species level.     

A combined polymerase chain reaction and restriction endonuclease enzyme assay for discriminating between Campylobacter coli and Campylobacter jejuni

Abstract A combined polymerase chain reaction and restriction endonuclease (RE) enzyme assay was developed to discriminate between Campylobacter coli and Campylobacter jejuni . Amplimers of the FlaA gene obtained by PCR were digested with Alu I and Hin fI to distinguish C. coli from C. jejuni . With Alu I digestion C. jejuni -specific bands were observed at 110, 140 and 160 bp and C. coli -specific bands at 293 and 147 bp. C. jejuni -specific bands of 349 and 109 bp were found by Hin fI digestion but Hin fI did not digest the Fla A amplimer of C. coli . This combined technique is fast and easy to perform, and distinguishes the two campylobacters unequivocally.     

A colorimetric assay for inorganic pyrophosphate that is also useful for measuring product accumulation in polymerase chain reactions

A novel coupled enzyme assay for measuring inorganic pyrophosphate (PP(i)) in biological samples is described. The total PP(i) is determined by a reaction with inosine 5'-monophosphate, catalyzed by hypoxanthine-guanine phosphoribosyl transferase, yielding hypoxanthine and phosphoribosyl pyrophosphate. The hypoxanthine is oxidized to uric acid by xanthine oxidase/xanthine dehydrogenase and can be measured by formation of formazan when a tetrazolium salt is used as the oxidant. The method is also useful for detecting and quantifying PP(i) released from nucleotides during polymerase chain reactions. This rapid and simple method for detecting amplified nucleic acids permits low-cost monitoring by eye or spectrophotometer.     

Genes that encode botulism neurotoxins A, B, E and F in neotropical bee honey identified with the polymerase chain reaction

Honey can be used for the treatment of wounds, sores and skin bums, but it might be contaminated with Clostridium botulinum spores. In order to evaluate Costa Rican raw honey samples, the detection of neurotoxin gene sequences (corresponding to the bacterium) C. botulinum A, B, E and F was done with the polymerase chain reaction. A total of 64 raw honey samples, coming from different Costa Rican sites were analyzed. Reference C. botulinum strains type A (ATCC 19397), type B (ATCC 7949), type E (ATCC 17786) and type F (ATCC 25764) were used as templates for testing the effectivity of the method. The process consisted in culturing the honey samples in prereduced triptose-peptone-glucose-yeast extract media (TGPY) for 5 days. After this, the bacteria lysate obtained was used for PCR. The amplicons, product of the reaction, were visualized using agarose gel 2%. From the 64 honey samples analyzed, none produced positive results in the PCR, since no amplicons were obtained. Even though, all the reference C. botulinum strains used as controls were visualized and showed the effectivity of the extraction method and of the PCR used. The results obtained show promising therapeutic uses for honey from Costa Rica, but further evaluations shall be done in order to be sure of the safety of the product.     

A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase

Polymerase chain reaction (PCR) primers that flank a 688-bp segment within the glutamate dehydrogenase gene (gdh) of Streptococcus suis type 2 could amplify efficiently the DNA of all 306 (100%) clinical S. suis isolates tested (pigs, n=305; human, n=1) encompassing all serotypes obtained from diverse organs, and geographic origins. When DNA from other bacteria were used as templates for amplification, no product was detected indicating specificity of the primers. Multiplex PCR was developed using the gdh gene primer pair and primers that targeted the gene encoding S. suis capsular biosynthesis (cps). This strategy enabled the detection of strains belonging to serotypes 1/2, 1, 2, 7, and 9, respectively. Using the multiplex-PCR technique, 12 out of 14 (86%) isolates that were previously identified as non-typable S. suis (based on biochemical reactions and serology) gave positive PCR results of which four were positive for serotype 7, three for serotype 2, and five for S. suis strains that belong to other serotypes. Retest results of all 14 isolates by several veterinary laboratories were identical with PCR and confirmed that the two non-PCR reactive isolates belonged to strains of other streptococcal species. These results indicated that PCR improved species determination and can thus be used as a reliable species-specific molecular diagnostic reagent for the accurate identification of S. suis isolates and a serotype-specific method for the detection of strains of serotypes 1/2, 1, 2, 7, and 9, respectively. The PCR method therefore has potential clinical and epidemiological applications.