Considerations for optimization of microRNA PCR assays for molecular diagnosis

<p>The remarkable stability of microRNAs in biofluids underlies their potential as biomarkers, but their small size presents challenges for detection by RT-qPCR. The heterogeneity of microRNAs, with each one comprising a series of variants or 'isomiRs', adds additional complexity. Presented here are the key considerations for use of RT-qPCR to measure microRNAs and their isomiRs, with a focus on plasma. Modified nucleotides can be incorporated into primer sequences to enhance affinity and provide increased specificity and sensitivity for RT-qPCR assays. Approaches based upon polyA tailing and use of a common oligo(dT)-based reverse transcription oligonucleotide will detect most isomiRs. Conversely, stem-loop RT oligonucleotides and sequence specific probes can enable detection of specific isomiRs of interest. Next generation sequencing of all the products of a microRNA RT-PCR reaction is a promising new approach for both microRNA quantification and characterization.</p>

Climate drives temporal replacement and nested���resultant richness patterns of Scottish coastal vegetation

Beta diversity quantifies spatial and/or temporal variation in species composition. It is comprised of two distinct components, species replacement and nestedness, which derive from opposing ecological processes. Using Scotland as a case study and a ��-diversity partitioning framework, we investigate temporal replacement and nestedness patterns of coastal grassland species over a 34-yr time period. We aim to 1) understand the influence of two potentially pivotal processes (climate and land-use changes) on landscape-scale (5 �� 5 km) temporal replacement and nestedness patterns, and 2) investigate whether patterns from one ��-diversity component can mask observable patterns in the other.<br/><br/>We summarised key aspects of climate driven macro-ecological variation as measures of variance, long-term trends, between-year similarity and extremes, for three important climatic predictors (minimum temperature, water-balance and growing degree-days). Shifts in landscape-scale heterogeneity, a proxy of land-use change, was summarised as a spatial multiple-site dissimilarity measure. Together, these climatic and spatial predictors were used in a multi-model inference framework to gauge the relative contribution of each on temporal replacement and nestedness patterns.<br/><br/>Temporal ��-diversity patterns were reasonably well explained by climate change but weakly explained by changes in landscape-scale heterogeneity. Climate was shown to have a greater influence on temporal nestedness than replacement patterns over our study period, linking nestedness patterns, as a result of imbalanced gains and losses, to climatic warming and extremes respectively. Important climatic predictors (i.e. growing degree-days) of temporal ��-diversity were also identified, and contrasting patterns between the two ��-diversity components revealed.<br/><br/>Results suggest climate influences plant species recruitment and establishment processes of Scotland's coastal grasslands, and while species extinctions take time, they are likely to be facilitated by climatic perturbations. Our findings also highlight the importance of distinguishing between different components of ��-diversity, disentangling contrasting patterns than can mask one another.

Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus , is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the Msp I satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus , up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation.

A rapid staining-assisted wood sampling method for PCR-based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR-based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining-assisted wood sampling method aiding PCR-based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5-mg wood cross-sections, of 0.5 �� 0.5 �� 0.015 cm dimension, that were cut from a wood sample of 0.5 �� 0.5 �� 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5-mg wood cross-sections (that adjoined the three 5-mg wood cross-sections, mentioned above) were cut. Nematode-staining-spots (NSSs) in each of the two stained sections were counted under a microscope at 100�� magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus ��� specific amplicon of 403 bp (DQ855275) was generated by PCR assay from 100.00% of 5-mg wood cross-sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx-detection methods such as PCR assay.

Determina����o dos gen��tipos de Listeria Monocytogenes por PCR em tempo real e avalia����o da sua citopatogenicidade

Disserta����o mest., Qualidade em An��lises, Universidade do Algarve, 2007

New method for the simultaneous identification of pathogenic human viruses using multiplex PCR

Tese de mestrado. Biologia (Biologia Molecular e Gen��tica). Universidade de Lisboa, Faculdade de Ci��ncias, 2014

Identification of Streptococcus gallolyticus subsp. macedonicus as the etiological agent in a case of culture-negative multivalve infective endocarditis by 16S rDNA PCR analysis of resected valvular tissue

Today, PCR using broad-range primers is being used increasingly to detect pathogens from resected heart valves. Herein is described the first case of multivalve infective endocarditis where 16S rDNA PCR was used to detect a single pathogen from two affected valves in a 61-year-old man. Triple heart valve replacement was required despite six weeks of appropriate antimicrobial therapy. The organism was confirmed as Streptococcus gallolyticus subsp. macedonicus, a member of the 'S. equinus/S. bovis' complex. To date, only one report has been made of human infection due to this organism. This may be due to the limited resolution of the routine diagnostic methods used and/or as a consequence of the complex nomenclature associated with this group of organisms.

Detec����o do v��rus do papiloma humano por PCR em tempo real : relev��ncia para medicina legal

Tese de mestrado, Medicina Legal e Ci��ncias Forenses, Faculdade de Medicina, Universidade de Lisboa, 2014

Detection of MegaTAL-induced HIV pol Mutation By Droplet Digital PCR

Thesis (Master's)--University of Washington, 2015

Accessing Planktothrix species diversity and associated toxins using quantitative real-time PCR in natural waters

Tese de Doutoramento em Biologia apresentada �� Faculdade de Ci��ncias da Universidade do Porto, 2015.

Development of triplet repeat primed PCR (TP-PCR) technique as a support of molecular test in Machado-Joseph disease

Disserta����o de Mestrado, Ci��ncias Biom��dicas, 3 de Fevereiro de 2016, Universidade dos A��ores.

Caracteriza����o de m��ltiplos polimorfismos de Pneumocystis Jirovecii por multiplex-PCR/Single base extension

Pneumocystis jirovecii �� conhecido por causar infec����es espec��ficas no aparelho respirat��rio de seus hospedeiros, principalmente em doentes imunocomprometidos, manifestando-se por uma pneumonia grave e por vezes fatal, normalmente designada por pneumonia por Pneumocystis. A caracteriza����o da diversidade gen��tica de P. jirovecii tem demonstrado que determinados polimorfismos de base ��nica poder��o ser reconhecidos como marcadores moleculares de elei����o para o estudo da distribui����o geogr��fica, vias de transmiss��o, resist��ncia/susceptibilidade a f��rmacos, factores de virul��ncia e gen��tica populacional de subtipos gen��ticos. Este estudo teve como objectivo a caracteriza����o de polimorfismos de P. jirovecii, atrav��s da metodologia PCR multiplex/Extens��o de base ��nica (do ingl��s single base extension), com a principal finalidade de constatar eventuais associa����es entre polimorfismos de base ��nica, gen��tipos multilocus, e dados cl��nicos e demogr��ficos da infec����o. Sessenta e seis esp��cimes pulmonares, previamente considerados positivos para P. jirovecii, obtidos entre 2001 e 2012, a partir de doentes portugueses imunocomprometidos, foram seleccionados de forma aleat��ria para este estudo multilocus. PCR multiplex foi utilizada para a amplifica����o simult��nea de tr��s regi��es gen��micas: subunidade grande do rRNA mitocondrial, super��xido dismutase e dihidropteroato sintetase. Cinco polimorfismos de base ��nica, previamente correlacionados com par��metros da doen��a, foram genotipados por extens��o de base ��nica: mt85, SOD110, SOD215, DHPS165 e DHPS171. Um total de 330 polimorfismos de base ��nica e 29 gen��tipos multilocus putativos de P. jirovecii foram identificados e caracterizados nos esp��cimes pulmonares analisados. Os padr��es de distribui����o dos polimorfismos foram analisados, sendo considerada a varia����o temporal e/ou geogr��fica das suas formas al��licas. Constatou-se grande diversidade genot��pica entre os isolados de P. jirovecii que poder�� ter influ��ncia a n��vel epidemiol��gico. Foram observadas associa����es estat��sticas entre mt85/gen��tipos multilocus e par��metros demogr��ficos e cl��nicos. A correla����o mais importante verificou-se entre mt85C e cargas parasit��rias baixas a moderadas, enquanto mt85T foi associado com cargas parasit��rias altas; MLG5, MLG9 e MLG13 foram associados com cargas parasit��rias baixas, moderadas e altas, respectivamente. Tais associa����es demonstram que potenciais marcadores moleculares da infec����o por P. jirovecii poder��o existir e que polimorfismos/gen��tipos espec��ficos poder��o determinar perfis epidemiol��gicos da pneumonia por Pneumocystis. A an��lise gen��tica cruzada permitiu verificar associa����es entre polimorfismos de base ��nica. Os polimorfismos SOD110T e SOD215C, SOD110C e SOD215T, DHPS165A e DHPS171C, DHPS165G e DHPS171T foram associados estatisticamente. Os gen��tipos multilocus mais prevalentes foram considerados para o teste recombinat��rio d1. Dois gen��tipos multilocus (MLG7 e MLG9) foram observados com elevada frequ��ncia, e a an��lise gen��tica indicou que estes se encontravam sobre-representados na popula����o de P. jirovecii estudada. Estas evid��ncias indicam que o fen��meno de desequil��brio de liga����o e a propaga����o clonal de subtipos gen��ticos �� frequente, considerando que a esp��cie P. jirovecii poder�� ser representada por uma popula����o com estrutura epid��mica. O presente trabalho confirmou a import��ncia do estudo de polimorfismos em P. jirovecii, sugerindo que a caracteriza����o multilocus poder�� fornecer informa����o relevante para a compreens��o dos padr��es, causas e controlo da infec����o, melhorando assim a investiga����o deste importante patog��neo.

Molecular tools in the diagnostic and epidemiology of infections caused by members of Mycobacterium avium Complex

Mycobacterium avium Complex (MAC) comprises microorganisms that affect a wide range of animals including humans. The most relevant are Mycobacterium avium subspecies hominissuis (Mah) with a high impact on public health affecting mainly immunocompromised individuals and Mycobacterium avium subspecies paratuberculosis (Map) causing paratuberculosis in animals with a high economic impact worldwide. In this work, we characterized 28 human and 67 porcine Mah isolates and evaluated the relationship among them by Multiple-Locus Variable number tandem repeat Analysis (MLVA). We concluded that Mah population presented a high genetic diversity and no correlations were inferred based on geographical origin, host or biological sample. For the first time in Portugal Map strains, from asymptomatic bovine faecal samples were isolated highlighting the need of more reliable and rapid diagnostic methods for Map direct detection. Therefore, we developed an IS900 nested real time PCR with high sensitivity and specificity associated with optimized DNA extraction methodologies for faecal and milk samples. We detected 83% of 155 faecal samples from goats, cattle and sheep, and 26% of 98 milk samples from cattle, positive for Map IS900 nested real time PCR. A novel SNPs (single nucleotide polymorphisms) assay to Map characterization based on a Whole Genome Sequencing analysis was developed to elucidate the genetic relationship between strains. Based on sequential detection of 14 SNPs and on a decision tree we were able to differentiate 14 phylogenetic groups with a higher discriminatory power compared to other typing methods. A pigmented Map strain was isolated and characterized evidencing for the first time to our knowledge the existence of pigmented Type C strains. With this work, we intended to improve the ante mortem direct molecular detection of Map, to conscientiously aware for the existence of Map animal infections widespread in Portugal and to contribute to the improvement of Map and Mah epidemiological studies.