RESUMO
O objetivo deste comunicado é desenvolver um método quantitativo PCR em tempo real, baseado em guia molecular (MB) (MB-qPCR) para detecção de infecção por espécies de Brucella, e avaliar seu potencial de utilização clínica. Os primers e as sondas MB foram desenhados para amplificação específica e determinação de sequência conservada do código do gene para os primeiros 58-aa da proteína de membrana externa OMP-2a, que é compartilhada em cinco espécies de Brucella epidêmicas. A avaliação metodológica foi realizada por análise de sensibilidade, especificidade, coeficiente de variação intra e inter, e a linearidade do qPCR. O potencial diagnóstico foi avaliado comparando-se o método qPCR desenvolvido com ensaios de exames bacteriológicos convencionais, incluindo os testes de soroaglutinação convencionais (SATs) e os testes do Rosa Bengala (RBPTs). O método exibiu alta sensibilidade (tão baixo quanto 50 cópias) e grande faixa de linearidade (102-108 cópias). Nenhuma reação cruzada foi encontrada com bactéria clínica comum. A sensibilidade diagnóstica foi superior ao exame bacteriológico, e a especificidade diagnóstica foi superior ao SAT ou ao RBPT. Um método MB-qPCR altamente sensível e específico para DNA de Brucella foi estabelecido com sucesso, provando ser uma ferramenta útil no diagnóstico molecular de brucelose.(AU)
Assuntos
Brucella/isolamento & purificação , Genoma Bacteriano , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
O objetivo deste comunicado é desenvolver um método quantitativo PCR em tempo real, baseado em guia molecular (MB) (MB-qPCR) para detecção de infecção por espécies de Brucella, e avaliar seu potencial de utilização clínica. Os primers e as sondas MB foram desenhados para amplificação específica e determinação de sequência conservada do código do gene para os primeiros 58-aa da proteína de membrana externa OMP-2a, que é compartilhada em cinco espécies de Brucella epidêmicas. A avaliação metodológica foi realizada por análise de sensibilidade, especificidade, coeficiente de variação intra e inter, e a linearidade do qPCR. O potencial diagnóstico foi avaliado comparando-se o método qPCR desenvolvido com ensaios de exames bacteriológicos convencionais, incluindo os testes de soroaglutinação convencionais (SATs) e os testes do Rosa Bengala (RBPTs). O método exibiu alta sensibilidade (tão baixo quanto 50 cópias) e grande faixa de linearidade (102-108 cópias). Nenhuma reação cruzada foi encontrada com bactéria clínica comum. A sensibilidade diagnóstica foi superior ao exame bacteriológico, e a especificidade diagnóstica foi superior ao SAT ou ao RBPT. Um método MB-qPCR altamente sensível e específico para DNA de Brucella foi estabelecido com sucesso, provando ser uma ferramenta útil no diagnóstico molecular de brucelose.(AU)
Assuntos
Brucella/isolamento & purificação , Genoma Bacteriano , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
The Internet of Things (IoT) involves a wide variety of heterogeneous technologies and resource-constrained devices that interact with each other. Due to such constraints, IoT devices usually require lightweight protocols that optimize the use of resources and energy consumption. Among the different commercial IoT devices, Bluetooth and Bluetooth Low Energy (BLE)-based beacons, which broadcast periodically certain data packets to notify their presence, have experienced a remarkable growth, specially due to their application in indoor positioning systems. This article proposes a family of protocols named Lightweight Protocol for Sensors (LP4S) that provides fast responses and enables plug-and-play mechanisms that allow IoT telemetry systems to discover new nodes and to describe and auto-register the sensors and actuators connected to a beacon. Thus, three protocols are defined depending on the beacon hardware characteristics: LP4S-6 (for resource-constraint beacons), LP4S-X (for more powerful beacons) and LP4S-J (for beacons able to run complex firmware). In order to demonstrate the capabilities of the designed protocols, the most restrictive (LP4S-6) is tested after implementing it for a telemetry application in a beacon based on Eddystone (Google's open beacon format). Thus, the beacon specification is extended in order to increase its ability to manage unlimited sensors in a telemetry system without interfering in its normal operation with Eddystone frames. The performed experiments show the feasibility of the proposed solution and its superiority, in terms of latency and energy consumption, with respect to approaches based on Generic Attribute Profile (GATT) when multiple users connect to a mote or in scenarios where latency is not a restriction, but where low-energy consumption is essential.
RESUMO
MicroRNAs (miRNAs) family members are usually different from each other in one-base variation. The high sequence homology poses a challenge for miRNA analysis with single-base selectivity. On the basis of G-quadruplex molecular beacons (G4MB) and duplex-specific nuclease (DSN), we developed a simple and highly selective amplification biosensor for miRNA detection. G4MB with a G4 motif stem is used as recognition probe. In the present of target miRNAs, G4MB hybridizes with target miRNA perfectly and forms a G4MB-miRNA duplex. Then, DSN subsequently cleaves the G4MB of the G4MB-miRNA duplex to recycle the target miRNA, which leads to fluorescence signal amplification. In the absence of target miRNAs, DSN can not digest the stem of G4MB because of the protection of G4 motif, which eliminates the false positive signal, and produces low fluorescence background. Importantly, the powerful discriminating abilities of both G4MB and DSN make the novel sensor suitable for miRNAs detection with high single-base selectivity. Comparing with traditional linear ssDNA probe-DSN-based method, the signal response of similar miRNA sequences with one-base difference has been reduced from 24% to 6% by using this G4MB-DSN-based method. Moreover, this simple sensor also exhibits a good applicability in cancer cell samples and a multiplex capability in one sample with different miRNA targets, making it a promising strategy for clinical diagnostics.
Assuntos
Técnicas Biossensoriais/métodos , Quadruplex G , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Linhagem Celular Tumoral , Sondas de DNA/química , Sondas de DNA/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Endonucleases/metabolismo , Células HeLa , HumanosRESUMO
A novel approach for highly sensitive and selective genotyping of single-nucleotide polymorphism (SNP) has been developed based on ligation-rolling circle amplification (L-RCA) and stemless molecular beacon. In this approach, two tailored DNA probes were involved. The stemless molecular beacon, formed through the inclusion interactions of γ-cyclodextrin (γ-CD) and bis-pyrene labeled DNA fragment, was served as signal probe. In the absence of mutant target, the two pyrene molecules were bound in the γ-CD cavity to form an excimer and showed a strong fluorescence at 475 nm. It was here named γ-CD-P-MB. The padlock DNA probe was designed as recognition probe. Upon the recognition of a point mutation DNA targets, the padlock probe was ligated to generate a circular template. An RCA amplification was then initiated using the circular template in the presence of Phi29 polymerase and dNTPs. The L-RCA products, containing repetitive sequence units, subsequently hybridized with the γ-CD-P-MB. This made pyrene molecules away from γ-CD cavity and caused a decrease of excimer fluorescence. As a proof-of-concept, SNP typing of ß-thalassemia gene at position -28 was investigated using this approach. The detection limit of mutated target was determined to be 40 fM. In addition, DNA ligase offered high fidelity in distinguishing the mismatched bases at the ligation site, resulting in positive detection of mutant target even when the ratio of the wildtype to the mutant is 999:1. Given these attractive characteristics, the developed approach might provide a great genotyping platform for pathogenic diagnosis and genetic analysis.