RESUMO
OBJECTIVE: To detect Japanese encephalitis virus (JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction (RT-PCR) detection system was developed. METHODS: By aligning the full-length sequences of JEV (G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay. RESULTS: With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/µL. The coefficients of variation of this real-time RT-PCR were all < 2.8%. The amplification efficiency of this method was between 90% and 103%. CONCLUSION: A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.
Assuntos
Culicidae/virologia , Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Japonesa (Espécie)/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: The primary problem in simultaneous bilateral auricle reconstruction is the fragility of the reconstructed ear structure. Postoperative pressure is strictly prohibited to ensure the operation's effectiveness. The study aimed to summarize the experience of perioperative postural management in simultaneous bilateral auricular reconstruction. METHOD: This study summarizes the experience of perioperative postural management, providing preoperative sleeping posture adaptability training, neck movement training, standardization of the head position angles and the head suspension time in surgery, using protective headrests, paying attention to the transfer and handover procedures, and using specially designed pillows. RESULTS: The comprehensive nursing approach in simultaneous bilateral auricular reconstruction significantly reduced complications, improved patient comfort, and optimized postoperative adaptation. Preoperative posture training, standardized intraoperative head positions, and vigilant postoperative care played pivotal roles, demonstrating positive outcomes in 46 cases. DISCUSSION: Perioperative position management can reduce the risk of complications and pressure injuries, improving patients' postoperative comfort, emotional state, tolerance, and adaptability. CONCLUSION: All ears were viable and in good shape after long-term follow-up. The experiences discussed in this study can be broadly applied to technically mature ear reconstruction teams.
Assuntos
Microtia Congênita , Pavilhão Auricular , Procedimentos de Cirurgia Plástica , Humanos , Procedimentos de Cirurgia Plástica/efeitos adversos , Orelha Externa/cirurgia , Cuidados Pós-Operatórios , Período Pós-Operatório , Pavilhão Auricular/cirurgia , Microtia Congênita/cirurgiaRESUMO
Bovine respiratory disease complex (BRDC) is a serious disease affecting feedlot cattle in China and likely other places worldwide. Bovine viral diarrhea virus (BVDV) and bovine parainfluenza virus type 3 (BPIV3) are principally responsible for causing BRDC, and are a major strain to the industrial economy. Eradication of these viruses/disease requires swift viral identification and treatment. Hence, this study established a fast and easy procedure of BVDV and BPIV3 identification that employs reverse transcription recombinase polymerase amplification (RT-RPA) and lateral flow dipstick (LFD), and uses primers and lateral flow (LF) probe targeting the 5'-UTR gene of BVDV and phosphoprotein P gene of BPIV3, respectively. Our assay was able to successfully amplify BVDV and BPIV3 RNA within 25 min at 35 °C using RT-RPA, with products visible on the LFD within 5 min at room temperature (RT). The lowest detection limits were 50 RNA molecules for BVDV and 34 RNA molecules for BPIV3 per reaction. We also demonstrated that the established dual RT-RPA LFD assay was precise and targeted, harboring excellent potential to become an onsite molecular diagnostic tool in the detection of BVDV and BPIV3. This method can detect BVDV (Pestivirus A, B) and BPIV3, and exhibit no cross-reaction with other viruses like the classical swine fever virus (CSFV) and infectious bovine rhinotracheitis virus (IBRV). The assay performance was further assessed with clinical samples, and demonstrated good performance in comparison to real-time RT-PCR (RT-qPCR). Moreover, the RT-RPA LFD assay was comparitively rapid and required minimal training.
Assuntos
Recombinases , Transcrição Reversa , Animais , Bovinos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Recombinases/genética , Sensibilidade e Especificidade , SuínosRESUMO
OBJECTIVE: Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis (TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease. METHODS: A reverse-transcription recombinase-aided amplification (RT-RAA) assay was developed. This assay can be completed in one closed tube at 39 °C within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type (WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay. RESULTS: The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units (pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay. CONCLUSION: A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.