dual detection

双重检测
  • 文章类型: Journal Article
    由于它们在室内和室外空气污染中的主要作用,即使在痕量水平,挥发性有机化合物引起了人们的极大兴趣,他们的监测需要灵敏的分析仪器。通常使用几种技术,如便携式传感器,质子转移反应质谱(PTR-MS)和热脱附气相色谱法(TD-GC)。后者与火焰电离检测器(FID)或质谱仪(MS)一起广泛使用。鉴于每个色谱图检测到大量分子,这些监测技术产生的数据通常会手动检查和重新处理。这个过程非常耗时并且可能导致人为错误。挑战是尽快提供可靠的结果。在这项研究中,测试了具有双检测FID和MS的在线TD-GC系统的性能。方法检测极限(MDL),60种VOC(烷烃,芳烃,对于FID和MS检测器,都计算了氧化和卤化)。MDL和准确度范围从0.006到0.618ppbv,FID从77%到100%,从0.018到0.760ppbv,从80%到100%。两种检测器均显示出良好的互补性,并允许开发两个程序以促进数据分析。这些算法旨在自主选择FID和MS检测器之间的最佳结果,并对室外和室内测量条件进行了评估。在野外活动中测量挥发性有机化合物是具有挑战性的,并且预计这些程序可以扩展到其他类型的双检测器系统或用于比较来自不同校准仪器的数据。
    Because of their major role in indoor and outdoor air pollution, even at trace levels, VOCs are of great interest, and their monitoring requires sensitive analytical instruments. Several techniques are commonly used, such as portable sensors, Proton Transfer Reaction Mass Spectrometry (PTR-MS) and Thermal Desorption Gas Chromatography (TD-GC). The latter is widely used off- and on-line with Flame Ionization Detectors (FID) or Mass Spectrometers (MS). Given the large number of molecules detected per chromatogram, the data generated by these monitoring techniques are usually checked and reprocessed manually. This process is extremely time consuming and could result in human error. The challenge is to provide reliable results as quickly as possible. In this study, the performances of an on-line TD-GC system with dual detection FID and MS were tested. The Method Detection Limits (MDL), linearities and accuracies of 60 VOCs (alkanes, aromatics, oxygenated and halogenated) were calculated both for FID and MS detectors. The MDLs and accuracies ranged from 0.006 to 0.618 ppbv and from 77 % to 100 % for FID, and from 0.018 to 0.760 ppbv and from 80 % to 100 % for MS. Both detectors showed good complementarity and allowed the development of two programs to facilitate data analysis. These algorithms were designed to autonomously select optimal results between FID and MS detectors, and were evaluated for outdoor and indoor measurement conditions. Measuring VOCs in field campaigns is challenging, and it is anticipated that these programs could be extended to other types of dual-detector systems or for the comparison of data from different calibrated instruments.
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  • 文章类型: Journal Article
    人类活动是环境污染物连续和逐渐开槽的原因,一些释放的有毒和危险化合物在自然条件下不能降解,造成了严重的安全问题。其中包括广泛存在的水溶性全氟烷基和多氟烷基物质(PFAS),有时被称为“永远的化学品”,因为它们不可能自然降解。因此,一个可靠的,表现力,并且应该开发简单的方法来监测和消除与这些化合物相关的风险。在这项研究中,我们提出了一个简单的,快递,和便携式检测方法的水溶性氟烷基化合物(PFOA和GenX)使用互补的方法:电化学阻抗谱(EIS)和表面增强拉曼光谱(SERS)。要实现我们的方法,我们开发了基于多孔硅的特殊衬底,该衬底具有顶部沉积的等离子体激元活性Au层,随后接枝-C6H4NH2化学部分以提供对带负电荷的水溶性PFAS的表面亲和力。随后的EIS利用使我们能够对浓度高达10-10M的PFOA和GenX进行半定量检测,因为PFAS的表面截留会导致电极-电解质电荷转移电阻的显着增加。然而,通过EIS区分是否连PFAS都被截留是不可能的,因此,随后对基板进行SERS测量(由于多孔Au层的存在,表面等离子体活性允许),清楚地表明特征C-F振动带的外观。
    Human activity is the cause of the continuous and gradual grooving of environmental contaminants, where some released toxic and dangerous compounds cannot be degraded under natural conditions, resulting in a serious safety issue. Among them are the widely occurring water-soluble perfluoroalkyl and polyfluoroalkyl substances (PFAS), sometimes called \"forever chemicals\" because of the impossibility of their natural degradation. Hence, a reliable, expressive, and simple method should be developed to monitor and eliminate the risks associated with these compounds. In this study, we propose a simple, express, and portable detection method for water-soluble fluoro-alkyl compounds (PFOA and GenX) using mutually complementary methods: electrochemical impedance spectroscopy (EIS) and surface-enhanced Raman spectroscopy (SERS). To implement our method, we developed special substrates based on porous silicon with a top-deposited plasmon-active Au layer by subsequently grafting -C6H4-NH2 chemical moieties to provide surface affinity toward negatively charged water-soluble PFAS. Subsequent EIS utilization allows us to perform semiquantitative detection of PFOA and GenX up to 10-10 M concentration because surface entrapping of PFAS leads to a significant increase in the electrode-electrolyte charge-transfer resistance. However, distinguishing by EIS whether even PFAS were entrapped was impossible, and thus the substrates were subsequently subjected to SERS measurements (allowed by surface plasmon activity due to the presence of a porous Au layer), clearly indicating the appearance of characteristic C-F vibration bands.
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  • 文章类型: Journal Article
    已经开发了第四代HIV免疫测定,以减少血清转换期间的检测窗口期,允许检测早期和已建立的感染。这项工作的目的是评估一种新开发的检测方法,在新型高通量DxI9000Access免疫测定分析仪上访问HIVAg/Ab组合(BeckmanCoulter,Inc.).该测定允许同时定性检测和区分HIV-1p24抗原和HIV-1/2抗体。将测定性能与两个金标准测定进行比较,雅培建筑师HIVAg/AbCombo和RocheElecsysHIVDuo,并在多中心研究中进行评估,使用广泛的样本组(n>9000,临床样本和病毒裂解物)代表抗体和抗原的遗传多样性,感染的早期阶段,负,和交叉反应样本。临床样品以及病毒裂解物的临床敏感性为100%。关于病毒裂解物的数据和血清转换面板上的早期检测显示了使用Access测定的更好结果。分析灵敏度显示p24检测的极限确定为约0.2IU/mL。总体特异性为99.91%,并且使用潜在的交叉反应样品没有发现干扰。总之,AccessHIVAg/Ab组合测定证明了其在DxI9000分析仪上准确诊断慢性和原发性HIV感染的能力,尽管这些病毒的遗传多样性很高。
    Fourth-generation HIV immunoassays have been developed to reduce the window period of detection during seroconversion period, allowing for the detection of early and established infections. The aim of this work was to evaluate a newly developed assay, Access HIV Ag/Ab combo on the novel high throughput DxI 9000 Access Immunoassay Analyzer (Beckman Coulter, Inc.). The assay allows for simultaneous qualitative detection and differentiation of HIV-1 p24 antigen and HIV-1/2 antibodies. Assay performance was compared to two gold standard assays, the Abbott Architect HIV Ag/Ab Combo and Roche Elecsys HIV Duo, and assessed in a multicenter study, using a wide panel of samples (n > 9000, clinical samples and viral lysates) representative of genetic diversity for both antibodies and antigens, early phases of infection, negative, and cross-reacting samples. The clinical sensitivity was 100 % for clinical samples as well as for viral lysates. Data on viral lysates and early detection on seroconversion panels showed a better result with the Access assay. Analytical sensitivity showed a limit of p24 detection determined around 0.2 IU/mL. The overall specificity was 99.91 %, and no interference was found using the potentially cross-reactive samples. In conclusion, the Access HIV Ag/Ab combo assay demonstrated its ability for accurate diagnosis of chronic as well as primary HIV infections on the DxI 9000 Analyzer, despite the high level of genetic diversity of these viruses.
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  • 文章类型: Journal Article
    水凝胶,作为柔性材料,在柔性传感器领域得到了广泛的应用。人体汗液中含有多种能反映人体生理状态的生物标志物。因此,通过水凝胶实现对汗液成分的检测并与人体运动传感相结合具有重要的现实意义和应用价值。基于贻贝启发的化学,将聚多巴胺(PDA)和金纳米粒子(AuNPs)涂覆在纤维素纳米晶体(CNCs)表面,得到CNC基纳米复合材料(CNCs@PDA-Au),这可以同时增强机械,电化学,和水凝胶的自修复性能。通过冻融循环将CNCs@PDA-Au与聚乙烯醇(PVA)水凝胶复合,得到纳米复合水凝胶(PVA/CNCs@PDA-Au)。PVA/CNCs@PDA-Au具有优异的机械强度(7.2MPa)和自修复性能(88.3%)。采用PVA/CNCs@PDA-Au设计的运动传感器具有快速的响应时间(122.9ms),宽应变传感范围(0-600.0%),出色的稳定性,和抗疲劳性。尿酸独特的电化学氧化还原特性,所设计的水凝胶传感器成功实现了对汗液中尿酸的检测,检测范围宽(1.0~100.0μmol/L),检测限低(0.42μmol/L)。在这项研究中,所设计的PVA/CNCs@PDA-Au纳米复合水凝胶成功实现了人体运动和汗液中尿酸的双重检测。
    Hydrogels, as flexible materials, have been widely used in the field of flexible sensors. Human sweat contains a variety of biomarkers that can reflect the physiological state of the human body. Therefore, it is of great practical significance and application value to realize the detection of sweat composition and combine it with human motion sensing through a hydrogel. Based on mussel-inspired chemistry, polydopamine (PDA) and gold nanoparticles (AuNPs) were coated on the surface of cellulose nanocrystals (CNCs) to obtain CNC-based nanocomposites (CNCs@PDA-Au), which could simultaneously enhance the mechanical, electrochemical, and self-healing properties of hydrogels. The CNCs@PDA-Au was composited with poly(vinyl alcohol) (PVA) hydrogel to obtain the nanocomposite hydrogel (PVA/CNCs@PDA-Au) by freeze-thaw cycles. The PVA/CNCs@PDA-Au has excellent mechanical strength (7.2 MPa) and self-healing properties (88.3%). The motion sensors designed with PVA/CNCs@PDA-Au exhibited a fast response time (122.9 ms), wide strain sensing range (0-600.0%), excellent stability, and fatigue resistance. With the unique electrochemical redox properties of uric acid, the designed hydrogel sensor successfully realized the detection of uric acid in sweat with a wide detection range (1.0-100.0 μmol/L) and low detection limit (0.42 μmol/L). In this study, the dual detection of human motion and uric acid in sweat was successfully realized by the designed PVA/CNCs@PDA-Au nanocomposite hydrogel.
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  • 文章类型: Journal Article
    全球范围内,乙型肝炎病毒(HBV)影响超过250万人,而丙型肝炎病毒(HCV)影响大约7000万人,构成重大公共卫生挑战。尽管有疫苗和治疗方法,缺乏全面的诊断覆盖导致许多病例未得到诊断和治疗。为了解决对敏感的需求,具体,和可访问的诊断,这项研究引入了一种多重环介导等温扩增测定法,该测定法具有侧流检测,可同时进行HBV和HCV检测。该测定实现了卓越的灵敏度,并能够在25分钟内同时在单管和单条上检测HBV和HCV,实现所需的临床敏感性(10和103基因组拷贝/反应为HBV和HCV,分别)。该方法在各种病毒基因型的临床样本中得到了验证,实现等效的检测限。此外,一种定制的便携式加热装置被开发用于现场使用。这里开发的检测方法,能够在带上直接检测病毒,显示了替代仅鉴定抗体并需要额外的qPCR进行病毒活性评估的当前方法的希望。这种经济、快速的检测方法符合即时检测需求,在资源有限的环境中增强病毒性肝炎诊断方面提供了重大进展。
    Globally, hepatitis B virus (HBV) affects over 250 million people, whereas hepatitis C virus (HCV) affects approximately 70 million people, posing major public health challenges. Despite the availability of vaccines and treatments, a lack of comprehensive diagnostic coverage has left many cases undiagnosed and untreated. To address the need for sensitive, specific, and accessible diagnostics, this study introduced a multiplex loop-mediated isothermal amplification assay with lateral flow detection for simultaneous HBV and HCV testing. This assay achieved exceptional sensitivity and was capable of detecting HBV and HCV concurrently in a single tube and on a single strip within 25 min, achieving the required clinical sensitivity (10 and 103 genomic copies/reaction for HBV and HCV, respectively). The method was validated in clinical samples of various viral genotypes, achieving an equivalent limit of detection. Additionally, a custom portable heating device was developed for field use. The assay developed here, capable of direct viral detection on the strip, shows promise in supplanting current methods that solely identify antibodies and necessitate additional qPCR for viral activity assessment. This economical and rapid assay aligns with point-of-care testing needs, offering significant advancements in enhancing viral hepatitis diagnostics in settings with limited resources.
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  • 文章类型: Journal Article
    作为一种重要的社会性昆虫,蜜蜂在农业生产中起着至关重要的作用,农业生产的可持续发展,和自然环境的平衡。然而,近年来,以色列急性麻痹病毒(IAPV)和慢性蜜蜂麻痹病毒(CBPV),蜜蜂麻痹的主要病原体,不断危害蜂群,给养蜂业造成一定损失。一些养蜂场位于野生或偏远山区,这些农场的样品不能及时送到实验室进行检测,从而限制了疾病的准确和快速诊断。
    在这项研究中,我们使用逆转录-重组酶聚合酶扩增-侧流试纸(RT-RPA-LFD)方法双重检测IAPV和CBPV.针对其保守基因分别设计了RPA引物和LFD检测探针。引物和探针进行筛选,以及正向和反向引物比率,反应时间,和温度进行了优化。根据优化试验的结果,RT-RPA的最佳反应温度为37°C,当与LFD结合时,用肉眼检测需要<20分钟。开发的RPA-LFD方法专门针对IAPV和CBPV,与其他常见的蜜蜂病毒没有交叉反应性。此外,RT-RPA-LFD方法的最低检测限为101拷贝/μL。
    基于这项研究,该方法适用于临床样品的检测,可用于IAPV和CBPV的现场检测。
    UNASSIGNED: As an important social insect, honey bees play crucial roles in agricultural production, sustainable development of agricultural production, and the balance of the natural environment. However, in recent years, Israeli acute paralysis virus (IAPV) and chronic bee paralysis virus (CBPV), the main pathogens of bee paralysis, have continuously harmed bee colonies and caused certain losses to the beekeeping industry. Some beekeeping farms are located in wild or remote mountainous areas, and samples from these farms cannot be sent to the laboratory for testing in a timely manner, thereby limiting the accurate and rapid diagnosis of the disease.
    UNASSIGNED: In this study, we used a reverse transcription-recombinase polymerase amplification-lateral flow dipstick (RT-RPA-LFD) method for the dual detection of IAPV and CBPV. RPA primers and LFD detection probes were designed separately for their conserved genes. Primers and probes were screened, and the forward and reverse primer ratios, reaction times, and temperatures were optimized. According to the results of the optimization tests, the optimal reaction temperature for RT-RPA is 37°C, and when combined with LFD, detection with the naked eye requires <20 min. The developed RPA-LFD method specifically targets IAPV and CBPV and has no cross-reactivity with other common bee viruses. In addition, the minimum detection limit of the RT-RPA-LFD method is 101 copies/μL.
    UNASSIGNED: Based this study, this method is suitable for the detection of clinical samples and can be used for field detection of IAPV and CBPV.
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  • 文章类型: Journal Article
    糖尿病与冠心病(CHD)发展倾向增加之间的病理生理联系已确立。现有证据证实,人体中低浓度的高敏C反应蛋白(hs-CRP)的少量增加可以决定冠心病的发展趋势。此外,糖化血红蛋白(HbA1c)是一种公认的评估糖尿病进展的生物标志物.鉴于糖尿病和冠心病之间的正相关,这项研究提出了一种前所未有的无标记电化学方法,用于检测总Hb和hs-CRP的%HbA1c,促进早期CHD预测和具有成本效益的即时诊断。此外,一种新型的氧化还原探针O-(4-硝基苯磷酰基)胆碱(C11H17N2O6P)用于CRP的电化学检测,以前在科学文献中没有记载的方法。校准曲线表明,对于0-30mg/mL的HbA1c的线性范围,在PBS(pH8)中的检测限(LOD)为5mg/mL,在模拟血液(SB)中为6mg/mL。同时,对于在PBS(pH7.4)和SB中测量的0.007mg/mL和0.008mg/mL的LOD报告为0-0.05mg/mL的CRP的线性范围。提出的电化学系统可以准确量化混合样品中的HbA1c和CRP,对复杂生物样品具有合理的特异性和实际适用性。
    The pathophysiological link between diabetes and heightened propensity for the development of coronary heart disease (CHD) is well-established. Prevailing evidence confirms that small increases in low concentrations of high-sensitivity C reactive protein (hs-CRP) in the human body can determine the tendency of developing CHD. Additionally, glycated hemoglobin (HbA1c) is a well-recognized biomarker to evaluate diabetes progression. Given the positive correlation between diabetes and CHD, this research presents a notably unprecedented label-free electrochemical approach for the dual detection of %HbA1c regarding Total Hb and hs-CRP, facilitating early CHD prediction and cost-effective point-of-care diagnostics. Furthermore, a novel redox probe O-(4-Nitrophenylphosphoryl)choline (C11H17N2O6P) was used for the electrochemical detection of CRP, a method not documented in scientific literature before. The calibration curves demonstrate a limit of detection (LOD) of 5 mg/mL in PBS (pH 8) and 6 mg/mL in simulated blood (SB) for a linear range of 0-30 mg/mL of HbA1c. Conjointly, a LOD of 0.007 mg/mL and 0.008 mg/mL for measurement in PBS (pH 7.4) and SB are reported for a linear range of 0-0.05 mg/mL of CRP. The electrochemical systems presented could accurately quantify HbA1c and CRP in mixed samples, demonstrating reasonable specificity and practical applicability for complex biological samples.
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  • 文章类型: Journal Article
    提高准确性,细胞内miRNA成像的灵敏度和速度对于癌症的早期诊断至关重要。为了实现这一目标,我们在此提出了一种通过基于DNA四面体的催化发夹组装(DCHA)对两种不同miRNA进行成像的策略。两个纳米探针,DTH-13和DTH-24是通过一锅法合成制备的。所得结构是用两组CHA发夹功能化的DNA四面体,分别响应miR-21和miR-155。使用这些结构化的DNA纳米颗粒作为载体,探针可以很容易地进入活细胞。miR-21或miR-155的存在可以触发DTH-13和DTH-24之间的CHA,导致FAM和Cy3的独立荧光信号。在这个系统中,由于DCHA的策略,敏感性和动力学显著增强.我们方法的传感性能在缓冲器中进行了彻底的研究,胎牛血清(FBS)溶液,活细胞,和临床组织样本.结果验证了DTH纳米探针作为癌症早期诊断工具的潜力。
    Improving the accuracy, sensitivity and speed of intracellular miRNA imaging is essential for early diagnosis of cancer. To achieve this goal, we herein present a strategy for imaging two distinct miRNAs by DNA tetrahedron-based catalytic hairpin assembly (DCHA). Two nanoprobes, DTH-13 and DTH-24, were prepared by one-pot synthesis. The resultant structures were DNA tetrahedrons functionalized with two sets of CHA hairpins, which respectively responded to miR-21 and miR-155. Using these structured DNA nanoparticles as the carriers, the probes could easily enter living cells. The presence of miR-21 or miR-155 could trigger CHA between DTH-13 and DTH-24, leading to independent fluorescence signals of FAM and Cy3. In this system, the sensitivity and kinetics were significantly enhanced owing to the strategy of DCHA. The sensing performance of our method was thoroughly investigated in buffers, fetal bovine serum (FBS) solutions, living cells, and clinical tissue samples. The results validated the potential of DTH nanoprobes as a diagnostic tool for early stages of cancer.
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  • 文章类型: Journal Article
    猫杯状病毒(FCV)和猫疱疹病毒I型(FHV-1)是引起猫呼吸道疾病的最常见的病毒病原体,经常发现这两种病原体合并感染。在兽医诊所,FCV和FHV-1的主要诊断方法是试纸条和聚合酶链反应(PCR)。然而,试纸的灵敏度不够,PCR是耗时的。因此,开发快速和高性能的临床诊断测试对于预防和治疗这些疾病至关重要。酶促重组酶扩增(ERA)是一种自动等温核酸扩增技术,可保持恒定的温度,既快速又高度准确。在这项研究中,使用Exo探针开发了用于FCV和FHV-1的差异检测的双重ERA方法。这种双重ERA方法表现出高性能,两种病毒的检测限均为101个拷贝,与猫细小病毒和F81细胞没有交叉反应。为了测试该方法在临床应用中的实用性,收集并测试来自具有呼吸道症状的猫的50个鼻咽拭子。FCV和FHV-1阳性率为40%(20/50,95%置信区间[CI],26.4至54.8%)和14%(7/50,95%CI,5.8至26.7%),分别。FCV和FHV-1的合并感染率为10%(5/50,95%CI,3.3至21.8%)。这些结果与使用定量实时PCR发现的结果一致。因此,这种双重ERA方法是用于FCV和FHV-1检测的新型有效的临床诊断工具。
    Feline calicivirus (FCV) and feline herpesvirus type I (FHV-1) are the most common viral pathogens responsible for cat respiratory diseases, and coinfection with these two pathogens is often found. In veterinary clinics, the main diagnostic methods for FCV and FHV-1 are test strips and polymerase chain reaction (PCR). However, the sensitivity of test strips are not sufficient, and PCR is time-consuming. Therefore, developing a rapid and high-performance clinical diagnostic test is imperative for the prevention and treatment of these diseases. Enzymatic recombinase amplification (ERA) is an automated isothermal nucleic acid amplification technique that maintains a constant temperature, and is both rapid and highly accurate. In this study, a dual ERA method was developed using the Exo probe for a differential detection of FCV and FHV-1. This dual ERA method demonstrated high performance with the detection limit of 101 copies for both viruses, and no cross-reactions with feline parvovirus virus and F81 cells. To test the utility of the method for clinical applications, 50 nasopharyngeal swabs from cats with respiratory symptoms were collected and tested. The positive rates of FCV and FHV-1 were 40% (20/50, 95% confidence interval [CI], 26.4 to 54.8%) and 14% (7/50, 95% CI, 5.8 to 26.7%), respectively. The rate of coinfection with FCV and FHV-1 was 10% (5/50, 95% CI, 3.3 to 21.8%). These results were in agreement with those found using quantitative real-time PCR. Therefore, this dual ERA method is a novel and efficient clinical diagnostic tool for FCV and FHV-1 detection.
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  • 文章类型: Journal Article
    鉴于受SARS-CoV-2污染的食物可能成为感染源,迫切需要开发一种快速、准确的食品中SARS-CoV-2核酸检测方法,以保证食品安全。这里,我们建议一个敏感的,具体,SARS-CoV-2的可靠分子检测方法。它具有控制扩增子污染的机制。来自加标冷冻虾的拭子被用作检测样品,通过在95°C下加热30s进行处理。这些预处理的样品用作后续扩增的模板。进行比色LAMP反应以在一个管中同时扩增SARS-CoV-2靶标和MS2噬菌体。通过比色LAMP检测MS2噬菌体作为内部对照,而SARS-CoV-2是用CRISPR/Cas12a系统检测到的。荧光结果可以用紫外灯目视检测。同时,在LAMP反应期间掺入尿嘧啶以提供扩增子污染证明机制。该测试可以在一个反应中检测到低至20个SARS-CoV-2拷贝。此外,检测可以在45分钟内完成。测试只需要一个加热块和一个紫外线灯,这显示了现场检测的潜力。
    Given the possibility that food contaminated with SARS-CoV-2 might become an infection source, there is an urgent need for us to develop a rapid and accurate nucleic acid detection method for SARS-CoV-2 in food to ensure food safety. Here, we propose a sensitive, specific, and reliable molecular detection method for SARS-CoV-2. It has a mechanism to control amplicon contamination. Swabs from spiked frozen shrimps were used as detection samples, which were processed by heating at 95 °C for 30 s. These preprocessed samples served as the templates for subsequent amplification. A colorimetric LAMP reaction was carried out to amplify both the SARS-CoV-2 target and the MS2 phage simultaneously in one tube. MS2 phage was detected by colorimetric LAMP as the internal control, while SARS-CoV-2 was detected with a CRISPR/Cas12a system. The fluorescence results could be visually detected with an ultraviolet lamp. Meanwhile, uracil was incorporated during the LAMP reaction to provide an amplicon contamination proof mechanism. This test could detect as low as 20 copies of SARS-CoV-2 in one reaction. Additionally, the detection could be finished in 45 min. The test only needs a heating block and an ultraviolet lamp, which shows the potential for field detection.
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