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客戶采用PuriMag Si-OH磁珠提取猴痘病毒DNA在《Analytica Chimica Acta》發表論文

Rapid and highly sensitive colorimetric LAMP assay and integrated device for visual detection of monkeypox virus

Yadan Peng 1 #, Ruolan Hu 1 #, Shuang Xue 2, Yugan He 1, Lili Tian 1, Zehan Pang 1, Yile He 1, Yuqi Dong 1, Yinghan Shi 1, Shuqi Wang 1, Bixia Hong 1, Ke Liu 1, Ruixue Wang 2, Lihua Song 1, Huahao Fan 1 3, Mengzhe Li 1, Yigang Tong 1 4
1 College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China;
2 College of Mechanical and Electrical Engineering, Beijing University of Chemical Technology, Beijing 100029, China
3 School of Life Sciences, Tianjin University, Tianjin, 300072, China
4 Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing 100029, China

Analytica Chimica Acta
Available online 14 May 2024, 342720

https://doi.org/10.1016/j.aca.2024.342720

Highlights
? A integrated visual nucleic acid self-testing device based on LAMP assay.
? Efficient enrichment of nucleic acids using Si OH magnetic beads without elution.
? Visual results can be obtained within 1 h using the sample-to-answer device.
? The LOD for monkeypox virus nucleic acid is as low as 137 copies/mL.
? The LAMP-based self-testing device is suitable for point-of-care testing.

Abstract
Background
The monkeypox virus (MPXV) is a linear double-stranded DNA virus with a large genome that causes tens of thousands of infections and hundreds of deaths in at least 40 countries and regions worldwide. Therefore, timely and accurate diagnostic testing could be an important measure to prevent the ongoing spread of MPXV and widespread epidemics.

Results
Here, we designed multiple sets of primers for the target region of MPXV for loop-mediated isothermal amplification (LAMP) detection and identified the optimal primer set. Then, the specificity in fluorescent LAMP detection was verified using the plasmids containing the target gene, pseudovirus and other DNA/RNA viruses. We also evaluated the sensitivity of the colorimetric LAMP detection system using the plasmid and pseudovirus samples, respectively. Besides, we used monkeypox pseudovirus to simulate real samples for detection. Subsequent to the establishment and introduction of a magnetic beads (MBs)-based nucleic acid extraction technique, an integrated device was developed, characterized by rapidity, high sensitivity, and remarkable specificity. This portable system demonstrated a visual detection limit of 137 copies/mL, achieving sample-to-answer detection within 1 h.

Significance
The device has the advantages of integration, simplicity, miniaturization, and visualization, which help promote the realization of accurate, rapid, portable, and low-cost testing. Meanwhile, this platform could facilitate efficient, cost-effective and easy-operable point-of-care testing (POCT) in diverse resource-limited settings in addition to the laboratory.

Graphical abstract

Fig. Schematic diagram of the integrated assay device. The integrated sample-to-answer device was fabricated with three modules, including nucleic acid extraction, LAMP reaction, and colorimetric readout.

背景
猴痘病毒（MPXV）是一種線性雙鏈DNA病毒，具有龐大的基因組，在全球至少40個國家和地區造成數萬例感染和數百人死亡。因此，及時準確的診斷檢測可能是防止MPXV持續傳播和廣泛流行的重要措施。

結果
在這里，我們為 MPXV 的目標區域設計了多組引物，用于環介導的等溫擴增 （LAMP） 檢測，并確定了最佳引物組。然后，使用含有靶基因、假病毒和其他 DNA/RNA 病毒的質粒驗證熒光 LAMP 檢測的特異性。我們還分別使用質粒和假病毒樣品評估了比色LAMP檢測系統的靈敏度。此外，我們使用猴痘假病毒模擬真實樣本進行檢測。在建立和引入基于磁珠（MBs）的核酸提取技術之后，開發了一種集成裝置，其特點是快速、高靈敏度和顯著的特異性。該便攜式系統展示了 137 拷貝/mL 的視覺檢測限，可在 1 小時內實現從樣品到答案的檢測。

意義
該器件具有集成化、簡單化、小型化、可視化等優點，有助于促進測試的準確、快速、便攜、低成本的實現。同時，除了實驗室之外，該平臺還可以在資源有限的各種環境中促進高效、經濟和易于操作的即時檢測（POCT）。

圖形摘要
無花果。集成檢測設備示意圖。該集成樣本到答案裝置由核酸提取、LAMP反應和比色讀出三個模塊組成。

2. Materials and methods
2.1. Materials and Reagents
The WarmStart? Colorimetric LAMP 2×Master Mix (DNA and RNA) and WarmStart? LAMP Kit (DNA and RNA) kits were purchased from New England Biolabs (New England, USA). Si-OH MBs (25 mg/mL) were manufactured by PuriMag Biotech Co., Ltd (Xiamen, China). Pseudovirus reference materials for MPXV mutant containing F3L gene (Cat No: NIM-RM4060) and B6R Gene (Cat No: NIM-RM 4059) were generated by the National Institute of Metrology (NIM, Beijing, China). A viral nucleic acid extraction kit was purchased from Nobelab Biotech Co., Ltd (Beijing, China). Nuclease-free water was purchased from Thermo Fisher Scientific (USA). Mineral oil was purchased from Sigma-Aldrich (St. Louis, MO, USA). EDTA-Na2, Tris, guanidine thiocyanate (GuSCN), and phosphate-buffered saline (1×PBS, 0.0067 M/pH7.4, without Calcium and Magnesium) were supplied by Solarbio Life Sciences (Beijing, China). Sodium hydroxide, sodium chloride, hydrochloric acid and anhydrous ethanol were obtained from Macklin Biochemicals (Shanghai, China). The lysis buffer contained 2.5 mM EDTA-Na2, 20 mM Tris, 4 M GuSCN and 2 M NaCl (pH6.5). Ultrapure water (18.2 MΩ×cm at 25°C) was generated by an ultrapure water system (Milli?-Q reference). The primers were designed by NEB? LAMP Primer Design Tool (https://lamp.neb.com/#!/), and plasmids containing B6R and F3L regions of MPXV were synthesized by RuiBo Biotech Co., Ltd (Beijing, China) (Table 1).

2.2. Integrated device design and fabrication
The integrated sample-to-answer device is fabricated with three main modules, including nucleic acid extraction, LAMP reaction, and colorimetric readout (Fig. 1A). The detachable module for nucleic acid extraction is disposable, and the heating module (Fig. 1A ) and the visualization result readout module (Fig. 1A ) are integrated into a single entity, capable of be recyclability. The device is compact to facilitate its application for outdoor mobile use or portable home testing.
The components and functions of these devices are shown in Fig. 1B. Firstly, pathogens were sampled from the skin surface using a swab, followed by agitation in lysis buffer. The resulting mixture containing MPXV DNA was transferred to the nucleic acid extraction module (Fig. 1B(a)). Secondly, the nucleic acid extraction module, comprising a magnetic lid and three distinct wells, was utilized. Three wells were responsible for viral lysis, washing and air-drying, respectively. The lysis well was prefilled with Si-OH MBs, and sealed with a tin foil sealing film to capture MPXV DNA. The washing well was preloaded with 1000 μL 80% ethanol (aq) to remove residues and inhibitors adsorbed to Si-OH MBs. The drying well was designed with multiple pores for fast air-drying. A customized magnetic lid with a hollow middle and sealed bottom, housing a small magnetic rod, was placed on lysis well () to concentrate Si-OH MBs adsorbed with nucleic acids to the bottom of the lid. The entire assembly was then transferred to the washing well (). Followed by the drying well () for are-drying of the Si-OH MBs. Thirdly, the Si-OH MBs with concentrated DNA were transferred to the LAMP reaction mixture and placed in the heating module. This reaction was conducted at 65°C for 40 min (Fig. 1B(c)). Finally, the results were observed by the naked eye in the colorimetric readout module (Fig. 1B(d)), comparing with the no template control (NTC) tube.

更多二氧化硅磁珠請參考 DNA提取硅羥基磁珠|RNA提取磁珠|核酸提取磁珠-生物磁珠專家 (purimagbead.com)