Integrated nucleic acid testing system to enable TB diagnosis in peripheral settings.
Integrated nucleic acid testing system to enable TB diagnosis in peripheral settings.
复制标题
DOI：
10.1039/d0lc00445f
复制
发表时间：
2020-10-27
期刊：
影响因子：
6.1
通讯作者：
Niemz A
中科院分区：
文献类型：
--
作者：
Lu HW;Sakamuri R;Kumar P;Ferguson TM;Doebler RW;Herrington KD;Talbot RP;Weigel KM;Nguyen FK;Cangelosi GA;Narita M;Boyle DS;Niemz A
为了促进治疗和限制结核病的传播，需要新的方法，以便能够在高负担、低资源的环境中快速和负担得起地诊断结核病。我们已经开发了一个原型集成核酸检测设备检测痰中结核分枝杆菌（M.tb）。该设备由一次性检测盒和紧凑、廉价的仪器组成，可自动进行病原体裂解、核酸提取、等温DNA扩增和侧向流检测。将液化和消毒的痰液样本手动注入检测盒中，所有其他步骤均自动化，在1.5小时内提供结果。细胞破碎和DNA提取在含有微型珠混合器（基于PureLyse®技术，克莱蒙BioSolutions LLC）的四端口主动阀内进行。将含DNA的引物与干燥的主混合物试剂组合，并等温扩增靶DNA。然后将放大的主混合物泵入侧向流动条室中进行检测。整个过程在一次性封闭系统检测盒中进行，以防止扩增子残留。对于结核分枝杆菌加标痰液的检测，系统提供的检测限为5*103菌落形成单位（CFU）/mL。所有阴性对照均未产生假阳性结果。相对于经验证的基于手动qPCR的比较方法，对来自TB病例和对照的45份临床痰液标本进行的检测显示，初步灵敏度为90%，特异性为96%。随着进一步的发展，本文所述的集成核酸测试装置可以在高TB负担国家的接近患者的低资源环境中在相同的临床遭遇中实现TB诊断和治疗开始。我们开发了一种核酸检测设备，可自动化病原体裂解，DNA提取，等温DNA扩增和侧流检测。
To facilitate treatment and limit transmission of tuberculosis (TB), new methods are needed to enable rapid and affordable diagnosis of the disease in high-burden low-resource settings. We have developed a prototype integrated nucleic acid testing device to detect Mycobacterium tuberculolsis (M.tb) in sputum. The device consists of a disposable cartridge and compact, inexpensive instrument that automates pathogen lysis, nucleic acid extraction, isothermal DNA amplification and lateral flow detection. A liquefied and disinfected sputum sample is manually injected into the cartridge, and all other steps are automated, with a result provided in <1.5 h. Cell disruption and DNA extraction is executed within a four-port active valve containing a miniature bead blender (based on PureLyse® technology, Claremont BioSolutions LLC). The DNA-containing eluate is combined with dry master-mix reagents and target DNA is isothermally amplified. Amplified master-mix is then pumped into a lateral flow strip chamber for detection. The entire process is performed in a single-use closed-system cartridge to prevent amplicon carryover. For testing of M.tb–spiked sputum the system provided a limit of detection of 5*103 colony forming units (CFU)/mL. None of the negative sputum-only controls yielded a false-positive result. Testing of 45 clinical sputum specimens from TB cases and controls relative to a validated manual qPCR-based comparator method revealed a preliminary sensitivity of 90% and specificity of 96%. With further development, the herein described integrated nucleic acid testing device can enable TB diagnosis and treatment initiation in the same clinical encounter in near-patient low-resource settings of high TB burden countries. We developed a nucleic acid testing device that automates pathogen lysis, DNA extraction, isothermal DNA amplification and lateral flow detection.
登录
查看更多内容
影响因子：
9.4
作者：
Luabeya, Angelique K.;Wood, Rachel C.;Cangelosi, Gerard A.
通讯作者：
Cangelosi, Gerard A.
影响因子：
4.6
作者：
Ferguson TM;Weigel KM;Lakey Becker A;Ontengco D;Narita M;Tolstorukov I;Doebler R;Cangelosi GA;Niemz A
通讯作者：
Niemz A
影响因子：
9.4
作者：
Blakemore, Robert;Story, Elizabeth;Alland, David
通讯作者：
Alland, David
DOI：
10.3390/bios8010017
发表时间：
2018-02-26
期刊：
Biosensors
影响因子：
--
作者：
Mauk MG;Song J;Liu C;Bau HH
通讯作者：
Bau HH
影响因子：
9.4
作者：
Iwamoto, T;Sonobe, T;Hayashi, K
通讯作者：
Hayashi, K