Integrated nucleic acid testing system to enable TB diagnosis in peripheral settings. Issue 21 (6th October 2020)

• Record Type: Journal Article
• Authors: Lu, Hsiang-Wei; Sakamuri, Rama; Kumar, Pranav; Ferguson, Tanya M.; Doebler, Robert W.; Herrington, Keith D.; Talbot, Ryan P.; Weigel, Kris M.; Nguyen, Felicia K.; Cangelosi, Gerard A.; Narita, Masahiro; Boyle, David S.; Niemz, Angelika
• Is Part Of: Lab on a chip. Volume 20:Issue 21(2020)
• Page Start: 4071
• Page End: 4081
• Publication Date: 2020-10-06
• Subjects: Miniature electronic equipment -- Periodicals; Combinatorial chemistry -- Periodicals; Biotechnology -- Periodicals; 543.0813
• DOI: 10.1039/d0lc00445f
• Languages: English
• ISSNs: 1473-0197

Abstract

Abstract : We developed a nucleic acid testing device that automates pathogen lysis, DNA extraction, isothermal DNA amplification and lateral flow detection. Abstract : 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 tuberculosis ( 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 × 10 3 colony forming units (CFU) per 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.