A modular paper-and-plastic device for tuberculosis nucleic acid amplification testing in limited-resource settings

Abstract

We present a prototype for conducting rapid, inexpensive and point-of-care-compatible nucleic acid amplification tests (NAATs) for tuberculosis (TB). The fluorescent isothermal paper-and-plastic NAAT (FLIPP-NAAT) uses paper-based loop mediated isothermal amplification (LAMP) for DNA detection. The cost of materials required to build a 12-test-zone device is $0.88 and the cost of reagents per reaction is $0.43. An inexpensive imaging platform enables filter-free fluorescence detection of amplified DNA using a cell-phone camera. FLIPP-NAAT can be operated by an untrained user and only requires a regular laboratory incubator as ancillary equipment. All reagents can be dry-stored in the device, facilitating storage and transportation without cold chains. The device design is modular and the assay demonstrated high specificity to Mycobacterium tuberculosis (Mtb), analytical sensitivity of the order of 10 copies of Mtb gDNA, and tolerance to complex samples. The clinical sensitivity and specificity of sputum-based FLIPP NAAT tests were 100% (zero false negatives) and 68.75% (five false positives), respectively (N = 30), using Xpert MTB/RIF assay as the reference standard. FLIPP-NAAT has the potential to provide affordable and accessible molecular diagnostics for TB in low- and middle-income countries, when used in conjunction with an appropriate sample preparation technique. Although demonstrated for the detection of TB, FLIPP-NAAT is a platform technology for amplification of any nucleic acid sequence.

Point-of-care diagnostics for infectious diseases: From methods to devices

“…More importantly, the clinical sensitivity and specificity of the device in clinical sputum samples were 100% (zero false negatives) and 68.75% (five false positives), respectively (N = 30), when utilized Xpert MTB/RIF assay as the reference standard. All those results demonstrate that the device has the potential to provide affordable and accessible molecular diagnostics of MTB at the POC [483] .…”

“…The easy-to-use device was able to detecting E. coli DNA consistently with a detection limit of as few as 2 × 10 3 copies mL −1 , which was suitable for use at home, in the field, and in resource poor regions [482] . In another work, Kaur et al developed a modular fluorescent paper-based LAMP device for the POC detection of MTB gDNA [483] . The device was very user-friendly and cheap with a 12-test-zone device cost of $0.88 and one reaction reagent cost of $0.43, which could be operated by an untrained user.…”

Advances in point-of-care nucleic acid extraction technologies for rapid diagnosis of human and plant diseases

“…Sputum is another oral fluid that is often used for the detection of a variety of human diseases such as tuberculosis (TB), influenza, and pneumonia ( Table 2 ). Unlike saliva, sputum is a highly viscous fluid ( Kaur et al, 2019 ). As a result, nucleic acid extraction from sputum samples involves additional pretreatment steps, including sample disinfection, liquefaction, and homogenization ( Reed et al, 2016 ).…”

“…However, conventional qPCR requires bulky, expensive, and sophisticated instruments. Alternatively, amplicon detection can also been achieved on cost-effective smartphone-based reader devices ( Borysiak et al, 2015 ; Choi et al, 2016 ; Kaur et al, 2019 ; Ma et al, 2019 ; Wang et al, 2020 ; Zhu et al, 2020 ). Smartphone-based nucleic acid detection platforms have been used for rapid screening of both human and plant diseases in resource-limited settings ( Hernández-Neuta et al, 2019 ; Kong et al, 2017 ).…”

“…However, most of these detection systems do not incorporate a sample preparation step to isolate biomarkers of interest from raw sample matrices such as blood, saliva, urine, and plant tissue ( Berry et al, 2014 ; Chan et al, 2016a ; Stumpf et al, 2016 ). Because of the complex nature of raw samples, many systems still depend on off-chip sample preparation ( Kaur et al, 2019 ; Liu et al, 2017 ; Magro et al, 2017b ; Rodriguez et al, 2015 ; Wang et al, 2020 ) or sample pretreatment steps such as plasma ( Kaarj et al, 2018 ; Yin et al, 2020 ) or serum separation ( Estrela et al, 2019 ; Tsai et al, 2019 ; Zhang et al, 2019 ) before the actual assay reactions. As a result, so far only a few number of truly integrated “sample-in-answer-out” systems have been demonstrated for practical use in real-world settings.…”