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.
Highlights
We present a prototype for conducting rapid, inexpensive and point-of-care-compatible nucleic acid amplification tests (NAATs) for tuberculosis (TB)
The top seal of the device consisted of a combination of pressure sensitive adhesive (PSA) and a transparency sheet cut to match the dimensions of the acrylic base, with an additional flap to ease peeling off
Sample preparation could be improved to suit the needs of FLIPP NAAT
Summary
We present a prototype for conducting rapid, inexpensive and point-of-care-compatible nucleic acid amplification tests (NAATs) for tuberculosis (TB). The WHO-endorsed GeneXpert (Xpert MTB/RIF assay) provides an unprecedented level of automation of the multiple steps involved in a NAAT and enables a minimally-trained user to conduct a NAAT, but the cost of this instrument remains prohibitively high in many settings[3] Another PCR-based test from a joint venture of Molbio diagnostics and Bigtec labs (Bangalore, India)[4] consists of two units -Trueprep Mag for sample preparation and Truelab Uno for DNA amplification. There exists significant room for technological innovation to provide easy-to-use, yet affordable solutions for TB-NAATs. Paper-based isothermal NAATs have recently emerged as popular platforms for low-cost nucleic acid detection. We present a new paper-based device for conducting isothermal NAATs at the point-of-care and demonstrate its utility in sensitive and specific detection of TB, directly from whole Mycobacterium tuberculosis (Mtb) genomic DNA (gDNA). In combination with an appropriate DNA extraction procedure, it promises to be an essential tool for conducting NAATs in places like rural India
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