Rapid amplification of Mycobacterium tuberculosis DNA on a paper substrate

Shetty, Prasad; Ghosh, Debanjan; Singh, Minakshi; Tripathi, A. Manish; Paul, Debjani

doi:10.1039/c6ra07529k

Cited by 35 publications

(13 citation statements)

References 25 publications

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“…Shetty et al . 12 used helicase-dependent amplification (HDA) in chromatography paper for detection of TB. The target DNA was an 84-base pair fragment from the insertion sequence 6110, amplified using PCR.…”

Section: Introductionmentioning

confidence: 99%

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

Kaur

Michael

Toley

2019

Sci Rep

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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.

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Section: Introductionmentioning

confidence: 99%

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

Kaur

Michael

Toley

2019

Sci Rep

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“…Orange-red food dye was obtained from Ajanta Industries (Gurugram, India); papers were dipped in a solution of 2 g/L and dried in an incubator at 37 °C; for preparation of mock sputum : methylcellulose (M-0262) and egg yolk emulsion (17148) were obtained from Sigma-Aldrich; mock sputum was prepared as a solution of 1.8% w/v methylcellulose and 10% egg yolk emulsion in DI water 21 ; for Mycobacterial culture : Tryptone soya broth 24392 (TM 018) was obtained from SRL and Middlebrook 7H9 Broth Base (M198-500G) was obtained from HiMedia. Tween-80 28940 (1628157) and glucose 51758 (0449130), obtained from SRL, were used as supplements; for E .…”

Section: Methodsmentioning

confidence: 99%

Paper Stacks for Uniform Rehydration of Dried Reagents in Paper Microfluidic Devices

Das

Dsouza

Kaur

et al. 2019

Sci Rep

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Spatially uniform reconstitution of dried reagents is critical to the function of paper microfluidic devices. Advancing fluid fronts in paper microfluidic devices drive (convect) and concentrate rehydrated reagents to the edges, causing steep chemical gradients and imperfect mixing. This largely unsolved problem in paper microfluidics is exacerbated by increasing device dimensions. In this article, we demonstrate that mixing of dried reagents with a rehydrating fluid in paper microfluidics may be significantly enhanced by stacking paper layers having different wicking rates. Compared to single-layer paper membranes, stacking reduced the “non-reactive area”, i.e. area in which the reconstituted reagents did not interact with the rehydrating fluid, by as much as 97% in large (8 cm × 2 cm) paper membranes. A paper stack was designed to collect ~0.9 ml liquid sample and uniformly mix it with dried reagents. Applications of this technology are demonstrated in two areas: (i) collection and dry storage of sputum samples for tuberculosis testing, and (ii) salivary glucose detection using an enzymatic assay and colorimetric readout. Maximizing the interaction of liquids with dried reagents is central to enhancing the performance of all paper microfluidic devices; this technique is therefore likely to find important applications in paper microfluidics.

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“…Unfortunately, its speed of long-target sequence amplification is a limiting factor. The strength of this method is its combination with paper-based microfluidics, as demonstrated by the detection of Mycobacterium tuberculosis [42].…”

Section: Isothermal Amplificationmentioning

confidence: 99%

LAMP-on-a-chip: Revising microfluidic platforms for loop-mediated DNA amplification

Zhang

Fohlerová

et al. 2019

TrAC Trends in Analytical Chemistry

186

126

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a b s t r a c tNucleic acid amplification for the detection of infectious diseases, food pathogens, or assessment of genetic disorders require a laboratory setting with specialized equipment and technical expertise. Isothermal deoxyribonucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), exhibit characteristics ideal for point-of-care (POC) applications, since their instrumentation is simpler in comparison with the standard method of polymerase chain reaction. Other key advantages of LAMP are robustness and the production of pyrophosphate in the presence of the target gene, enabling to detect the reaction products using the naked eye. Polymerase inhibitors, presented in clinical samples, do not affect the amplification process, making LAMP suitable for a simple sample-to-answer diagnostic systems with simplified sample preparation. In this review, we discuss the trends in miniaturized LAMP techniques, such as microfluidic, paper-based, and digital with their advantages and disadvantages, especially for POC applications alongside our opinion of the future development of miniaturized LAMP.

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Rapid amplification of Mycobacterium tuberculosis DNA on a paper substrate

Abstract: We have amplified DNA from Mycobacterium tuberculosis on a paper substrate in 10 minutes using helicase dependent amplification technique.

Cited by 35 publications

References 25 publications

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

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

Paper Stacks for Uniform Rehydration of Dried Reagents in Paper Microfluidic Devices

LAMP-on-a-chip: Revising microfluidic platforms for loop-mediated DNA amplification

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