A versatile-deployable bacterial detection system for food and environmental safety based on LabTube-automated DNA purification, LabReader-integrated amplification, readout and analysis

Abstract

Contamination of foods is a public health hazard that episodically causes thousands of deaths and sickens millions worldwide. T o ensure food safety and quality, rapid, low-cost and easy-to-use detection methods are desirable. Here, the LabSystem is introduced for integrated, automated DNA purification, 10 amplification and detection. It consists of a disposable, centrifugally -driven DNA purification platform (LabT ube) and the subsequent amplification and detection in a low-cost UV/vis-reader (LabReader). For demonstration of the LabSystem in the context of food safety, purification of Escherichia coli (nonpathogenic E. coli and pathogenic verotoxin-producing E. coli (VT EC)) in water and milk, and the product-spoiler Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice was integrated and 15 optimized in the LabT ube. Inside the LabReader, the purified DNA was amplified, readout and analyzed using both qualitative isothermal loop-mediated DNA amplification (LAMP) and quantitative real-time PCR. For the LAMP -LabSystem, the combined detection limits for purification and amplification of externally lysed VT EC and A. acidoterrestris is 10 2 -10 3 cell-equivalents. In the PCR-LabSystem for E. coli cells, the quantification limit is 10 2 cell-equivalents including LabT ube-integrated lysis. T he 20 demonstrated LabSystem only requires a laboratory centrifuge (to operate the disposable, fully closed LabT ube) and the low-cost LabReader for DNA amplification, readout and analysis. Compared with commercial DNA amplification devices, the LabReader improves sensitivity and specificity by the simultaneous readout of four wavelengths and the continuous readout during temperature cycling. T he use of a detachable eluate tube as an interface affords semi-automation of the LabSystem, which does not 25 require specialized training. It reduces hands-on time from about 50 to 3 min with only two handling steps: sample input and transfer of the detachable detection tube.

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“…The use of centrifugal forces to enhance automation of DNA extraction from whole blood samples and purification of His-tagged proteins has been demonstrated by Kloke et al, using the so-called LabTube devices [18]. Similar devices have also been employed for the detection of foodborne microorganisms [19]. Due to their complex architecture, LabTube cartridges require multiple manufacturing procedures, often including injection moulding, LED scanning, stereolithography, and thermal sealing.…”

A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification

“…Whilst the system is low cost ($200) the use of PCR tubes limits the integration of sample preparation which is currently being developed in the next iteration of the proposed device [ 11 ]. The LabTube and LabReader system automates sample preparation using a centrifugal method within a modified 50 mL centrifuge tube [ 12 ]. The purified DNA is automatically transferred to a PCR tube.…”

“…The purified DNA is automatically transferred to a PCR tube. This tube is manually transferred to the LabReader which can run two isothermal or PCR reactions simultaneously [ 12 ]. This is fewer than the presented platform and excitation filters are required between the LEDs and the PCR tube [ 12 ].…”

“…This tube is manually transferred to the LabReader which can run two isothermal or PCR reactions simultaneously [ 12 ]. This is fewer than the presented platform and excitation filters are required between the LEDs and the PCR tube [ 12 ]. The cost of the LabReader is expected to be one order of magnitude lower than the GeneXpert which is significantly more expensive than the presented low-cost platform [ 12 , 13 ].…”

“…This is fewer than the presented platform and excitation filters are required between the LEDs and the PCR tube [ 12 ]. The cost of the LabReader is expected to be one order of magnitude lower than the GeneXpert which is significantly more expensive than the presented low-cost platform [ 12 , 13 ]. Jiang et al utilized solar heating to enable PCR in resource limited settings with a smartphone reader [ 14 ].…”