Lab in a Pasteur pipette: low-cost, rapid and visual detection of Bacillus cereu using denaturation bubble-mediated strand exchange amplification

Driven by a bright prospect for rapid, portable and cost-effective point-of-care testing, an assembled Pasteur pipette device to integrate nucleic acid extraction, amplification and detection was developed to detect B. cereus in a sample-to-answer format. Denaturation Bubble-mediated Strand Exchange Amplification (SEA) was chosen to perform isothermal amplification because it requires only a pair of primers and one Bst DNA polymerase. The established SEA can detect as low as 1.0 x 10(-13) M genomic DNA of B. cereus, which was comparable with the previously reported method for B. cereus detection. The assembled Pasteur pipette allows sample-to-answer diagnostic in a simple, low-cost, portable, and disposable format. The inherent function of Pasteur pipette enables direct liquid handling without the need of extra pipettes, syringes or pumps. Visual readout was achieved by using a pH sensitive dye, further simplifying result judgment process. The detection limit for B. cereus is 1.0 x 10(4) CFU/mL in pure cultures, while the detection limit in artificially contaminated milk is 1.0 x 10(5) CFU/mL without enrichment and 1.0 x 10(0) CFU/mL following 12 h enrichment. Considering that typical cell counts in food samples associated to food poisoning are 1.0 x 10(5) to 1.0 x 10(8) CFU per gram/milliliter B. cereus, our Pasteur pipette is enough sensitive for answer-to-sample diagnosis of B. cereus even directly from foods without enrichment. The whole diagnostic procedure could be completed within 50 min, dramatically decreasing the detection time. In a word, the assembled Pasteur pipette device, combined with a homemade metal bath, possesses great potential for sample-to-answer application in resource-limited settings. (C) 2019 Elsevier B.V. All rights reserved.