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Lab in a Pasteur pipette: low-cost, rapid and visual detection of Bacillus cereu using denaturation bubble-mediated strand exchange amplification
- Source :
- Analytica Chimica Acta. 1080:162-169
- Publication Year :
- 2019
- Publisher :
- Elsevier BV, 2019.
-
Abstract
- 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 × 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 × 104 CFU/mL in pure cultures, while the detection limit in artificially contaminated milk is 1.0 × 105 CFU/mL without enrichment and 1.0 × 100 CFU/mL following 12 h enrichment. Considering that typical cell counts in food samples associated to food poisoning are 1.0 × 105 to 1.0 × 108 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.
- Subjects :
- DNA, Bacterial
Paper
Pasteur pipette
Loop-mediated isothermal amplification
Food Contamination
DNA-Directed DNA Polymerase
02 engineering and technology
01 natural sciences
Biochemistry
Analytical Chemistry
Geobacillus stearothermophilus
Bacillus cereus
Bacterial Proteins
Limit of Detection
Animals
Environmental Chemistry
Denaturation (biochemistry)
Coloring Agents
Spectroscopy
Detection limit
Bacillus (shape)
Chromatography
biology
Chemistry
010401 analytical chemistry
Pipette
021001 nanoscience & nanotechnology
biology.organism_classification
Bacterial Load
0104 chemical sciences
Milk
Visual detection
Cereus
Colorimetry
0210 nano-technology
Nucleic Acid Amplification Techniques
Subjects
Details
- ISSN :
- 00032670
- Volume :
- 1080
- Database :
- OpenAIRE
- Journal :
- Analytica Chimica Acta
- Accession number :
- edsair.doi.dedup.....336b080e84f1d42ee18f9b4e1e86123b