Researchers say they believe they have found a cheap method to identify bacteria in a few hours on a mobile-phone-sized device.
The bacterial identification method, called ON-rep-seq, examines strain-specific fragments of the bacterial genome, allowing results that earlier required DNA sequencing of the whole bacterial genome. The method also returns results faster than approaches like pulsed field gel electrophoresis (PFGE), which had been the gold standard for strain-level typing of microorganisms, according to the Danish research team.
The team said the method could change the approach used for investigating foodborne disease outbreaks by making analysis less time and cost consuming. It is based on nanopore sequencing, which is a real-time DNA sequencing approach. ON-rep-seq cannot discriminate between strains that differ solely with single nucleotide polymorphisms (SNPs), the researchers reported.
“Our new method allows identification and typing of hundreds of samples in less than two hours, and we expect that this will even be reduced to real time in a short period of time,” said Lukasz Krych, associate professor in the department of food science at the University of Copenhagen.
Method uses DNA sequencing device
Despite developments in culture-independent methods for detecting microorganisms, culture-based methods remain important in microbiology and especially in foodborne illness investigations. Currently, bacterial detection and identification based on bacterial DNA requires expensive instrumentation and hours of work by trained specialists. If there is a suspected Salmonella outbreak, to locate its origin, investigators have to analyze many samples and analysis has to be precise to distinguish one bacterial strain from another.
The smallest sequencer from Oxford Nanopore Technologies, called MinION, is a hand-held, USB-powered device that became commercially available in 2015. Despite the revolution in DNA sequencing the system offered, the data generated are not perfect because of sequencing errors and analysis remained relatively expensive.
Scientists from the food science department at the University of Copenhagen say they have found a way to use this technology to analyze hundreds of bacteria at a time, cutting costs to less than $2 per bacterium, while increasing accuracy to more than 99 percent. DNA library preparation for 96 isolates takes less than five hours, according to the study published in the Communications Biology journal.
Quickly find disease-causing bacteria
The research project was carried out with Polish company GenXone S.A., which helped set up a bioinformatics pipeline to perform analysis of sequencing data.
“Our method can be used both within food safety, where you can quickly find disease-causing or health-promoting bacteria, and also in the health sector, where you will be able to perform certain analyses that you are not even considering today because of the price and time-consuming nature of traditional analysis,” said Josue Leonardo Castro Mejia, a researcher on the study.
The method was tested on 38 different bacterial species and three strain-level groups identifying all bacteria down to the species-level and discriminating strains with a sensitivity similar to a whole-genome sequencing (WGS)-based approach. Several companies are testing it in their systems for establishing rapid screening programs.
Five Listeria monocytogenes, four Salmonella (three serovar Typhimurium and one Oranienburg), and two Bacillus cereus strains were used for strain-level discrimination.
The DNA enrichment method combines an optimized version of repetitive extragenic palindromic PCR (Rep-PCR) with a consecutive dual-stage Rep-PCR-2 step during which sample-specific barcodes are incorporated. The modified version of Rep-PCR allows for reproducible amplification followed by a dual-stage Rep-PCR-2 step allowing tagging of up to 96 samples in one reaction.
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