By Ben Butkus
Having sequenced the genome of Escherichia coli O104, the antibiotic-resistant E. coli strain responsible for killing at least 25 people in Germany and sickening thousands more worldwide, separate teams from Life Technologies and BGI said this week that they have developed PCR-based tests to detect the bacterium and help public health officials identify and track future outbreaks.
Life Tech's test is based on in-house sequencing analysis it conducted last week in collaboration with scientists at Germany's University Hospital Münster using the company's Ion Personal Genome Machine.
The TaqMan E. coli O104 test kit, which is optimized for use on the company's 7500 Fast qPCR instrument and is for research use only, is currently available in Europe and is being further tested by various reference laboratories in Germany in order to provide independent verification of its sensitivity and specificity, a Life Tech official said this week.
Meantime, BGI's test, also based on sequencing analysis performed last week on the Ion PGM, has a high degree of sensitivity and specificity, according to the institute, which it determined using a combination of computational analysis using publicly available sequence data, and through experimental analysis of dozens of E. coli strains, including the culprit of the recent outbreak.
BGI said that its kit is now available from the institute, even though it continues to validate the test on samples from infected individuals. The institute has also made the complete test protocol available online for laboratories wishing to develop their own tests.
After the emergent E. coli strain reared its head in Germany in early May, scientists scrambled to identify the strain that was responsible and its source. The latter is currently still a mystery, but last week, the independent research teams from Life Tech/University Hospital Münster and BGI announced that they had successfully sequenced the bug, identifying it as a hybrid of two strains — enteroaggregative E. coli and enterohemorrhagic E. coli — and a carrier of antibiotic-resistance genes.
As reported this week by PCR Insider sister newsletter In Sequence, both research teams credited the speed of the Ion PGM for producing preliminary sequence data in a matter of days, allowing them to turn their attention to developing a molecular test for the bacteria and to conduct more in-depth sequence analyses on other sequencing platforms.
Life Tech announced the availability of its test first this week. Nir Nimrodi, general manager of food safety and animal health at Life Tech, told PCR Insider this week that the company was well-positioned to quickly identify the E. coli strain and develop a molecular test based on the sequence data.
"What is new here is having the [Ion PGM], a system that is capable of generating sequencing data in hours, and accurately," Nimrodi said. "You could have done sequencing this fast in the past, but it was never as accurate."
"And the other thing is that we were really able to … bring the sequencing data into our bioinformatics group; to bring in the qPCR team; and quickly bring everything together and send it back to the customer," Nimrodi added. "I think this is setting new standards in how we can help governments, or a food company, tackle [the identification of] positive samples."
Nimrodi also noted that Life Tech and University Hospital Münster have made their sequencing data available to the public so that laboratories can develop their own homebrew tests.
Because it is not being used as an in vitro diagnostic test in humans, Life Tech's test did not require any specific approval or even validation. AOAC approval "is not required, but desirable," Nimrodi said; however, since that takes months to obtain, the company has already begun the process of independently vetting the assay with "a couple of reference labs" in Germany.
Although Life Tech's internal testing has led it to believe that its test is "more reliable than others," the company will wait to make any specific claims about sensitivity or specificity until it is independently evaluated.
The assay is optimized for use on Life Tech's Applied Biosystems Fast 7500 real-time PCR system, but it can be used on other PCR platforms, Nimrodi said. "Preferably they'd do it on the 7500, because that would give them the fastest time to result thanks to the high throughput; and because everything works well together," he said. "You also need the master mixes and the sample prep. Obviously you can't take a bean sprout or cucumber and put it in the 7500; you need the sample prep. And we can provide the entire suite of products."
Meantime, BGI said this week that it has developed "a straightforward PCR diagnostic protocol" to rapidly identify E. coli O104. The protocol consists of two pairs of amplification primers that target the enteroaggregative- and hemorrhagic-associated genes from the strain.
BGI yesterday also disclosed the most recent genome assembly of the bacterium based on a combination of preliminary Ion PGM data obtained last week and subsequent Illumina HiSeq data.
However "the primers for the PCR testing kit were designed based on the preliminary draft obtained from Ion Torrent," Bicheng Yang, public communication officer for BGI, wrote in an e-mail to PCR Insider.
"After [defining] the target sequences, we compared them against … publicly available whole-genome sequences of bacteria strains in silico to evaluate the specificity, and this [took] almost zero time … on our computer cluster. The experimental validation was carried [out] on six PCR machines in parallel, which quickly provided us evidence on specificity and sensitivity."
BGI said that it computationally analyzed 4,547 strains of E. coli from 2,183 species using publicly available whole-genome sequences; and experimentally analyzed 323 DNA samples from 93 species including 55 E. coli strains with different phenotypes as well as the current infectious strain.
The institute said that using its test, no other strain other than E. coli O104 had positive amplification results of both target regions. Further, the test was able to detect the bacterium using a DNA concentration as low as about 1 picogram.
BGI and the Beijing Institute of Microbiology and Epidemiology, BGI's partner in developing the assay, said that they will provide the designed and synthesized primers free to any disease control or research agency worldwide.
According to Yang, the protocol "has no particular requirements on reagents or instrument." The institutes said that the assay can provide results within two to three hours after receipt of a sample.
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