By Ben Butkus
Scientists from Japan's Riken have developed a nucleic acid amplification method that eliminates the need for RNA extraction and thermal cycling, and have demonstrated its ability to rapidly and sensitively detect in clinical samples the influenza strain responsible for a 2009 pandemic.
The method, called RT-SmartAmp, combines isothermal amplification and reverse transcription in a single step, and could serve as the basis of a simple and rapid diagnostic test in the event of a future flu pandemic, according to the researchers.
To that end, the scientists are now working on a small, fluorescence-based visual detection device as well as a simpler sample preparation protocol in order to adapt RT-SmartAmp for true point-of-care use, a research team member told PCR Insider this week.
The researchers, primarily from the Riken Omics Science Center in Yokohama, described the technical development and clinical validation of their RT-SmartAmp assay for detecting influenza A(H1N1) in a paper published last week in PLoS One.
RT-SmartAmp is based on an isothermal amplification technique called Smart Amplification Process — originally shortened to SMAP but later changed to SmartAmp — which some of the same Riken scientists invented and first described in a Nature Methods paper in 2007.
The SmartAmp method is "based on the concept that DNA amplification itself is the signal for the presence of a specific target sequence," Toshihisa Ishikawa, a research scientist at the Riken Omics Science Center and corresponding author on the PLoS One paper, wrote in an e-mail to PCR Insider.
"Differing from the widely used PCR, the SmartAmp reaction is an isothermal DNA amplification," Ishikawa said. "In isothermal DNA amplification by the SmartAmp method, the initial step of copying a target sequence from DNA is a prerequisite."
This step is accomplished through the use of four different primers termed TP, FP, OP1, and OP2. According to Ishikawa, the FP and TP primers hybridize the template genomic DNA. Next, both of the products primed for the FP and TP primers are detached from the template DNA by strand-displacing DNA polymerase, whose extensions are primed by the OP1 and OP2 primers.
The resulting single-stranded DNA products become templates in the second step for opposing FP and TP primers, and are generated by the strand-displacement activity of the DNA polymerase, being primed from the flanking region of OP primers adjacent to the target sequence.
"The resulting DNA products are referred to as intermediate products that play key roles in the subsequent amplification steps," Ishikawa said. "The formation of those intermediate products is the rate-limiting step in SmartAmp-based isothermal DNA amplification."
Through additional isothermal self-priming and elongation processes, these intermediate products then form concatenated DNA products that can be detected by conventional gel electrophoresis, with DNA ladder patterns that are distinct from those typically formed from PCR products.
In addition, a fifth primer, called BP, is covalently linked to a so-called Exciton dye — also invented at Riken — thus creating a hybridization-sensitive fluorescent primer that enables real-time monitoring of the DNA amplification reactions.
Lastly, in order to apply the technique to a viral RNA template — a region of the hemagglutininin, or HA, gene in the case of their influenza assay — the researchers use a commercially available reverse transcriptase called AMV RT that synthesizes a first cDNA strand from the target viral RNA negative strand encoding HA, thus creating two DNA intermediates in the subsequent SmartAmp reaction.
Though rather complex in its chemistry, the upshot is that the final RT-SmartAmp assay "combines the reverse transcriptase and isothermal DNA amplification reactions in a single step, such that the required detection time is only about 40 minutes, and tangled RNA extraction is not required," the researchers wrote in their paper.
This differs markedly from PCR which, while shown to be extremely sensitive for detecting viral RNA targets, requires separate laborious and time-consuming RNA extraction and reverse transcription steps, according to the researchers.
In order to clinically validate their assay, Ishikawa and colleagues used it to test a total of 225 swab samples collected from outpatients with influenza-like illness at three hospitals and 11 clinics in the Tokyo and Chiba areas of Japan during the H1N1 pandemic period of October 2009 to January 2010.
They compared the ability of their assay to detect the virus against the widely accepted gold standard of lateral flow immunochromatographic testing, as well as viral genome sequence analysis. Among the 255 collected samples, the RT-SmartAmp method correctly detected 140 samples as positive for infection by the virus, compared to 110 positives with the immune-based method.
Further, RT-SmartAmp was able to detect more than 70 percent of the infection-positive cases within 24 hours of the onset of fever, and about 20 percent of the infection-positive cases in less than six hours after the onset of fever.
They also compared the sensitivity of their assay with that of another established commercial qRT-PCR assay, the Roche Real-Time Ready Influenza A (H1N1) test, and found that RT-SmartAmp was comparably able to detect virus at a minimum of about 50 copies per 25-µL reaction mixture.
Ishikawa conceded that other, simpler isothermal amplification methods — in particular reverse-transcription loop-mediated isothermal amplification, or LAMP — have been shown to be effective tools to detect the 2009 pandemic influenza strain.
In fact, according to Ishikawa, LAMP-based methods are primarily used for rapid and accurate pathogen detection, while the SmartAmp method is particularly useful for SNP and mutation detection. As such, the researchers believe that SmartAmp could serve as the ideal basis of a diagnostic test for future pandemic strains that have mutated to become resistant to antiviral compounds.
"It is well known that the His-to-Tyr substitution at amino acid residue 275 confers [to] the 2009 [pandemic] virus oseltamivir resistance, and this mutation was detected in patients infected with 2009 influenza A(H1N1) viruses in our clinical study," Ishikawa said.
Further, "it has been reported that the oseltamivir-resistant 2009 [pandemic] influenza A(H1N1) viruses were as pathogenic and transmittable as their drug-sensitive counterparts," Ishikawa added. "Since oseltamivir has been heavily used in pharmaceutical treatments in Japan, the oseltamivir resistance rate is expected to greatly increase in the second [pandemic] virus wave. Therefore, the next challenge for the RT-SmartAmp assay is to develop a kit for rapid detection of the mutation causing oseltamivir resistance."
In addition, the RT-SmartAmp assay currently requires the use of either a real-time PCR platform capable of fluorescence detection or, if the fluorescent primers are left out, a gel electrophoresis setup. As such, the assay is not truly ideal for point-of-care use, which is a crucial factor in a viral outbreak situation.
Ishikawa said the researchers are currently developing a "small … visual detection device to detect highly pathogenic H5N1 virus" using the RT-SmartAmp assay.
What’s more, as with any nucleic acid amplification method, sample preparation is a bottleneck, the researchers acknowledged. In their PLoS One study, the scientists used a pre-treatment containing 5 percent SDS to dissolve the viral membranes and facilitate RNA extraction.
"Therefore, the SDS had to be removed from the sample by spin column chromatography," the researchers wrote. "This step should be improved in future technology development. Sample preparation starting from clinical specimens, such as nasopharyngeal swabs, needs to be coupled with amplification and detection to achieve the final goal of point-of-care technologies."
Ishikawa declined to comment on the patent status of the RT-SmartAmp technique, and he said that Riken currently has no outlet for commercializing the method.
He did note that in November 2010, an undisclosed Japanese company acquired regulatory approval from the Pharmaceutical and Medical Devices Agency of Japan for a RT-SmartAmp-based detection kit for influenza A(H1N1) pandemic 2009 virus. However, that kit is not yet commercially available.
According to the website of DNAForm, a Riken spinout established in 1998 to commercialize several technologies developed in the laboratory of Riken researcher and SmartAmp co-inventor Yoshihide Hayashizaki, the company is responsible for the "licensing and diffusion" of the original SmartAmp method, and for the "manufacturing and marketing of genetic detection [assay reagents] for SmartAmp."
Further, according to an archived DNAForm press release, the company helped develop the SmartAmp method along with Hayashizaki's lab.
It is unclear whether DNAForm is also working with Riken to commercialize the RT-SmartAmp method or associated diagnostic kits. Representatives from the company could not be reached for comment prior to publication of this article.
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