Meridian Bioscience has developed a simple, equipment-free, rapid nucleic acid purification method for use with a variety of clinical specimens, and demonstrated that it enables similar analytical and clinical sensitivity as current commonly used methods such as silica-based purification.
According to the company, the new method is expected to further simplify the workflow in front of several of its isothermal amplification-based molecular diagnostic assays that use complex clinical sample types such as stool, urine, and certain respiratory samples.
More particularly, company officials said this week that the new method is expected to make it easier for customers to perform several assays currently in clinical trials or development at Meridian, including tests for Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma pneumoniae.
Meridian unveiled its new sample prep method at the Association for Molecular Pathology annual meeting in October. There, Ahmer Kodvawala, a scientist in Meridian's research and product development division, presented a poster detailing the new sample prep method and comparing it to other, more established techniques.
The method uses the concept of size exclusion chromatography to process sample volumes of 200 µL to 350 µL. The process works via gravity flow and does not require any laboratory equipment, such as a centrifuge.
As described in the poster, the technique comprises mixing 200 µL of clinical specimen with 50 µL of internal control and 50 µL of lysis buffer; then loading 200 µL of lysate onto the universal sample prep, or USP, column.
Once the sample enters the column, 200 µL of buffer is added to move the DNA through the column, followed by an additional 200 µL of buffer and collection of eluate into a testing tube. The eluate can then be added directly to lyophilized beads for amplification using Meridian's Illumigene assays, which are based on loop-mediated isothermal amplification, or LAMP; or to master mix for PCR amplification.
Scientists from the company compared the analytical performance of its technology to a Qiagen DNeasy Blood and Tissue kit in front of Meridian's Illumigene Mycoplasma assay, which does not yet have US regulatory approval, to detect an M. pneumonia strain spiked into transport medium.
They also purified culture-enriched Group B Strep clinical samples with the USP and DNeasy blood kit in front of the Illumigene Group B Streptococcus assay and in-house PCR assays; and processed urine samples positive for C. trachomatis using the USP method, testing for the bacteria using both a PCR-based test and a Meridian LAMP-based assay that does not yet have regulatory approval.
Finally, to test clinical performance, they used the USP method and Illumigene C. difficile test to purify and assay 50 frozen stool specimens that had been previously tested using Cepheid's Xpert C. difficile assay.
The Meridian scientists reported that the yield and quality of DNA from M. pneumonia clinical specimens was similar for the USP method and Qiagen kit. In addition, the USP method and silica-based methods performed similarly for the detection of GBS in enriched culture. Meridian also showed that the clinical performance of its Illumigene C. difficile test with USP was concordant to Cepheid's Xpert assay for 19 of 20 positives and 28 of 30 negatives. The discrepant positive result was negative when retested using the Xpert assay, suggesting that DNA degradation had occurred during storage; while the two discrepant negative results were found to be true positives using follow-up in-house PCR assays, the researchers reported.
According to the poster, the average sample processing time of the USP procedure was just under five minutes. In comparison, the Qiagen DNeasy workflow usually takes 30 to 40 minutes, Kodvawala said.
Since the AMP presentation, Meridian has conducted further comparative tests of its methods. Last week, the company noted that it had successfully completed beta trials for its Illumigene tests for C. trachomatis and N. gonorrhoeae on both swab and urine samples, and noted that the assays for the first time employed the USP method. According to the company, the beta trial compared the performance of the new assays to "two market-leading molecular platforms on a statistically significant cohort of symptomatic patients," but provided no further details.
"Essentially Meridian Bioscience's … mentality is to simplify molecular testing … and that's why we choose LAMP technology," Slava Elagin, executive vice president of research and product development at Meridian Bioscience, told PCR Insider this week. Meridian has a license from Japan's Eiken Chemical to use the LAMP technology in clinical diagnostic applications. All of Meridian's Illumigene tests run on the company's US Food and Drug Administration-approved Illumipro-10 Integrated Incubator/Reader, and take about one hour to complete.
"All of our technologies center around LAMP right now," Elagin added. "And in some cases, depending on the complexity of the test, we are using standard nucleic acid purification methods. But our vision is to ensure that our nucleic acid purification methods will … be as simple as our amplification platform. That's why we started working on this universal sample prep method."
Meridian currently has three Illumigene molecular diagnostic assays approved by the US Food and Drug Administration: for C. difficile, Group B strep, and Group A strep. In addition, the company has in the pipeline the aforementioned CT/NG assays; and has submitted to the FDA the M. pneumonia test and is preparing to begin clinical trials for an assay for Bordetellla pertussis.
Currently, Meridian offers a different integrated sample prep method with its C. difficile, and its Strep tests don't require special sample prep because the sample is simply transferred from culture, Elagin said.
"So far, our sample purification is such [that a] user doesn't need any off-the-shelf purification reagents," he said. "But once we start to talk about sample types like urine and swabs [in transport medium], in that case, we will not be able to do the same simple manual steps. So that’s another reason to develop this universal sample prep."
Elagin noted that the USP method might also be useful in combination with the Illumigene C. difficile assay. "There is no measured advantage in performance, but at the same time we can simplify the procedure even farther, and go from a total of 10 minutes of preparation time … to about 5 minutes."
He added, though, that the company has not yet decided if it will offer the USP method, once it is commercialized, in front of the C. diff assay.
Meridian plans to commercialize two to three molecular tests per year for the foreseeable future, Elagin said. "Our current pipeline for 2013 is already disclosed, and we are ahead of schedule for the Mycoplasma test and Bordetella test. We will begin clinical trials for chlamydia and gonorrhea also. I'm not sure if we will have clearance for CT/NG in 2013, but most likely we will."
He added that Meridian is the early stages of development for a number of other molecular tests, but declined to disclose them at this point in time.
"We want to provide quick, interactive tests [that provide] meaningful information … so a physician can act on the results," Elagin said. "For instance, we don't have a molecular test for influenza, because in some cases it's not a high-value test that will yield meaningful information for physicians to act upon the results.
"We are looking at this sample prep method as a new vehicle for us, because we can expand our technology and expand the sample types that we can use in conjunction with our [Illumigene] technology," he added.