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Australian Researchers Develop Multiplex PCR Method for Genotyping Infectious Disease Agents

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By Ben Butkus

Researchers from Western Australia University and Australian government-owned pathology lab PathWest Laboratory Medicine are using multiplex PCR and microfluidic lab chips to develop a series of molecular assays suitable for diagnosing and typing a range of infectious agents in resource-poor areas of the world, the project's principal investigator said this week.

In addition, the researchers have published a research paper demonstrating their method's ability to rapidly genotype Mycobacterium tuberculosis isolates from clinical samples — a capability that could serve as a supporting laboratory technique in tuberculosis control programs, they said.

The method, called multiplex amplified nominal tandem repeat analysis, or MANTRA, is an offshoot of a previously developed technique called variable number tandem repeat, or VNTR, amplification and detection.

PathWest, which acts as the state of Western Australia's reference laboratory for tuberculosis, introduced VNTR genotyping a few years back, Timothy Inglis, a medical microbiologist with PathWest and WAU, wrote in an e-mail to PCR Insider.

In VNTR analysis, researchers use PCR or other amplification and detection techniques to identify VNTRs from the genome of an infectious agent and use them to identify potential clusters of genetically related isolates. When used for tuberculosis typing, the technique is referred to as mycobacterial interspersed repetitive unit, or MIRU, analysis, and is an attractive alternative to the long turnaround times of other tuberculosis typing methods such as insertion sequence 6110 typing and spoligotyping.

In a paper published online last week in the Journal of Clinical Microbiology, Inglis and colleagues wrote that VNTR and MIRU analysis have become "an important addition to the M. tuberculosis genotyping repertoire;" and that an "increasing number of Australian public health laboratories now offer an M. tuberculosis VNTR genotyping service, and use an online VNTR data interpretation Web browser."

In general, the researchers wrote, public health laboratories with both PCR capability and the ability to perform subsequent gene fragment length analysis can now perform VNTR analysis, "but at the cost of limited sequencing capacity."

The researchers noted that the US Centers for Disease Control and Prevention previously developed an alternative version of VNTR genotyping using microfluidic lab chips for endpoint analysis, but that the method has since "sparked limited interest, possibly due to the multiple PCR products that need fragment analysis."

Inglis further told PCR Insider that VNTR genotyping has probably not been further developed for TB typing because "most mycobacterial genotyping work is performed in well-equipped reference labs in developed countries where relatively small numbers of isolates can be analyzed under optimal conditions" and because of "conventional wisdom that multiplex amplification of mycobacteria would produce misleading results."

The MANTRA method developed by Inglis and colleagues is a 15-target, fully multiplexed version of VNTR genotyping that uses Agilent Technologies' 2100 Bioanalyzer coupled with the company's DNA 1000 LabChip "in order to achieve more immediate throughput than either monoplex format PCR or a DNA sequencer on genescan mode," the researchers wrote in their paper.

Using the method, the researchers were able to identify epidemiologically and genotypically distinct isolate cultures of M. tuberculosis from 34 initial samples whose genotype was previously unknown.

The main difference between MANTRA and VNTR analysis is that MANTRA "is not meant to generate a definitive VNTR description," Inglis said. "It is designed primarily and solely for use as a field assay to be used at the sharp end of disease control in resource-limited settings."

Inglis added that his group intends to follow preliminary MANTRA analysis with a "definitive second method that adds specificity, but requires access to reference lab back-up. The extracts used for MANTRA could be shipped out for subsequent MIRU without further extraction."

Indeed, the researchers noted in their paper that MANTRA is "intended for comparative use … a reminder that the technique does not aim to be a standalone genotyping method." What's more, the technique would at this point still require "further confirmation of apparent genotypic clustering by a distinct second method such as spoligotyping or IS6110 typing," they wrote.

Inglis also said that MANTRA has potential as a standalone genotyping method, but it would first need "careful evaluation" against in-use reference methods.

Nevertheless, the new method is important, Inglis said, because TB control often needs genotyping and diagnostic methods that can give a quick and relatively accurate first assessment of the situation.

"Molecular epidemiology continues to look for the single method that has all the attributes we need in a single hit," Inglis said. "In the meantime, we often have to make choices between two or more less-than-ideal genotyping methods. Sometimes it is necessary to support hospital infection control and public health interventions by using two different genotyping methods; one to give us a preliminary sense of whether there is any genotypic clustering, and the second to act as a confirmatory subtyping method."

The researchers have also previously demonstrated the use of MANTRA to confirm melioidosis infections in Sri Lanka, data that was published in a previous JCM paper. Now, working under the moniker "Laboratories without Walls," Inglis and colleagues are developing a range of assays based on MANTRA "as quickly as possible" to be deployed in "capacity-building projects, humanitarian assistance, and emerging infectious disease response" in resource-poor countries and remote communities in Western Australia.

All the assays will be based on nucleic acid amplification technology using a range of chemistry-platform combinations, and will include tests for epidemic influenza, arboviruses (the causative agent of Dengue fever), malaria, and generalized sepsis, Inglis said.

As far as potential widespread commercialization of the method goes, Inglis said that it "should not be difficult. The Bioanalyzer and DNA 1000 kits are already widely available, and the method we published could be set up quickly."

Inglis added that PathWest has "one of the largest clinical molecular microbiology labs in Australia and uses it to good effect." Agilent is one of a "small number" of companies that are partnering with the lab to develop clinical assays, he said.

"My group … has a wide collaborative network, and recognizing that we make fastest progress by working with the best the biotech industry has to offer, we have concentrated on field applications," Inglis added.

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