A Novel Approach for Automation of MIRU-VNTR Typing of Mycobacterium Tuberculosis

Strain typing is critical to improving our understanding and control of tuberculosis (TB) transmission and pathogenicity. MIRU-VNTR (Mycobacterial Interspersed Repetitive Unit – Variable Number of Tandem Repeat) typing is a well-described molecular method for TB strain genotyping. It is based on mini-satellite allele numbering and requires PCR amplification, electrophoresis, and manual allele calling. The conventional process is long and cumbersome and is associated with several sources of error. Thus, an alternative method is needed to simplify the protocol and increase reliability. QIAxcel Advanced is an automated high-throughput capillary electrophoresis system, which can also automate allele calling. This presentation will describe our assessment of the QIAxcel Advanced system and its comparison to conventional methods for TB strain typing.

We analyzed 15 MIRU-VNTR loci in TB isolates of different genotypes, according to an epidemiological panel described by P. Supply et al., 2006. The samples were processed manually using conventional methods and automatically using the QIAxcel Advanced. The automated procedure included a 30 minute electrophoresis program specially designed for this application, only 1 µl of input sample, and automatic allele calling by the software. We assessed the standard deviation between and within runs, and according to DNA concentration, as well as potential influences on allele calling.

We found that QIAxcel Advanced calculated proper allele calling for 98.6% of processed amplicons, providing genotype patterns for various lineages, with an observed size limitation. The automated system provided results with much shorter turnaround and hands-on times, and at a lower cost than the conventional method. Our results demonstrate that the QIAxcel Advanced system provides reliable MIRU-VNTR typing results with a faster, automated process and would be well-suited for implementation in laboratories performing TB genotyping.

QIAGEN is not affiliated with the Fondation Mérieux’s Emerging Pathogens Laboratory. The views expressed herein are those of the speaker, and do not necessarily express the views of QIAGEN.

Marie Gauthier

Marie Gauthier is a research assistant specialized in infectious diseases at Fondation Mérieux’s Emerging Pathogens Laboratory in France. Her early research involved the design and validation of a high-density DNA microarray for HBV genotyping and detection of resistance mutations. Since 2009, she has focused on tuberculosis research and projects in the field of epidemiology. She spent two years in Haiti working in MDR-TB (multi-drug-resistant TB) surveillance, implementing MDR-TB detection and launching a BSL-3 laboratory. She now studies molecular markers for TB resistance and strain genotyping, with the aim of providing GABRIEL network laboratories methods to conduct epidemiological surveys for evaluating drug-resistant TB prevalence and trends. Her particular goal is the development of appropriate methods adapted to the field and the transfer of this knowledge.