Elsevier

Tuberculosis

Volume 101, December 2016, Pages 160-163
Tuberculosis

Quick and cheap MIRU-VNTR typing of Mycobacterium tuberculosis species complex using duplex PCR

https://doi.org/10.1016/j.tube.2016.10.003Get rights and content

Abstract

While minisatellites are usually typed using capillary sequencers or qiaplex systems in developed countries, many low-resource regions cannot afford it. We propose an optimized agarose gel electrophoresis method to genotype Mycobacterium tuberculosis species complex minisatellites in their standardized format (24 MIRU-VNTR). It is based on duplex PCRs combining VNTR loci harboring distinct amplicon sizes whatever the repetition number of each locus. This method performs well both on DNA extracts of good quality and on thermolysates while reducing workload and reagents costs.

Introduction

Even if Whole Genome Sequencing is in the path for becoming a surveillance tool, Mycobacterium tuberculosis species complex (MTC) outbreak surveillance relies still today in most of the countries on the standardized typing of 24 minisatellite loci (MIRU-VNTR for Mycobacterial Interspersed Repetitive Units-variable Number of Tandem Repeats) [5] preferentially in combination with spoligotyping [1]. The faster protocol to perform 24 MIRU-VNTR-typing is based on gel or capillary electrophoresis-based sequencers [5], and requires only 6 or 8 PCR per sample because of the use of quadri- or triplex PCRs. Even though prices are decreasing, fluorescent molecules for capillary sequencing remain relatively costly so that many laboratories, especially in resource-limited countries, perform simplex PCR and run them on agarose gels using standard electrophoresis devices. As a consequence, they perform 24 PCR per sample and run them on as many gels. The analysis of 96 samples requires around 2 months for a fully trained technician.

We aimed at proposing duplex PCRs to reduce both workload and reagent costs.

Section snippets

Materials and methods

We mined MIRU-VNTR diversity in 1) a publication of reference from Wirth et al. [6]; and 2) in the RIVM database [1], [4]. We defined as “most common size range” for each MIRU-VNTR locus all amplicon sizes for which the two studies had at least two instances each. These ranges were used to pair the duplexes with minimal overlap. In case of overlap, we associated loci so that a difference of at least 16 bp remained between any paired amplicons.

DNA samples of the Quality Control studies were

Results and discussion

We aimed at proposing duplex PCRs to reduce both workload and reagent costs.

We first identified common ranges of each MIRU-VNTR locus using both published data from Wirth et al. [6] and the diversity identified in the RIVM database 2004–2008 including 3454 isolates [1], [4]. We separated them in 3 categories according to their length (short, intermediate and long) (Table 1). We identified 34 possible pairs coupling VNTR with different size ranges and exhibiting differences of at least 16 bp

Conclusion

The technique we propose allows speeding up genotyping of Mycobacterium tuberculosis complex isolates from which DNA extracts are of good quality. It reduces workload and reagent costs by more than 30%. The protocol is not more complex than that of simplex, but requires rigor to appropriately pair primers at the advised concentrations. As it reduces the number of tubes to handle, it helps reducing errors due to large experiment management.

The same type of duplexes can easily be transferred to

Funding

Memona Yasmin stay in Université Paris-Sud laboratory was funded by Erasmus [grant number 744079L]. G. Refrégier and Christophe Sola acknowledge recurrent support from CNRS-Université Paris-Sud.

Conflict of interest

We declare no financial or other type of conflict of interest.

Acknowledgements

We acknowledge University of Paris-Sud financial support. We thank J. Zhang and M. Gomgnimbou for help in daily laboratory management, and Gilson International (Villiers le Bel, France) for technical support.

References (7)

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