PyroTyping, a novel pyrosequencing-based assay for Mycobacterium tuberculosis genotyping

B Molina-Moya,C Arnold,S Samper,A Lacoma,J Domínguez,N García-Sierra,J Ruiz-Manzano,S Blanco,L Haba,C Prat

doi:10.1038/s41598-017-06760-5

Abstract

We developed a novel method, PyroTyping, for discrimination of Mycobacterium tuberculosis isolates combining pyrosequencing and IS6110 polymorphism. A total of 100 isolates were analysed with IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, mycobacterial interspersed repetitive units – variable number tandem repeats (MIRU-VNTR), and PyroTyping. PyroTyping results regarding clustering or discrimination of the isolates were highly concordant with the other typing methods performed. PyroTyping is more rapid than RFLP and presents the same discriminatory power, thus, it may be useful for taking timely decisions for tuberculosis control.

Highlights

Tuberculosis (TB) is one of the leading causes of death among curable infectious diseases, and worldwide spread of Mycobacterium tuberculosis isolates, the causative agent, poses a threat to the global control of TB1
The aim of the present study was to develop a novel molecular method based on PCR amplification and pyrosequencing (PyroTyping) for discrimination of isolates based on the polymorphism of the IS6110 insertion site
Results between restriction fragment length polymorphism (RFLP), spoligotyping, and PyroTyping for the 100 M. tuberculosis isolates were concordant: the three methods agreed on clustering for 74 isolates (Fig. 2a), and on discrimination for 26 isolates (Fig. 2b) (Table 1)

Summary

Introduction

Tuberculosis (TB) is one of the leading causes of death among curable infectious diseases, and worldwide spread of Mycobacterium tuberculosis isolates, the causative agent, poses a threat to the global control of TB1. In order to improve TB control, it is important to track the spread of M. tuberculosis isolates, identify index cases, and detect outbreaks For these purposes, several genotyping methods have been developed. RFLP is laborious and time-consuming, the time until results are available may be too long for decision-making Another extensively used genotyping method is spoligotyping, based on the polymorphism of the clustered regularly interspaced short palindromic repeats (CRISPR) locus in the M. tuberculosis genome. Isolates share the same RFLP pattern, the IS6110 copies are located in the same position in the genome, and the sequence of the IS6110-5′ flanking regions would be identical In this case, pyrosequencing profiles would be identical. RFLP and spoligotyping were performed for all the isolates, and MIRU-VNTR was performed for the 86 isolates for which DNA was available (78 isolates corresponding to 24 case studies and the six MDR isolates)

Objectives

Methods

Results