Abstract
BackgroundMultiplex ligation-dependent probe amplification (MLPA) is a powerful tool to identify genomic polymorphisms. We have previously developed a single nucleotide polymorphism (SNP) and large sequence polymorphisms (LSP)-based MLPA assay using a read out on a liquid bead array to screen for 47 genetic markers in the Mycobacterium tuberculosis genome. In our assay we obtain information regarding the Mycobacterium tuberculosis lineage and drug resistance simultaneously. Previously we called the presence or absence of a genotypic marker based on a threshold signal level. Here we present a more elaborate data analysis method to standardize and streamline the interpretation of data generated by MLPA. The new data analysis method also identifies intermediate signals in addition to classification of signals as positive and negative. Intermediate calls can be informative with respect to identifying the simultaneous presence of sensitive and resistant alleles or infection with multiple different Mycobacterium tuberculosis strains.ResultsTo validate our analysis method 100 DNA isolates of Mycobacterium tuberculosis extracted from cultured patient material collected at the National TB Reference Laboratory of the National Center for Tuberculosis and Lung Diseases in Tbilisi, Republic of Georgia were tested by MLPA. The data generated were interpreted blindly and then compared to results obtained by reference methods. MLPA profiles containing intermediate calls are flagged for expert review whereas the majority of profiles, not containing intermediate calls, were called automatically. No intermediate signals were identified in 74/100 isolates and in the remaining 26 isolates at least one genetic marker produced an intermediate signal.ConclusionBased on excellent agreement with the reference methods we conclude that the new data analysis method performed well. The streamlined data processing and standardized data interpretation allows the comparison of the Mycobacterium tuberculosis MLPA results between different experiments. All together this will facilitate the implementation of the MLPA assay in different settings.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-572) contains supplementary material, which is available to authorized users.
Highlights
Multiplex ligation-dependent probe amplification (MLPA) is a powerful tool to identify genomic polymorphisms
Combined resistance and lineage identification is especially interesting in areas where there is a high prevalence of multidrug resistant TB (MDR-TB) and resistance is associated with specific lineages [2,3,4]
We have previously developed and validated a bead-based multiplex ligation-dependent probe amplification (MLPA) assay and demonstrated its potential to simultaneously identify a range of drug resistance markers, discriminate within the genetic group Mycobacterium tuberculosis (MTB) complex (MTBC), and detect and identify the clinically most relevant non-tuberculous mycobacterial species in cultured isolates [10,11,12]
Summary
Multiplex ligation-dependent probe amplification (MLPA) is a powerful tool to identify genomic polymorphisms. We have previously developed a single nucleotide polymorphism (SNP) and large sequence polymorphisms (LSP)-based MLPA assay using a read out on a liquid bead array to screen for 47 genetic markers in the Mycobacterium tuberculosis genome. After initial diagnosis and before administered standardized empirical therapy timely detection of resistance mutations in the genome of Mycobacterium tuberculosis (MTB) can help clinical decision-making and support infection control efforts. SNP- and LSP-based assays are ideal for combined drug resistance testing and genotyping of MTB. We have previously developed and validated a bead-based multiplex ligation-dependent probe amplification (MLPA) assay and demonstrated its potential to simultaneously identify a range of drug resistance markers, discriminate within the genetic group MTB complex (MTBC), and detect and identify the clinically most relevant non-tuberculous mycobacterial species in cultured isolates [10,11,12]
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