A rapid multiplex assay for nucleic acid-based diagnostics

Deshpande, Alina; Gans, Jason; Graves, Steven W.; Green, Lance; Taylor, Laura K.; Kim, Heung-Bok; Kunde, Yuliya A.; Léonard, Pascale M.; Li, Po-E; Mark, Jacob A.; Jiang, Song; Vuyisich, Momchilo; White, Paul S.

doi:10.1016/j.mimet.2009.12.001

Cited by 40 publications

(69 citation statements)

References 19 publications

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“…MLPA assays or MLPA-like assays have been developed and reported by others for TB [20], other infectious agents [13–15], human DNA or RNA [16–18]. However, the bead-based tuberculosis-specific MLPA is unique in having a multiplexing capacity of up to 50 markers and combined detection of drug resistance and MTB lineage in a single assay.…”

Section: Introductionmentioning

confidence: 99%

“…As we perform a consistency check some of these calls will be identified, since many TB lineage markers are mutually exclusive, but for drug resistance markers this type of checking is not appropriate as mutations may occur in any lineage [19] and multiple mutations can be present in different combinations. To overcome this issue different approaches have been applied; for example adjusting the threshold value for all markers individually, making use of the signal-to-noise-ratio [15], reference genes [18], or calculating allelic ratios [20]. Here we have applied intra-sample normalization and inter-sample correction to allow low confidence calls to be identified and flagged for scrutiny.…”

Section: Introductionmentioning

confidence: 99%

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Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates

et al. 2014

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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.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates

et al. 2014

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“…This multiplex oligonucleotide ligation-PCR (MOL-PCR) was used for detection of 13 signatures of the biothreat agents Bacillus anthracis, Yersinia pestis, and Francisella tularensis. 40 Such PCR-based BBMAs have been previously described and diagrammed in more detail elsewhere. 68,74,82,102,107 All the aforementioned genotyping BBMAs can be used for host SNP genotyping.…”

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confidence: 99%

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

et al. 2013

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Abstract. Bead-based multiplex assays (BBMAs) are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several to 50-500 analytes within a single, small sample volume). Currently, few assays are commercially available for veterinary applications, but they are available to identify and measure various cytokines, growth factors and their receptors, inflammatory proteins, kinases and inhibitors, neurobiology proteins, and pathogens and antibodies in human beings, nonhuman primates, and rodent species. In veterinary medicine, various nucleic acid and protein-coupled beads can be used in, or for the development of, antigen and antibody BBMAs, with the advantage that more data can be collected using approximately the same amount of labor as used for other antigen and antibody assays. Veterinary-related BBMAs could be used for detection of pathogens, genotyping, measurement of hormone levels, and in disease surveillance and vaccine assessment. It will be important to evaluate whether BBMAs are "fit for purpose," how costs and efficiencies compare between assays, which assays are published or commercially available for specific veterinary applications, and what procedures are involved in the development of the assays. It is expected that many veterinary-related BBMAs will be published and/or become commercially available in the next few years. The current review summarizes the BBMA technology and some of the currently available BBMAs developed for veterinary settings. Some of the human diagnostic BBMAs are also described, providing an example of possible templates for future development of new veterinary-related BBMAs.

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“…Last but not least, compared to GeneXpert (estimated unitary cost, $17 U.S.), the estimated cost of spoligoriftyping could fall below $10, i.e., allowing worldwide implementation in specialized TB labs for an effective MDR-TB control strategy. Together with other tests, such as the MLPA-TB assay (3a, 29), high-throughput nucleic acid-based tests may become interesting alternative methods to phenotypic DST, especially if they become applicable not only to DNA extracted from culture but also to clinical samples (11).…”

Section: Discussionmentioning

confidence: 99%

“Spoligoriftyping,” a Dual-Priming-Oligonucleotide-Based Direct-Hybridization Assay for Tuberculosis Control with a Multianalyte Microbead-Based Hybridization System

et al. 2012

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We developed “spoligoriftyping,” a 53-plex assay based on two preexisting methods, the spoligotyping and “rifoligotyping” assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.

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A rapid multiplex assay for nucleic acid-based diagnostics

Cited by 40 publications

References 19 publications

Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates

Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

“Spoligoriftyping,” a Dual-Priming-Oligonucleotide-Based Direct-Hybridization Assay for Tuberculosis Control with a Multianalyte Microbead-Based Hybridization System

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