Forensic typing of autosomal SNPs with a 29 SNP-multiplex—Results of a collaborative EDNAP exercise

Sánchez, Juan José Martínez; Børsting, Claus; Balogh, Kinga; Berger, Burkhard; Bogus, Magdalena; Butler, John M.; Carracedo, Ángel; Court, Denise Syndercombe; Dixon, Lindsey; Filipović, B; Fondevila, M.; Gill, Peter; Harrison, C.; Hohoff, Carsten; Huel, René; Ludes, Bertrand; Parson, Walther; Parsons, Thomas J.; Petkovski, Elizabet; Phillips, C.; Schmitter, H.; Schneider, Peter M.; Vallone, Peter M.; Morling, Niels

doi:10.1016/j.fsigen.2007.12.002

Cited by 52 publications

(27 citation statements)

References 11 publications

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“…Hence, we developed the multiplex SNaPshot assay to simultaneously identify pathogenic tuberculosis mycobacteria and the five most common NTM species, namely, M. avium, M. intracellulare, M. chelonae, M. gordonae, and M. kansasii, in a single assay. Although the multiplex SNaPshot method for single-nucleotide polymorphism analysis is one of the most stimulating SNP genotyping techniques in genetic research (3,2,7,24,29), this is the first attempt to employ it in identification mycobacterial species. In our study, 468 mycobacterial isolates from clinical specimens were analyzed by multiplex SNaPshot assay.…”

Section: Discussionmentioning

confidence: 99%

Rapid Method for Identification of Six Common Species of Mycobacteria Based on Multiplex SNP Analysis

et al. 2010

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A multiplex method using the SNaPshot technique was developed to screen for six common mycobacterial species: Mycobacteria tuberculosis, M. avium, M. intracellulare, M. chelonae, M. kansasii, and M. gordonae. A total of 468 mycobacterial clinical isolates were subjected to analysis for the presence of the six mycobacterial species by the multiplex SNaPshot method. Of the 468 mycobacterial isolates, 464 (99.15%) could be correctly identified by this assay. The multiplex SNaPshot technique is a promising discriminatory tool for rapid and accurate identification of frequently encountered clinical mycobacterial species.Even though Mycobacterium tuberculosis continues to be a serious health concern worldwide, it has been increasingly recognized that nontuberculous mycobacteria (NTM) are important human pathogens (4,16,23). NTM are ubiquitous organisms, with nearly 100 different species found in soil and water that can act as opportunistic pathogens in humans, causing a wide variety of skin and soft tissue infections, lymphadenitis, and lung disease (6, 15). The early differentiation of M. tuberculosis from NTM and the identification of species among NTM are crucial for immediate implementation of the appropriate therapy because susceptible drugs vary widely among different species (9).Conventionally, identification of mycobacteria is carried out by time-consuming biochemical tests that are not always accurate (5,17,25). Chromatographic techniques such as highperformance liquid chromatography (HPLC), gas-liquid chromatography (GLC), and thin-layer chromatography (TLC) are labor-intensive, difficult, or expensive (21,26). DNA sequence analysis of the 16S rRNA gene region is now regarded as the gold standard for the identification of mycobacteria (13,22,25,27). However, equipment and running costs are high. Simple genotypic assays for the identification of mycobacteria, such as Accuprobe (Gen-Probe Inc., San Diego, CA) (1), , and Genotype Mycobacterium (Hain Diagnostika) (19) are available commercially. Even though these tests are simple, they are often suited for small test volumes and are too expensive for high-throughput laboratories to use in a routine clinical diagnostic setting.In this study, we developed a novel multiplex SNaPshot method using fluorescently labeled terminators and capillary electrophoresis to screen for six common clinically encountered mycobacterial species (M. tuberculosis, M. avium, M. intracellulare, M. chelonae, M. kansasii, and M. gordonae) based on eight single nucleotide polymorphisms (SNPs) located in conserved regions of the 16S rRNA and Hsp65 genes. MATERIALS AND METHODSReference strains and clinical isolates. Thirteen reference strains of mycobacteria and 468 clinical isolates (Table 1) were subjected to the multiplex SNaPshot analysis. The reference strains and 20 clinical isolates were kindly provided by Beijing Thoracic Tumor and Tuberculosis Research Institute. A total of 448 clinical isolates were obtained in our microbiology laboratory, which is the reference laboratory for tub...

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Section: Discussionmentioning

confidence: 99%

Rapid Method for Identification of Six Common Species of Mycobacteria Based on Multiplex SNP Analysis

et al. 2010

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“…The SBE-based SNP typing assay of the SNPforID 52-plex was validated for forensic genetic investigations [4] and it performed better than the most commonly used STR kits when partly degraded DNA samples were investigated [2,4]. Nevertheless, the unspecific addition of dA nucleotides at the end of the PCR products by the Taq polymerase [11] and the fact that the signal strength of the four colours in the SNaPshot 1 kit (AB) is unbalanced makes the interpretation of the results challenging [12].…”

Section: à19mentioning

confidence: 99%

“…The usefulness of SNP typing in forensic genetics has previously been pointed out in several publications [1][2][3]. Short DNA fragments with SNPs can be amplified, which makes it possible to analyze SNPs in partly degraded DNA samples [1,2,4].…”

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Autosomal SNP typing of forensic samples with the GenPlex™ HID System: Results of a collaborative study

Tomàs

Axler-DiPerte

Budimlija

et al. 2011

Forensic Science International: Genetics

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“…The allelic state is then determined after separation of the extension products and the detection of fluorescence by capillary electrophoresis. This SNP typing method is currently widely used in the fields of forensic and population genetics (8,9,25,44,47) and has recently raised interest in clinical research (2,5,17,31,39). It was also applied in a large-scale study for the elucidation of the M. tuberculosis population substructure and evolution (26,27), and a recent study reports on its use for the identification of six common mycobacterial species (M. tuberculosis, M. avium, M. intracellulare, M. chelonae, M. kansasii, and M. gordonae) (55).…”

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Identification and Genotyping of Mycobacterium tuberculosis Complex Species by Use of a SNaPshot Minisequencing-Based Assay

et al. 2010

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The aim of the present study was to investigate the use of the SNaPshot minisequencing method for the identification of Mycobacterium tuberculosis complex (MTBC) isolates to the species level and for further genotyping of M. tuberculosis isolates. We developed an innovative strategy based on two multiplex allelespecific minisequencing assays that allowed detection of eight species-specific and eight lineage-specific single nucleotide polymorphisms (SNPs). Each assay consisted of an eightplex PCR amplification, followed by an eightplex minisequencing reaction with the SNaPshot multiplex kit (Applied Biosystems) and, finally, analysis of the extension products by capillary electrophoresis. The whole strategy was developed with a panel of 56 MTBC strains and 15 negative controls. All MTBC strains tested except one M. africanum clinical isolate were accurately identified to the species level, and all M. tuberculosis isolates were successfully further genotyped. This two-step strategy based on SNaPshot minisequencing allows the simultaneous differentiation of closely related members of the MTBC, the distinction between principal genetic groups, and the characterization of M. tuberculosis isolates into one of the seven prominent SNP cluster groups (SCGs) and could be a useful tool for diagnostic and epidemiological purposes.Although tuberculosis (TB) is an age-old disease, it still represents a major health problem worldwide, accounting for nearly 2 million deaths annually (World Health Organization, Tuberculosis Facts 2009 [http://www.who.int/tb/publications /factsheets/en/]). Besides the need for an improved therapy and for new vaccines, effective TB control requires the initiation of appropriate therapy as well as an increased understanding of its epidemiology.The causative agents of TB in humans and animals, including Mycobacterium tuberculosis, M. africanum, M. bovis, M. canettii, M. microti, M. caprae, and M. pinnipedii, form the Mycobacterium tuberculosis complex (MTBC). Although they differ widely in terms of their host tropisms, phenotypes, and pathogenicities, all members of the MTBC are closely related genetically (51). M. tuberculosis species, the most common pathogen in humans, can be further divided into genetic groups that also show differences in their levels of virulence, immunogenicities, and geographical distributions (21). On the one hand, it is important to differentiate MTBC species to distinguish between strict human and zoonotic TB and to initiate an appropriate therapy (15). In particular, the distinction between M. tuberculosis and M. bovis is necessary, as the latter species is naturally resistant to the antituberculous drug pyrazinamide (48). On the other hand, genotyping of M. tuberculosis isolates is useful as a means of addressing evolutionary questions but also as a means of surveying the transmission dynamics of this pathogen and identifying new outbreaks.Identification of MTBC isolates to the species level is so far routinely performed by analysis of the phenotypic and biochemical ch...

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Forensic typing of autosomal SNPs with a 29 SNP-multiplex—Results of a collaborative EDNAP exercise

Cited by 52 publications

References 11 publications

Rapid Method for Identification of Six Common Species of Mycobacteria Based on Multiplex SNP Analysis

Rapid Method for Identification of Six Common Species of Mycobacteria Based on Multiplex SNP Analysis

Autosomal SNP typing of forensic samples with the GenPlex™ HID System: Results of a collaborative study

Identification and Genotyping of Mycobacterium tuberculosis Complex Species by Use of a SNaPshot Minisequencing-Based Assay

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