Multiplex PCR for Typing Strains of
            <i>Toxoplasma gondii</i>

Ajzenberg, Daniel; Dumètre, Aurélien; Dardé, Marie‐Laure

doi:10.1128/jcm.43.4.1940-1943.2005

Cited by 88 publications

(62 citation statements)

References 17 publications

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“…Using multilocus analysis, all Tunisian isolates examined in the present study, except one, harbored recombinant I/II and/or I/III strain, which is in concordance with the very few results reported in Ugandan HIV patients and other patients of African origin. 10,11,21 Sequencing of different alleles revealed a greater homology than those previously reported in African free-ranging chickens. 22,23 Although the very limited clinical data in the present study cannot allow hypothesis testing about strain virulence, it is important to note the presence of strains with close homology to the natural recombinant I/III P-Br strain that was incriminated in severe cases in South America.…”

Section: Discussionmentioning

confidence: 46%

“…Recently, multiplex PCR of microsatellites for typing was developed. 21 However, this method has some limitations; a special sequencer and analysis software are needed, and it has a low level of analytic sensitivity (at least 50 parasites in a sample). In contrast, the multilocus PCR-RFLP genotyping method developed by Khan and others 14 offers the advantages that it is simple, can be conducted on small amounts of different types of samples, and has good sensitivity (detecting between 5 and 10 parasites).…”

Section: Discussionmentioning

confidence: 99%

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Direct Genotypic Characterization of Toxoplasma gondii Strains Associated with Congenital Toxoplasmosis in Tunisia (North Africa)

Boughattas¹,

Ben-Abdallah²,

Siala³

et al. 2010

The American Society of Tropical Medicine and Hygiene

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Here, we determined the Toxoplasma gondii genotype in amniotic fluid, placenta, and cerebrospinal fluid samples from 14 congenital toxoplasmosis cases in Tunisia, North Africa. Direct genotypic characterization of T. gondii strains was performed by polymerase chain reaction (PCR) amplification of six genetic markers (3'SAG2, 5' SAG2, SAG3, BTUB, GRA6, and APICO) and thereafter, was analyzed by restriction fragment-length polymorphism (RFLP). Samples were sequenced to resolve strain type whenever there were unclear enzyme digestion results. Multilocus analysis revealed that only one specimen harbored the type I allele in all studied loci, whereas the 13 others gave mixed genotype results with different alleles at different markers. Seven specimens produced RFLP profile of the recombinant strains I/III, and three produced a profile of I/II recombinant strains. The last three specimens produced complex digestion patterns. In these cases, sequence analysis revealed double peaks at known polymorphic sites, indicating the presence of multiple alleles.

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Section: Discussionmentioning

confidence: 46%

Section: Discussionmentioning

confidence: 99%

Direct Genotypic Characterization of Toxoplasma gondii Strains Associated with Congenital Toxoplasmosis in Tunisia (North Africa)

Boughattas¹,

Ben-Abdallah²,

Siala³

et al. 2010

The American Society of Tropical Medicine and Hygiene

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“…From each animal, DNA extraction was performed from a mixture of several tissue types (mainly spleen, lymph node, and liver) and repeated from the liver only. The analysis of length polymorphism at seven microsatellite markers was performed using the method of Ajzenberg et al (2005), with minor modifications. We used six microsatellite markers (B18, TUB2, TgM-A, W35, B17, and M33) for genotyping and a seventh marker (M48) for further characterization (Blackston et al, 2001;Ajzenberg et al, 2005).…”

Section: Haresmentioning

confidence: 99%

“…The analysis of length polymorphism at seven microsatellite markers was performed using the method of Ajzenberg et al (2005), with minor modifications. We used six microsatellite markers (B18, TUB2, TgM-A, W35, B17, and M33) for genotyping and a seventh marker (M48) for further characterization (Blackston et al, 2001;Ajzenberg et al, 2005). The primers were synthesized by Applied Biosystems (Warrington, UK): the forward primers were 59-end labeled with either 6-carboxyfluorescein (B18, TUB2, and M48), hexachlorofluorescein (TgM-A, W35, and B17), or 2,79,89-benzo-59-fluoro-29,4,7-trichloro-5-carboxyfluorescein (M33).…”

Section: Haresmentioning

confidence: 99%

Natural Toxoplasma Gondii Infections in European Brown Hares and Mountain Hares in Finland: Proportional Mortality Rate, Antibody Prevalence, and Genetic Characterization

Jokelainen

Isomursu

Näreaho

et al. 2011

Journal of Wildlife Diseases

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ABSTRACT:In material examined postmortem in Finland from May 2006 to April 2009, acute generalized toxoplasmosis was the immunohistochemically confirmed cause of death in 14 (8.1%) of 173 European brown hares (Lepus europaeus) and four (2.7%) of 148 mountain hares (Lepus timidus). Sera from 116 of the European brown hares and 99 of the mountain hares were screened with a commercial direct agglutination test for Toxoplasma gondii-specific IgG antibodies at a dilution of 1:40. All sera from cases of fatal toxoplasmosis had high titers of antibodies reactive to T. gondii. In contrast, none of 107 European brown hares and four (4%) of 96 mountain hares that died of other causes were antibody-positive. The proportional mortality rates and the T. gondii antibody prevalences among noncases differed significantly between the two host species (P,0.05). Direct genetic characterization of the causative agent was performed on DNA extracted from formalin-fixed, paraffin-embedded tissue of the hares with fatal toxoplasmosis. Based on the results with six microsatellite markers (B18, TUB2, TgM-A, W35, B17, and M33; all six in 15 cases and four in three cases), all the cases were caused by T. gondii genotype II; the size of the PCR product at the seventh marker (M48) varied (213-229 base pairs). The presence of T. gondii genotype II, which is endemic in Europe, is now confirmed in Finnish wildlife: Natural infections with T. gondii parasites belonging to this widespread genotype caused fatal generalized toxoplasmosis in the two species of wild hares.

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“…The commonly used multilocus methods for genotyping Toxoplasma gondii strains employ PCR-restriction fragment length polymorphism (PCR-RFLP) analysis at 10 markers (16), sequencing at 8 introns of 5 loci (14), and length polymorphism analysis of 5 microsatellite (MS) markers (4). Advantages and disadvantages of these different markers have been reviewed elsewhere (16).…”

mentioning

confidence: 99%

Genotyping of Toxoplasma gondii Isolates with 15 Microsatellite Markers in a Single Multiplex PCR Assay

et al. 2010

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We developed an easy-to-use method for genotyping Toxoplasma gondii isolates in a single multiplex PCR assay with 15 microsatellite markers. This method was validated by testing 26 reference isolates that had been characterized with other sets of markers.The commonly used multilocus methods for genotyping Toxoplasma gondii strains employ PCR-restriction fragment length polymorphism (PCR-RFLP) analysis at 10 markers (16), sequencing at 8 introns of 5 loci (14), and length polymorphism analysis of 5 microsatellite (MS) markers (4). Advantages and disadvantages of these different markers have been reviewed elsewhere (16). Based on our experience within the Toxoplasma Biological Resource Center (BRC ToxoBS) and the National Reference Center for Toxoplasmosis, it appears that screening of a large number of clinical isolates for T. gondii strain typing should rely on an easy-to-use method with two different levels of discrimination. The first step of discrimination is the performed at the typing level: that is, employing the ability of markers to distinguish the major clonal lineages from atypical strains. In areas with a marked clonal population structure such as Europe, the genetic screening of clinical isolates should rapidly identify basic type II or III strains, which represent the majority of isolates, and atypical strains, which are the exception to the rule (3, 11). In this context, the identification of atypical strains is of clinical and epidemiological importance because atypical strains are usually associated with severe disease outcomes and contamination of individuals by non-European strains either during residence abroad or after consumption of imported meat (5, 9, 10). The second level of discrimination is the fingerprinting level, representing a high degree of discriminatory power for differentiating closely related strains belonging to the same lineage. This high-resolution analysis is required for identifying laboratory contaminations for diagnosis issues and for establishing a common source of infection among different infected individuals in an outbreak (7,8).Microsatellite (MS) sequences are tandem repeats of short (1 to 6 bp) DNA motifs that are ubiquitous in eukaryotic genomes and undergo length changes due to insertion or deletion of one or multiple repeat units. The most commonly proposed mutation mechanism for MS sequences is strand slippage, occurring predominantly during replication (13). The numbers of repeat motifs differ in a population, thereby creating multiple alleles at an MS locus. MS loci are amplified by PCR using fluorescently labeled forward and unlabeled reverse primers. The dye-labeled products are separated by size using automated electrophoresis and identified by fluorescence detection. We previously designed a multiplex PCR assay with 5 MS markers that were able to reach the typing level but not the fingerprinting level of discrimination between strains (4). Here we developed an easy-to-use and rapid genotyping method which aimed to ensure both levels of genetic discrimina...

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Multiplex PCR for Typing Strains of Toxoplasma gondii

Cited by 88 publications

References 17 publications

Direct Genotypic Characterization of Toxoplasma gondii Strains Associated with Congenital Toxoplasmosis in Tunisia (North Africa)

Direct Genotypic Characterization of Toxoplasma gondii Strains Associated with Congenital Toxoplasmosis in Tunisia (North Africa)

Natural Toxoplasma Gondii Infections in European Brown Hares and Mountain Hares in Finland: Proportional Mortality Rate, Antibody Prevalence, and Genetic Characterization

Genotyping of Toxoplasma gondii Isolates with 15 Microsatellite Markers in a Single Multiplex PCR Assay

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