The Patterns of Restriction Fragment of Several Enzymes to Distinguish Toxoplasma gondii Isolates Virulent and Avirulent Strains using GRA1 and GRA7 Genetic Marker (original) (raw)
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International Journal of One Health, 2021
Background and Aim: Toxoplasma gondii is a unicellular coccidian parasite distributed globally and is an important zoonotic pathogen. Approximately 30% of the human population worldwide is chronically infected with T. gondii. The pathogenicity of this species depends on the type originating from the clonal population. Techniques for more accurately determining the type of T. gondii have recently been developed using genetic markers. Specifically, T. gondii has been typed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This study aimed to identify sets of PCR-RFLP markers that have high power to discriminate genotyping of T. gondii and are easy to use and are easy to use. The objective of this study was to characterize virulent strain isolates of T. gondii by PCR-RFLP using 10 markers with DdeI. Materials and Methods: T. gondii tachyzoites (RH virulent strain) were derived from culture cells at the Indonesian Research Center for Veterinary Sciences. Genotyping was performed on T. gondii DNA extracted from cell cultured tachyzoites using 10 genetic markers of PCR-RFLP, namely, B1#1, B1#2, B1#3, SAG1#1, SAG1#2, P30, BAG1, ROP1, GRA1, and GRA7, with digestion using the restriction enzyme DdeI. Results: The 10 genes were amplified by PCR. Among them, three genetic markers, B1#3, ROP1, and GRA1, were genotyped by the PCR-RFLP using restriction enzyme DdeI. Overall, the findings showed that the specific RFLP profile of digestion of gene regions by DdeI could be used as a specific marker for the virulent biotype causative of toxoplasmosis. In addition, virulent strains of T. gondii can be easily detected by these markers. Conclusion: Three pairs of primers (B1#3, ROP1, and GRA1) with DdeI have proven useful for the diagnosis of acute toxoplasmosis (virulent strain biotype I). This proposed method is relatively simple, rapid, cheap, and can be performed in most laboratories, providing a practical approach for the routine analysis of T. gondii strains.
International Journal for Parasitology, 2001
The position of mobile genetic elements (MGE) within eukaryotic genomes is often highly variable and we have exploited this phenomenon to develop a novel approach to strain differentiation in Toxoplasma gondii. Two PCR based strategies were designed in which speci®c primers were used to amplify T. gondii MGE's revealing information on element size and positional variation. The ®rst PCR strategy involved the use of a standard two primer PCR while the second strategy used a single speci®c primer in a step-up PCR protocol. This approach was applied to T. gondii reference strains which were either acute virulent or avirulent to mice. The use of a standard two primer PCR reaction revealed the presence of a virulence related marker in which all avirulent strains possessed an additional 688 bp band. The single primer PCR strategy demonstrated that all virulent strains had identical banding patterns suggesting invariance within this group of strains. However, all avirulent strains had different banding patterns indicating the presence of a number of individual lineages within this group. The applicability and sensitivity of MGE-PCR in epidemiological studies was demonstrated by direct ampli®cation of T. gondii from sheep tissue samples. All sheep isolates, tested in this way, gave identical banding patterns suggesting the presence of an endemic Toxoplasma strain on this farm. q
Journal of Clinical Microbiology, 2001
Genetic analysis of the SAG2 locus was performed to determine the prevalence of the different genotypes of Toxoplasma gondii (strain types I, II, and III) associated with human toxoplasmosis in Spain. This determination was made directly from primary clinical samples, obviating the previous process of isolation in mice or cell culture. A total of 34 isolates of T. gondii, collected from immunocompromised patients and congenital infection cases, were analyzed. Restriction fragment length polymorphism in PCR-amplified SAG2 products was used to group strains into one of the three genotypes of T. gondii. Complete characterization of the SAG2 gene was successful in 76.5% of the cases, demonstrating the feasibility of direct genotype analysis from clinical samples of different origins. Strains of T. gondii type II were the most prevalent in immunocompromised patients, with 52% of cases, while strains of type I were present in 75% of the congenital infection cases. These data differ from previous reports that show type II strains to be mostly associated with all kinds of human toxoplasmosis. These differences might be an effect of selection in the process of culture and isolation of the samples performed by other researchers prior to strain characterization.
PCR–RFLP based genotyping of Indian isolates of Toxoplasma gondii
Journal of Parasitic Diseases, 2016
Toxoplasma gondii is an apicomplexan parasite capable of infecting a wide variety of warm-blooded animals, including birds and humans and is zoonotically important too. Felidae serve its definitive hosts and most infections are inoccous while in various intermediate hosts (e.g. sheep), it is responsible for abortion, still births. Humans which are immune compromised are also susceptible to toxoplasmosis. Most of the epidemiological studies have revealed it to be belonging to three clonal types with exceptions in South Africa having atypical isolates. Current genotyping was carried out at 11 genetic loci (SAG1, 5 0-SAG2, 3 0-SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358 and PK1) using multiplexnested polymerase chain reaction restriction fragment length polymorphism (Mn-PCR-RFLP). SAG1, alt SAG2, SAG3, BTUB, GRA6, C22-8, C29-2, L358 and PK1 could differentiate our strain/isolates as type I (T. gondii RH) and type III (T. gondii isolates from Chennai and Izatnagar). 5 0 SAG2 and 3 0 SAG2 in combination confirmed these as above mentioned genotypes. Further, the T. gondii RH was assigned Toxo DB#10 and local isolates of T. gondii were assigned Toxo DB#2. The present study is the first report on existence of Type III T. gondii lineage from animal population of Indian subcontinent based on PCR-RFLP.
Iranian Journal of Parasitology
Background: Soil is one of the environmental sources of Toxoplasma gondii oocysts. The other hand, genotype of the parasite is one of the important factors for its pathogenicity. Due to the importance of toxoplasmosis on public health, this study aimed to isolation and genotyping of T. gondii in environmental soil samples of Mazandaran Province, north of Iran. Methods: Overall, 192 soil samples were collected from different areas in Mazandaran Province from Apr to Sep 2014. The flotation method was used for recovering oocysts. Then, soil samples were investigated for DNA detection of T. gondii using nested PCR of RE gene, genotyping with Semi-nested PCR of GRA6 gene and restriction fragment length polymorphism (RFLP) analysis. Results were analyzed using Chi-squared test. A significant difference was considered with a P<0.05. Results: From 192 soil samples, T. gondii DNA was detected in 150 samples (78.1%). Then genotype of 23 samples was determined (91.3% type I and 8.7% type II...
Molecular Detection and Genotyping of Toxoplasma gondii from Clinical Samples
Toxoplasmosis - Recent Advances, 2012
Toxoplasmosis-Recent Advances 104 2. Molecular diagnostics 2.1. Methodology Conventional PCR was, in the beginning, the molecular detection method of choice for the majority of laboratories dealing with the diagnosis of toxoplasmosis and it was based on both in-house protocols and commercial kits (Lavrard et al., 1995). To increase the sensitivity of molecular diagnostics of toxoplasmosis nested PCR was introduced, although in recent years real-time PCR has shown a significantly higher sensitivity as well as specificity
DNA extraction and PCR assays for detection of Toxoplasma gondii
APMIS, 2004
For detection of Toxoplasma gondii we compared the sensitivity of two different DNA extraction methods and three different PCR assays. Sensitivities of DNA extraction by QIAamp DNA mini Kit or MagNa pure followed by PCR, nested PCR and oligochromatography or Light Cycler PCR using either SYBR green chemistry or TaqMan probe were compared. No significant difference between extraction methods was found using pure T. gondii tachyzoites. Spiked blood samples, 10 4 to 10 parasites per sample, generated no difference in sensitivity between the two DNA extraction methods when analysed by nested PCR detected by oligochromatography or analysed by Light Cycler PCR TaqMan. In spiked blood samples Light Cycler PCR SYBR green was unable to detect the parasite and a reduction in sensitivity was observed with the TaqMan assay. Conventional PCR was more sensitive when DNA was extracted from the spiked samples using the QIAamp DNA mini Kit. Conventional and nested PCR were found to be more sensitive than Light Cycler PCR TaqMan using the QIAamp DNA mini Kit. It was not possible to use Light Cycler PCR SYBR green in blood samples. Conventional PCR was more sensitive for detection of T. gondii in spiked blood samples using QIAamp DNA mini Kit DNA extraction, suggesting that the choice of DNA extraction method may affect PCR assays differently.
Acta parasitologica / Witold Stefański Institute of Parasitology, Warszawa, Poland, 2014
PCR-RFLP and nucleotide sequencing based genotyping of Toxoplasma gondii Indian isolates (Izatnagar and Chennai isolates and Chennai clone) vis-a vis RH-IVRI strain was conducted by targeting GRA6 as genetic marker. The 791 bp GRA6 product was PCR amplified from the genomic DNA of different T. gondii Indian isolates, including the RH-IVRI strain. Tru1I restriction endonuclease based PCR-RFLP of GRA6 sequence produced polymorphic digestion pattern that discriminated the virulent RH-IVRI strain (as type I) from the moderately virulent local isolates as type III. The PCR amplicon of T. gondii GRA6 from RH-IVRI strain as well as from the local isolates were cloned in cloning vector and custom sequenced. The nucleotide and deduced amino acid sequences of T. gondii isolates were aligned with that of the type I, II and III strains (RH, BEVERLEY, ME49, C56, TONT and NED) available in public domain and analyzed in silico using MEGA version 4.0 software. Nucleotide sequencing and phylogenetic...