Dna was extracted and the b1 t gondii gene was amplified by pcr. Infected definitive hosts cats shed oocysts in feces that. Transcriptomic insights into the early hostpathogen. A 289bp long dna fragment was obtained using the primersg cytb1 5. Oligonucleotide primers and a fluorescencelabeled taqman probe were designed to amplify the t.
The annealing temperature and concentrations of the primers, mgcl 2, and. Toxoplasma gondii, molecular detection, pcr, varies krmc. X0002 ultrasensitive qualitative detection of toxoplasma gondii by real time pcr x0002 is included on p0028 feline diarrhea panel, on p0036. The protozoan toxoplasma gondii is estimated to be carried by one third of the worlds population with infection typically occurring by eating infected meat, contact with a cat that has itself recently been infected, or by transmission from mother to fetus. The infection is the result of toxoplasma crossing the placental barrier in the case of an acute maternal infection. Toxoplasma gondii is an obligate intracellular protozoan parasite that is capable of infecting a variety of intermediate hosts including humans. Quantitative detection of toxoplasma gondii dna in human body. If not ordering electronically, complete, print, and send a microbiology test request t244 with the specimen. Toxoplasma gondii is a species of parasitic protozoa in the genus toxoplasma. Molecular diagnosis of toxoplasma gondii infection in libya. Pcrbased detection of toxoplasma gondii dna in blood and.
In the nested pcr, two sets of primers are used in two successive pcrs. Sensitive and rapid detection of infection with toxoplasma gondii in transplanted immunocompromised patients is crucial for a good prognosis. Toxoplasma gondii, molecular detection, pcr, varies aumc. Toxoplasma gondii, a widely prevalent protozoan parasite, causes serious toxoplasmosis infections in humans and other animals. Toxoplasma gondii detection by the polymerase chain reaction pcr is based on the amplification of the pathogen genome specific region using specific toxoplasma gondii primers. In this study, a new multiplex taqman realtime pcr for detection of t. Toxoplasma is a crescent shaped sporozoan that lives as an intracellular parasite in various tissues of vertebrates and completes its life cycle in a single host. Diagnosis of swine toxoplasmosis by pcr and genotyping of.
Toxoplasma gondii, molecular detection, pcr, blood duke. Toxoplasma gondii is an obligate intracellular protozoan parasite with a global distribution in humans and other warmblooded animals. Toxoplasma gondii infections are prevalent in humans and animals throughout libya. Test classification this test was developed and its performance characteristics determined by mayo clinic in a manner consistent. Although sexual reproduction of the parasite toxoplasma gondii exclusively occurs in the cat intestine, knowledge about the alteration of gene expression in the intestine of cats infected with t. The toxo taqman probe sequence was designed using the software primer. A new set of primers for the detection of toxoplasma gondii in.
Molecular methods are used increasingly for the detection of toxoplasma gondii infection. A realtime quantitative pcr assay to quantify the toxoplasma gondii apicoplast was studied. Invasion of host cells by apicomplexan parasites such as toxoplasma gondii is critical for their infectivity and pathogenesis. Development and validation of a realtime pcr assay for. Test code ptox toxoplasma gondii, molecular detection, pcr, varies performing laboratory mayo clinic laboratories in rochester. Detection of toxoplasma gondii oocysts in fresh vegetables. Toxoplasma gondii can be responsible for congenital toxoplasmosis leading to mild or severe sequelae, and for lifethreatening infections in immunocompromised hosts. Realtime pcr for quantitative detection of toxoplasma gondii. Toxoplasma gondii is a protozoan parasite that infects humans and a wide variety of animals particularly felids, 7 and it is estimated that nearly onethird of the human population has. Detection of toxoplasma gondii dna by pcr in blood samples. The primers and probe were designed using primer express software.
Toxoplasma gondii, molecular detection, pcr, blood. Author summary toxoplasma gondii infects essentially all warmblooded animals due to its broad species and tissue tropism. Pcr detection of toxoplasma gondii in blood has been suggested as a possibly efficient method for the diagnosis of ocular toxoplasmosis ot and furthermore for genotyping the strain involved in the. The forward primer toxof, reverse primer toxor, and taqman probe for realtime pcr amplification were designed with the primerexpress software pe. The protozoan toxoplasma gondii is one of the most common infectious pathogenic parasites and can cause severe medical complications in infants and immunocompromised individuals. Toxoplasma gondii, an obligate intracellular parasite, is an important opportunistic pathogen of immunosuppressed patients.
Diagnosis of toxoplasma gondii infections in newborn farm. Multiplex pcr for typing strains of toxoplasma gondii. Realtime rtpcr for the qualitative detection of toxoplasma gondii this qualitative duplex test simultaneously amplifies a target sequence in the toxoplasma gondii genome, and an endogenous. Two dna fragments are used currently for detecting t. To our surprise, we were unable to detect parasites in the brain either through rtpcr using t. It is a coccidian parasite of cats as final hosts, and all nonfeline warm. Endogenous control primer probe mix brown pre pcr pack pre pcr heat. Toxoplasma gondii, molecular detection, pcr, varies. Triplex pcr using new primers for the detection of toxoplasma gondii.
Toxoplasma gondii, molecular detection, pcr, varies west. Toxoplasma gondii dna pcr national university hospital. Toxoplasma pcr university of washington laboratory. Direct detection of toxoplasma gondii from clinical specimens. An internal control is added to ensure the extraction was performed correctly and the pcr reaction was not inhibited. Almost onethird of the human population carry toxoplasma.
The annealing temperature and concentrations of the primers. Development of a realtime pcr assay for detection of. An internal control is added to ensure the extraction was performed correctly. How a chronic infection that causes a prolonged th1 expansion. This method is simple, rapid, reproducible, and adapted to a large set of isolates. In contrast, polymerase chain reaction pcr assays for toxoplasma have the. This study developed a rapid, sensitive, and specific conventional triplex pcr for the detection of the b1 gene and its1 region of t. Toxoplasma gondii is an obligate, intracellular protozoan parasite with a wide distribution in europe. Extraction of toxoplasma gondii dna from specimen followed by amplification and detection using realtime, quantitative pcr. Sepsis is a severe syndrome that arises when the host response to an insult is exacerbated, leading to organ failure and frequently to death. B 1 gene sequence was amplified using two pairs of primers. Primers were designed to amplify a 305 bp product specific to t.
Toxoplasma gondii, molecular detection, pcr, varies mayo. The polymerase chain reaction pcr technique was used to identify t. Diagnosis of toxoplasmosis and typing of toxoplasma gondii. A multiplex pcr assay was designed for multilocus strain typing of toxoplasma gondii based on length polymorphism of five microsatellite markers. Toxoplasma gondii qpcr as an approach to detecting and simultaneously quantifying pathogens is the method of choice given its advantages in speed, sensitivity and clear cut answersscoring when. Taqman probe sequence was designed using the software primer express ver.
Aging with toxoplasma gondii results in pathogen clearance. Realtime pcr detection of toxoplasma gondii in tissue samples of. The protozoan toxoplasma gondii is estimated to be carried by one third of the. For detection of the parasitic protozoa of toxoplasma gondii. Primers expected molecular weight of pcr products and pcr conditions for b1 gene. Key basic research program of china 973 program grant nos. Current diagnosis is based on detection of toxoplasmaspecific igm and igg. It is a coccidian parasite of cats as final hosts, and all nonfeline warmblooded animals including humans as intermediate hosts.
This test has not been cleared or approved for diagnostic use by the u. Toxoplasma gondii elisa kits the elisa enzymelinked immunosorbent assay is a widely used application for detecting and quantifying proteins and antigens from various samples. Lamb as a potential source of toxoplasma gondii infection. Triplex pcr using new primers for the detection of. Performance testing of pcr assay in blood samples for the. A member of the ferlin calcium sensor family is essential. Identification of toxoplasma gondii by rapid realtime pcr. Development and validation of a realtime pcr assay for the. Development of triplex realtime pcr and detection of toxoplasma. Time pcr detection system, including cfx manager v3. Molecular detection and genotyping of toxoplasma gondii. The relative positions of the primers and taqman probe in the b1 gene for realtime pcr and nested pcr are shown.
Toxoplasma gondii oocyst detection and an estimation of their numbers was performed by conventional pcr and real time qpcr, using specific primers for a 183bp sequence of the t. Catalog id ptox toxoplasma gondii, molecular detection, pcr, varies important note. In toxoplasma, secretion of essential egress, motility, and invasion. We report here the development of a realtime pcrbased assay for the detection of t. In aids patients and transplant patients, this infection may. Investigation of false positives associated with loop.
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