[Results and contributing prospects of molecular probes and genetic amplification in vitro (PCR) in toxoplasmosis].
Toxoplasmosis is one of the latest parasitic diseases to have benefited from molecular biological study (1989). To our knowledge we were one of the first to have cloned several DNA probes using genomic DNA from Toxoplasma gondii. One of these probes, TGR1E, has been sequenced and has enabled the selection and chemical synthesis of 4 oligonucleotides (22 to 25 nucleotides respectively) for use in the Polymerase Chain reaction (PCR). Completely specific for Toxoplasma gondii, they confer remarkable sensitivity allowing detection of quantities of DNA as small as 2 fg (f = 10(-15) g) corresponding to less than 1/50th of the DNA contents in a single tachyzoïte. The method is currently being applied using the in situ hybridization technique. One hundred samples (sera, CSF, broncho-alveolar washings, biopsies), mostly from immunocompromised patients, have been studied by the Southern-blot method. The results are still preliminary but appear very encouraging, especially since serological diagnostic studies during acquired or post-therapeutic immunodeficiencies (transplants) are usually impossible. Nevertheless, as it has already been reported in various other applications, the risk of false positive results requires that PCR be performed with highly rigorous technique, particularly in order to avoid any contamination by foreign DNA. An analysis of restriction profiles by the Southern method has also been carried out in 6 different strains of Toxoplasma gondii. Although a certain number of DNA fragments are common to all strains, some fractions of greater than 1.6 kb could characterize certain isolates. This protocol which is still preliminary is currently in progress and will be carried out in a larger number of cloned strains.(ABSTRACT TRUNCATED AT 250 WORDS)
Ambroise-Thomas
P
P
Département de parasitologie-mycologie médicale et moléculaire, Faculté de Médecine, Université Joseph Fourier-La Tronche Unité Associée CNRS URA 1344.
Cristina
N
N
D001483:Base Sequence; D015139:Blotting, Southern; D015342:DNA Probes / Q000138:chemical synthesis; D005189:False Positive Reactions; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D014123:Toxoplasmosis / Q000175:diagnosis / Q000453:epidemiology / Q000235:genetics*
D015342:DNA Probes
Résultats et perspectives apportés par les sondes moléculaires et par l'amplification génique in vitro (PCR) dans la toxoplasmose.