Detection of Toxoplasma oocysts from soil by modified sucrose flotation and PCR methods.
A detection method of Toxoplasma gondii oocysts from soil was evaluated using the sucrose flotation technique with modification involving addition of 0.1% gelatin into washing and floating solutions. PCR was performed on untreated samples and after treatment with polyvinylpyrrolidone (PVP), heating and cooling, and NaCl. The addition of gelatin in the sucrose solution yielded a higher number of oocysts. A very thin band was observed when DNA extract was diluted to 1:1024, indicating the presence of PCR inhibitor in the soil. PCR performed on untreated DNA, on PVP-treated, and on PVP-treated with heating and cooling without added bovine serum albumin (BSA) showed a band only at higher dilutions (1:1024 and 1:512) but at a much lower dilution (1:8) with BSA. In contrast, DNA treated with all three agents showed a band at a much lower dilution (1:64), even without added BSA, and no dilution was required when BSA was added. The PCR inhibitors present in the soil were removed by employing various treatment procedures during DNA extraction, and BSA in PCR. Furthermore, the detection limit with the method was 1 oocyst/g of soil, indicating that this method is useful in epidemiological studies.
Matsuo
Junji
J
Department of Medical Technology, Faculty of Health Science, Kobe University School of Medicine, Kobe, Japan.
Kimura
Daisuke
D
Rai
Shiba Kumar
SK
Uga
Shoji
S
D000818:Animals; D002415:Cats; D002458:Cell Fractionation / Q000379:methods; D002498:Centrifugation; D016054:DNA, Protozoan / Q000032:analysis*; D005243:Feces / Q000469:parasitology; D007564:Japan; D033661:Oocysts / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012822:Silicon Dioxide / Q000032:analysis; D012987:Soil / Q000469:parasitology*; D012996:Solutions; D013395:Sucrose; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*
D016054:DNA, Protozoan; D012987:Soil; D012996:Solutions; D013395:Sucrose; D012822:Silicon Dioxide