title: [Laboratory-based evaluation of loop-mediated isothermal amplification (LAMP) to detect Cryptosporidium oocyst and Giardia lamblia cyst in stool specimens].
abstract: To establish an alternative and more sensitive test method to detect oocyst of Cryptosporidium parvum and cyst of Giardia lamblia in clinical stool specimens, loop-mediated isothermal amplification (LAMP) was evaluated. Minimum cell concentrations at which LAMP assay could detect C. parvum oocyst and G. lamblia cyst were determined as 6.25 x 10(-1) and 3.12 x 10(-1) cells/assay when the stool specimens were spiked with the respective parasites. The results indicated 400 times higher sensitivities or more when compared to the microscopic readings. Twenty and nineteen diarrhea stool specimens spiked with C. parvum oocyst or G. lamblia cyst were assayed by LAMP. The results indicated that 14 (70%) and 16 (84%) samples successfully resulted in positive readings. But the remaining 6 and 3 samples were read as negative probably due to residual stool color. However, further dilutions of DNA extraction samples and addition of bovine serum albumin to LAMP reaction mixture showed positive effects on the occurrence of false-negative readings. With these results, we can conclude that the LAMP assay provides us an accurate and highly sensitive test method to detect C. parvum oocyst and cyst of G. lamblia, in place of labor-intensive and experience-dependent microscopic examination, in clinical laboratories.
authors.affiliation: Clinical Laboratories, University Hospital of the Ryukyus, Nakagami-gun, Okinawa-pref. 903-0215, Japan. tamarky@med.u-ryukyu.ac.jp
authors.forename: Tamami T
authors.initials: TT
authors.lastname: Nago
authors.forename: Yoshino T
authors.initials: YT
authors.lastname: Tokashiki
authors.forename: Kyoko
authors.initials: K
authors.lastname: Kisanuki
authors.forename: Isamu
authors.initials: I
authors.lastname: Nakasone
authors.forename: Nobuhisa
authors.initials: N
authors.lastname: Yamane
mesh_terms: D000818:Animals; D003458:Cryptosporidium / Q000235:genetics / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000302:isolation & purification; D005243:Feces / Q000469:parasitology*; D016829:Giardia lamblia / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan