Loop-mediated isothermal amplification (LAMP): early detection of Toxoplasma gondii infection in mice.
BACKGROUND
Toxoplasmosis is a widespread zoonotic parasitic disease that occurs in both animals and humans. Traditional molecular assays are often difficult to perform, especially for the early diagnosis of Toxoplasma gondii infections. Here, we established a novel loop-mediated isothermal amplification targeting the 529 bp repeat element (529 bp-LAMP) to detect T. gondii DNA in blood samples of experimental mice infected with tachyzoites of the RH strain.


RESULTS
The assay was performed with Bst DNA polymerase at 65°C for 1 h. The detection limit of the 529 bp-LAMP assay was as low as 0.6 fg of T. gondii DNA. The sensitivity of this assay was 100 and 1000 fold higher than that of the LAMP targeting B1 gene (B1-LAMP) and nested PCR targeting 529 bp repeat element (529 bp-nested PCR), respectively. The specificity of the 529 bp-LAMP assay was determined using the DNA samples of Trypanosoma evansi, Plasmodium falciparum, Paragonimus westermani, Schistosoma japonicum, Fasciola hepatica and Angiostrongylus cantonensis. No cross-reactivity with the DNA of any parasites was found. The assay was able to detect T. gondii DNA in all mouse blood samples at one day post infection (dpi).


CONCLUSIONS
We report the following findings: (i) The detection limit of the 529 bp-LAMP assay is 0.6 fg of T. gondii DNA; (ii) The assay does not involve any cross-reactivity with the DNA of other parasites; (iii) This is the first report on the application of the LAMP assay for early diagnosis of toxoplasmosis in blood samples from experimentally infected mice. Due to its simplicity, sensitivity and cost-effectiveness for common use, we suggest that this assay should be used as an early diagnostic tool for health control of toxoplasmosis.
Institute of Parasitic Diseases, Zhejiang Academy of Medical Sciences, Hangzhou, China.
Qing-Ming
QM
Kong
Shao-Hong
SH
Lu
Qun-Bo
QB
Tong
Di
D
Lou
Rui
R
Chen
Bin
B
Zheng
Takashi
T
Kumagai
Li-Yong
LY
Wen
Nobuo
N
Ohta
Xiao-Nong
XN
Zhou
D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000097:blood* / Q000235:genetics; D003959:Diamines; D042241:Early Diagnosis; D005260:Female; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D055029:Inverted Repeat Sequences / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000379:methods* / Q000592:standards; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction; D011804:Quinolines; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*; D014123:Toxoplasmosis / Q000175:diagnosis* / Q000469:parasitology
D052160:Benzothiazoles; D017931:DNA Primers; D016054:DNA, Protozoan; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I