The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China.
The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies with high sensitivity, efficiency, and rapidity, deoxyribonucleic acid (DNA) under isothermal condition in simple incubators. Two primer sets for the LAMP method were designed using the nucleotide sequences of 18S rRNA gene of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 isolated in China. The primers were used to detect parasite DNA extracted from infected blood and purified parasites by LAMP. The specific ladder bands were amplified from the autologous genomic DNA of two Babesia species, respectively, and did not cross-react with the genomic DNA of Theileria sp. China 1, Theileria sp. China 2, B. bovis, Theileria sp. (Japan) and sheep. The LAMP was sensitive enough to detect 0.02 pg and 0.2 pg genomic DNA of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005, respectively, from 10-fold serially diluted samples corresponding to the amount of DNA present in 50 microl of 0.000002% and 0.00002% parasitemic erythrocytes. Furthermore, DNA extracted from blood of intact (non-splenectomized) sheep experimentally infected with Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 was amplified by the LAMP from week 1 to 9 and week 2 and 3 post-infection, respectively, demonstrating the high sensitivity of these primers. Of 365 samples collected from Gansu province, 14.3% (52/365) were positively detected by the LAMP. Of 145 samples collected on filter papers (Whatman) from the grazing sheep in Xinjiang province, 3.5% (5/145) were positive. These results show that the LAMP could be an alternative diagnostic tool for the detection of babesial infection in sheep and goats.
Guan
Guiquan
G
Key Laboratory of Veterinary Parasitology of Gansu Province, Department of Veterinary Parasitology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou Gansu 730046, PR China.
Chauvin
Alain
A
Luo
Jianxun
J
Inoue
Noboru
N
Moreau
Emmanuelle
E
Liu
Zhijie
Z
Gao
Jinliang
J
Thekisoe
Oriel M M
OM
Ma
Miling
M
Liu
Aihong
A
Dang
Zhisheng
Z
Liu
Junlong
J
Ren
Qiaoyun
Q
Jin
Yurong
Y
Sugimoto
Chihiro
C
Yin
Hong
H
D000818:Animals; D001403:Babesia / Q000235:genetics / Q000302:isolation & purification*; D001404:Babesiosis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D001483:Base Sequence; D002681:China; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation / Q000379:methods / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012313:RNA / Q000737:chemistry; D012337:RNA, Ribosomal, 18S / Q000737:chemistry; D012680:Sensitivity and Specificity; D016415:Sequence Alignment / Q000662:veterinary; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000469:parasitology
C034477:RNA primers; D012337:RNA, Ribosomal, 18S; D012313:RNA