Development of loop-mediated isothermal amplification (LAMP) for detection of Babesia gibsoni infection in dogs.
Diagnosis of canine babesiosis, caused by Babesia gibsoni is difficult, especially in chronically infected dogs. A loop mediated isothermal amplification (LAMP) assay was developed and standardized by using four oligonucleotide primers targeting the hypervariable region of 18S rRNA gene (GenBank Acc. no. KC461261). The primers specifically amplified B. gibsoni DNA, while no amplification was detected with DNA from non-infected dogs as well as from dogs infected with Babesia canis vogeli, Hepatozoon canis, Ehrlichia canis and Trypanosoma evansi. The assay could detect 1.35 × 10(-7) parasitaemia and 10(-4) dilution of recombinant plasmid, equivalent to 12 pg of target DNA. All the samples were tested by nested PCR as well as LAMP assay. LAMP was found to be 10 times more sensitive than nested PCR targeting the same gene. Out of 75 suspected field samples, collected from different parts of the country, LAMP could detect B. gibsoni in 43 samples, while nested PCR and microscopy could detect 37 and 23 samples, respectively. High sensitivity, specificity and rapidity of LAMP assay may be exploited for screening large number of samples in a field setting.
Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243 122, India.
Mrityunjay
M
Mandal
Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243 122, India. Electronic address: banerjeeparth62@gmail.com.
Partha S
PS
Banerjee
Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243 122, India.
Saroj
S
Kumar
Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243 122, India.
Hira
H
Ram
Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243 122, India.
Rajat
R
Garg
Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243 122, India.
Abhijit M
AM
Pawde
D000818:Animals; D001403:Babesia / Q000145:classification / Q000302:isolation & purification*; D001404:Babesiosis / Q000175:diagnosis*; D004269:DNA, Bacterial / Q000235:genetics; D004283:Dog Diseases / Q000175:diagnosis / Q000469:parasitology*; D004285:Dogs; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000662:veterinary*; D012329:RNA, Bacterial / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity; D013045:Species Specificity
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012337:RNA, Ribosomal, 18S
Babesia gibsoni; Detection threshold; LAMP; Nested PCR