Establishment Of Loop-mediated Isothermal Amplification For Detecting Of Babesiosis

Babesia is an Apicomplexa protozoan spread by ticks and parasitized in animals' red blood cell. Cattle babesiosis occurs all over the world. China has 14 provinces (regions) which have reported the existence of the disease, endemic in the southern provinces, which cause a great damage to livestock and our national economy. In China, it is reported that five species of Babesia in cattle, that is Babesia bovis, Babesia bigemina, Babesia major, Babesia ovata, Babesia U sp. Cattle, infected with the parasitic disease, are often suffered from anemia, fever, hemoglobinuria, ataxia and other symptoms, even cause death. Hitherto, prevention strategy of this disease is very important.At present, the methods to detection of Babesia bovis including investigation of blood smear, serological examination, conventional PCR detection, etc, have low sensitivity of cross-infection, and unneglected false positive rate of defects. Still, some methods require sophisticated equipment, such as real-time quantitative PCR technique. Loop-mediated isothermal amplification (LAMP) is a novel isothermal nucleic acid amplification method proposed by Notomi in 2000. As sensitivity as 109 copies could be obtained within a short time in a constant temperature condition eploying LAMP. taking cytochrome b of Babesia bovis as a target gene, this study aimed to establish a simple, efficient and fast-detected LAMP method, which could be used for animal babesiosis diagnosis, especially for early diagnosis.We obtained the cytochrome b gene sequence of Babesia bovis using specific primers designed according to the sequence of cyt b of Babesia bovis in NCBI, while that of Babesia bigemina was separated from B. bigemina genomic DNA using specific primers designed according to the sequence of cyt b from B. bovis and found the conserved regions by alignment. Two specific primers were designed online for genus-specific LAMP, according to the principle of LAMP primer designation, followed by the establishement of the LAMP detection method of Babesia. Taking Babesia bovis genomic DNA as the template for LAMP amplification, a 254-bp gene segment of cyt b was amplified and cloned into pMD18-T vector. Using this method, the relative bands could not be detected, when taking the genomic DNA of some species, such as Bovine Theileria sergenti, Babesia martel, Toxoplasma gondii, Sheep Theileria, which showed a expected specifity. And the minimum detection contents in the genomic DNA from both B. bigemina and B. boivs were 0.85 fg/μL and 0.14 fg/μL, respectively, which showed a higher sentivity.Babesia bigemina was currently one of the major common diseases to cow health. A species-specific LAMP method was established to detected B. bigemina. According to the cyt b gene of B. bigemina, four specific primers was designed, followed by the establishment of LAMP, which showed excited specificity. And Babesia bigemina cyt b gene in Babesia could be detected with a minimum content of 0.085 fg/μL. The successful establishment of the detection system could provide a strong technical for the detection, identification and epidemiological survey of the cattle infected with Babesia. a Babesia bovis LAMP detection method was also established in this study. According to the published Babesia bovis cyt b DNA, four specific primers was designed, followed by the establishment of LAMP, which showed excited specificity. And B. bovis cyt b gene in Babesia could be detected with a minimum content of 0.014 fg/μL.. The successful establishment of the detection system could provide a strong technical support for the detection, identification and epidemiological survey of the cattle infected with B. bovisAs stated above, Babesia is one of the major pathogenic pathogens in tropical and subtropical regions. China has 14 provinces (regions) that report the existence of the disease, endemic in the southern provinces. The established Babesia LAMP detection methods showed sound specificity and high sensitivity for epidemio-logical investigation. This method could be applied for detection of Babesiosis in the fields, and possessed a bright applicant perspective.