Detection of Babesia bovis using loop‑mediated isothermal amplifcation (LAMP) with improved thermostability, sensitivity and alternative visualization methods

Bovine babesiosis is one of the most economically important tick-borne diseases in tropical and subtropical countries. A conventional microscopic diagnosis is typically used because it is inexpensive and expeditious. However, it is highly dependent on well-trained microscopists and tends to be
incapable of detecting subpatent and chronic infections. Here, we developed a novel nucleic acidbased amplifcation method using loop-mediated isothermal amplifcation (LAMP) in conjunction with a colori-fuorometric dual indicator for the rapid and accurate detection of Babesia bovis based
on the mitochondrial cytochrome b gene. We aimed to improve the thermostability, sensitivity, specifcity, and alternative visualization of LAMP-based methods. We assessed its diagnostic performance compared to two conventional PCR agarose gel electrophoresis (PCR-AGE) methods.
The thermostability of LAMP reaction mixtures and DNA templates in variable conditions was also assessed. In addition, we evaluated alternative visualization methods using diferent light sources including neon, LED, and UV lights. We found that the LAMP-neon was ten times more sensitive than the PCR-AGE, while the LAMP-LED and LAMP-UV were 1,000 times more sensitive. The current LAMP
method showed no cross-amplifcation with uninfected cattle DNA or other common blood parasites in cattle, including Babesia bigemina, Theileria orientalis, Anaplasma marginale, and Trypanosoma evansi. In addition, the developed LAMP method has good thermostability and the potential for
on-site utility as B. bovis DNA could still be detected up to 72 h after initial preparation. Our fndings
suggested that the developed LAMP method provides an alternative approach for B. bovis detection
with sensitivity higher than PCR-AGE diagnostics, high specifcity, and the fexibility to use neon, LED,
and UV light sources for positive signal observations.