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

AbstractBovine 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 acid-based amplification method using loop-mediated isothermal amplification (LAMP) in conjunction with a colori-fluorometric 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, specificity, 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 different 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-amplification 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 findings suggested that the developed LAMP method provides an alternative approach for B. bovis detection with sensitivity higher than PCR-AGE diagnostics, high specificity, and the flexibility to use neon, LED, and UV light sources for positive signal observations.