ZnO Nanolower-Based NanoPCR as an Efficient Diagnostic Tool for Quick Diagnosis of Canine Vector-Borne Pathogens

Abstract

: Polymerase chain reaction (PCR) is a unique technique in molecular biology and biotechnology for amplifying target DNA strands, and is also considered as a gold standard for the diagnosis of many canine diseases as well as many other infectious diseases. However, PCR still faces many challenges and issues related to its sensitivity, specificity, efficiency, and turnaround time. To address these issues, we described the use of unique ZnO nanoflowers in PCR reaction and an efficient ZnO nanoflower-based PCR (nanoPCR) for the molecular diagnosis of canine vector-borne diseases (CVBDs). A total of 1 mM of an aqueous solution of ZnO nanoflowers incorporated in PCR showed a significant enhancement of the PCR assay with respect to its sensitivity and specificity for the diagnosis of two important CVBDs, Babesia canis vogeli and Hepatozoon canis. Interestingly, it drastically reduced the turnaround time of the PCR assay without compromising the yield of the amplified DNA, which can be of benefit for veterinary practitioners for the improved management of diseases. This can be attributed to the favorable adsorption of ZnO nanoflowers to the DNA and thermal conductivity of ZnO nanoflowers. The unique ZnO nanoflower-assisted nanoPCR greatly improved the yield, purity, and quality of the amplified products, but the mechanism behind these properties and the effects and changes due to the different concentrations of ZnO nanoflowers in the PCR system needs to be further studied.

Keywords: Babesia canis vogeli; Hepatozoon canis; PCR; ZnO nanoflowers; canine vector-borne diseases; nanoPCR; nanomaterial-assisted polymerase chain reaction.

Figure 1

XRD patterns of ZnO nanoflowers when compared to the standard card of ZnO Powder diffraction file (PDF)#36-1451 showing exact similarity to the standard card patterns.

Figure 2

Scanning electron micrographs of ZnO: (a) low magnification with a diameter of 3.00 µm, (b) high magnification with a diameter of 1.00 µm.

Figure 3

Agarose gel electrophoresis images generated by PCR set A: (a) agarose gel electrophoresis for B. canis vogeli DNA (619bp), B1—sample with ZnO nanoflowers, M—2000kb DNA marker, B2—sample without ZnO nanoflowers; (b) agarose gel electrophoresis for H. canis DNA (666bp), H1—sample without the ZnO nanoflowers, M—2000kb DNA marker, H2—sample with ZnO nanoflowers.

Figure 4

Agarose gel electrophoresis images generated by PCR set B (modified thermal cycling conditions): (a) agarose gel electrophoresis for B. canis vogeli DNA(619bp), B3—sample with the ZnO nanoflowers, M—2000kb DNA marker, B4—sample without ZnO nanoflowers; (b) agarose gel electrophoresis for H. canis DNA (666bp), H3—sample with the ZnO nanoflowers, M—2000kb DNA marker, H4—sample without ZnO nanoflowers.