Development and Application of nanoPCR Method for Detection of Feline Panleukopenia Virus

Abstract

Feline panleukopenia (FP) is a severe viral illness caused by the feline panleukopenia virus (FPV), putting sectors like companion cat breeding and endangered feline conservation at risk. The virus has a high morbidity and fatality rate and is found all over the world. We created a novel FPV assay using nanoPCR technology and assessed the method's specificity and sensitivity. The approach amplified a 345 bp nucleic acid fragment with a minimum detection limit of 7.97 × 10² copies/μL, which is about 100 times greater than traditional PCR. We collected anal swabs from 83 cats suspected of FPV infection for practical application, and the FPV-positive rate determined by the nanoPCR approach was 77.1%. In conclusion, the approach is more sensitive than conventional PCR and more convenient and cost-effective than qPCR methodology and may be utilized for the clinical detection of FPV.

Keywords: VP2 gene; clinical testing; epidemiology; feline panleukopenia virus; nanoPCR.

Figure 1

Optimization of particle diameter, (a) particle concentration, (b) primer concentration, and (c) annealing temperature (d): (a) Lane M, DL 2000 Marker; Lane 1, negative control; Lanes 2–6, 10, 15, 20, 30, and 40 nm; (b) Lane M, DL 2000 Marker; Lane 1, negative control; Lanes 2–6, 0.1, 0.2, 0.3, 0.4, and 0.5 mM; (c) Lane M, DL 2000 Marker; Lane 1, negative control; Lanes 2–11, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 μM; (d) Lane M, DL 2000 Marker; Lane 1, negative control; Lanes 2–9, 55.0, 54.2, 52.9, 51.0, 48.6, 46.9, 45.7, and 45.0 °C (please find the WB full membrane in Figure S1).

Figure 2

The sensitivity of nanoPCR (a) and conventional PCR (b): (a) Lane M, DL 2000 Marker; Lane 1, negative control; Lanes 2–11, 7.97 × 10¹⁰, 7.97 × 10⁹, 7.97 × 10⁸, 7.97 × 10⁷, 7.97 × 10⁶, 7.97 × 10⁵, 7.97 × 10⁴, 7.97 × 10³, 7.97 × 10², and 7.97 × 10¹ copies/μL; (b) Lane M, DL 2000 Marker; Lane 1, negative control; Lanes 2–11, 7.97 × 10¹⁰, 7.97 × 10⁹, 7.97 × 10⁸, 7.97 × 10⁷, 7.97 × 10⁶, 7.97 × 10⁵, 7.97 × 10⁴, 7.97 × 10³, 7.97 × 10², and 7.97 × 10¹ copies/μL (please find the WB full membrane in Figure S1).

Figure 3

The specificity of nanoPCR. Lane M, DL 2000 Marker; Lane 1, negative control; Lanes 2–5, FPV, FHV, FCoV, and FCV (please find the WB full membrane in Figure S1).

Figure 4

The detection results of some FPV clinical samples. Lane M, DL 2000 Marker; Lane 1, negative control; Lanes 2–10, FPV clinical samples (please find the WB full membrane in Figure S1).

Figure 5

Phylogenetic analysis of FPV with CPV based on the VP2 amino acid sequence by the Neighbor-Joining (N-J) method. The green background is the FPV branch, and the yellow background is the CPV branch. The red fonts represent some of the clinical samples in this study.