Development and application of a multiplex PCR method for the simultaneous detection and differentiation of feline panleukopenia virus, feline bocavirus, and feline astrovirus

Abstract

A multiplex polymerase chain reaction (mPCR) assay was developed to detect and distinguish feline panleukopenia virus (FPV), feline bocavirus (FBoV) and feline astrovirus (FeAstV). Three pairs of primers were designed based on conserved regions in the genomic sequences of the three viruses and were used to specifically amplify targeted fragments of 237 bp from the VP2 gene of FPV, 465 bp from the NP1 gene of FBoV and 645 bp from the RdRp gene of FeAstV. The results showed that this mPCR assay was effective, because it could detect at least 2.25-4.04 × 10⁴ copies of genomic DNA of the three viruses per μl, was highly specific, and had a good broad-spectrum ability to detect different genotypes of the targeted viruses. A total of 197 faecal samples that had been screened previously for FeAstV and FBoV were collected from domestic cats in northeast China and were tested for the three viruses using the newly developed mPCR assay. The total positive rate for these three viruses was 59.89% (118/197). From these samples, DNA from FPV, FBoV and FeAstV was detected in 73, 51 and 46 faecal samples, respectively. The mPCR testing results agreed with the routine PCR results with a coincidence rate of 100%. The results of this study show that this mPCR assay can simultaneously detect and differentiate FPV, FBoV and FeAstV and can be used as an easy, specific and efficient detection tool for clinical diagnosis and epidemiological investigation of these three viruses.

Fig. 1

Specificity of multiplex PCR for the detection of positive and negative controls using mixed primers. Lanes 1-13: 1, FPV; 2, FBoV; 3, FeAstV; 4, FPV and FBoV; 5, FPV and FeAstV; 6, FBoV and FeAstV; 7, FeAstV, FBoV and FPV; 8, FCV; 9: FHV-1; 10, FCoV; 11, FeLV; 12, FIV; 13, negative control; M, DL2000 DNA marker

Fig. 2

Sensitivity of multiplex PCR and routine PCR for the detection of tenfold serially diluted plasmid DNA from FPV, FBoV and FeAstV. (a, b, c) Routine PCR for FeAstV, FBoV and FPV; (d) mPCR. Lanes 1-10 (a, b and c), FeAstV-, FBoV- and FPV-positive plasmid concentrations ranging from 2.25 × 10⁹ to 2.25 × 10⁰copies/μl, 3.13 × 10⁹ to 3.13 × 10⁰copies/μl and 4.04 × 10⁹ to 4.04 × 10⁰copies/μl. Lanes 1-10 in (d) are reactions performed with tenfold serial dilutions of mixtures of plasmids containing sequences from the three viruses (2.25 × 10⁹ - 2.25 × 10⁰copies/μl, 3.13 × 10⁹ - 3.13 × 10⁰copies/μl, and 4.04 × 10⁹ - 4.04 × 10⁰copies/μl of each virus/sample). M, DL2000 DNA marker

Fig. 3

Prevalence of FPV, FBoV and FeAstV in diarrheic and healthy cats. (a) Total positive rates of these three viruses in diarrheic and healthy cats. The difference was evaluated using the chi-square test. Probability (p) values are shown on the bar chart. A p-value <0.05 was considered statistically significant. (b) Monoinfection and coinfection rates of these three viruses in diarrheic and healthy cats