Simultaneous detection of feline parvovirus and feline bocavirus using SYBR Green I-based duplex real-time polymerase chain reaction

Abstract

Since both feline parvovirus (FPV) and feline bocavirus (FBoV) can cause diarrhea in cats, it is difficult to distinguish them clinically. This study aimed to develop a SYBR Green I-based duplex real-time polymerase chain reaction (PCR) assay for distinguishing FPV and FBoV-1 on the basis of the melting temperature of the PCR product. A total of 132 fecal samples from different domestic and feral cats were collected, and the results of SYBR Green I-based duplex real-time PCR assay were compared with those of the traditional PCR assay for a comprehensive evaluation. The melting temperatures were found to be 86 °C and 77.5 °C for FBoV-1 and FPV, respectively, and no specific melting peaks for other non-targeted feline viruses were observed. The data obtained from this assay had a good linear relationship; the detection limits of FPV and FBoV-1 were 2.907 × 10¹ copies/μL and 3.836 × 10¹ copies/μL, respectively. In addition, the experiment exhibited high reproducibility. The positive detection rates of the SYBR Green I-based duplex real-time PCR assay for FPV and FBoV-1 were 16.67% (22/132) and 6.82% (9/132), respectively, and the positive detection rate for co-infection with FPV and FBoV-1 was 3.03% (4/132). This result was much more sensitive than that of the traditional PCR method. Thus, the developed SYBR Green I-based assay is a sensitive, rapid, specific, and reliable method for the clinical diagnosis of FPV and FBoV-1 and can provide technical support for the simultaneous detection of co-infection with these viruses in the future.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-021-02947-w.

Keywords: Duplex real-time polymerase chain reaction; Feline bocavirus; Feline parvovirus; Simultaneous detection.

Fig. 1

Standard curve analysis. a Standard curve of the SYBR Green I-based duplex real-time qPCR assay for FPV (concentrations ranged from 2.907 × 10⁸ copies/μL to 2.907 × 10¹ copies/μL; y = − 3.226x + 36.155; R² = 0.999). b Standard curve of the SYBR Green I-based duplex real-time qPCR assay for FBoV-1 (concentrations ranged from 3.836 × 10⁸ copies/μL to 3.836 × 10¹ copies/μL; y = − 3.295x + 38.133; R² = 0.998)

Fig. 2

Melting curve analysis. a Melting curve of FPV (Tm = 78 ± 0.5 °C). b Melting curve of FBoV-1 (Tm = 86 ± 0.5 °C). Tm, melting peak temperature

Fig. 3

Melting curve analysis of the SYBR Green I-based duplex real-time qPCR for FPV (Tm = 77.5 °C) and FBoV-1 (Tm = 86 °C). Tm, melting peak temperature

Fig. 4

Sensitivity analysis. a Amplification curve of the SYBR Green I-based duplex real-time qPCR assay of FPV. The lowest limit of detection of the assay was 2.907 × 10¹ copies/μL. b Amplification curve of the SYBR Green I-based duplex real-time qPCR assay of FBoV-1. The lowest limit of detection of the assay was 3.836 × 10¹ copies/μL. c Amplification curve of the SYBR Green I-based duplex real-time qPCR assay of FPV and FBoV-1. The lowest limit of detection of the assay was 10¹ copies/μL

Fig. 5

Specificity analysis. There are no specific curves of FAstV, FCV, FHV, FCoV, and RNase-free H₂O