Development of a multiplex PCR to detect and discriminate porcine circoviruses in clinical specimens

Abstract

Background: A diagnostic method to simultaneously detect and discriminate porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3) in clinical specimens is imperative for the differential diagnosis and monitoring and control of PCVs in the field.

Methods: Three primer pairs were designed and used to develop a multiplex PCR assay. And 286 samples from 8 farms in Hubei province were tested by the developed multiplex PCR assay to demonstrate the accuracy.

Results: Each of target genes of PCV1, PCV2 and PCV3 was amplified using the designed primers, while no other porcine viruses genes were detected. The limit of detection of the assay was 10 copies/μL of PCV1, PCV2 OR PCV3. The results of the tissue samples detection showed that PCV1, PCV2 and PCV3 are co-circulating in central China. The PCV1, PCV2 and PCV3 singular infection rate was 52.4% (150/286), 61.2% (175/286) and 45.1% (129/286), respectively, while the PCV1 and PCV2 co-infection rate was 11.2% (32/286), the PCV1 and PCV3 co-infection rate was 5.9% (17/286), the PCV2 and PCV3 co-infection rate was 23.4% (67/286), and the PCV1, PCV2 and PCV3 co-infection rate was 1.7% (5/286), respectively, which were 100% consistent with the sequencing method and real-time PCR methods.

Conclusions: The multiplex PCR assay could be used as a differential diagnostic tool for monitoring and control of PCVs in the field. The results also indicate that the PCVs infection and their co-infection are severe in Hubei province, Central China.

Keywords: Multiplex PCR; PCV1; PCV2; PCV3; Porcine circovirus; Viral discrimination.

Fig. 1

Geographical distribution of PCV3 in China (red regions, till June 2018) and the position of pig farms (red stars) in this study

Fig. 2

Electrophoresis of multiplex PCR products in optimization conditions. Lane M, DL2000 DNA marker; lane 1, PCV1; lane 2, PCV2; lane 3, PCV3; lane 4, ddH₂O

Fig. 3

Specificity of multiplex PCR for the detection of PCVs. Lane M, DL2000 DNA marker; lane 1, PCV1; lane 2, PCV2; lane 3, PCV3; lane 4, TTSuV1; lane 5, TTSuV2; lane 6, PRV; lane 7, PPV; lane 8, RV; lane 9, JEV; lane 10,PEDV; lane 11, PDCoV; lane 12, ddH₂O

Fig. 4

Sensitivity of multiplex PCR for the detection of PCVs. Lane M, DL2000 DNA marker; lanes 1–10 are: 1, 10⁷; 2, 10⁶; 3, 10⁵; 4, 10⁴; 5, 10³; 6, 10²; 7, 10¹; 8, 10⁰; 9, 10^− 1; 10, 10^− 2 copies/μL