Evaluation of a multiplex PCR method for the detection of porcine parvovirus types 1 through 7 using various field samples

Abstract

Porcine parvoviruses (PPVs) are small, nonenveloped DNA viruses that are widespread in the global pig population. PPV type 1 (PPV1) is a major causative agent of reproductive failure and has been recognized since the 1960s. In recent decades, novel PPVs have been identified and designated as PPVs 2 through 7 (PPV2~PPV7). Although the epidemiological impacts of these newly recognized parvoviruses on pigs are largely unknown, continuous surveillance of these PPVs is needed. The aim of this study was to develop an improved and efficient detection tool for these PPVs and to assess the developed method with field samples. Using 7 sets of newly designed primers, a multiplex polymerase chain reaction (mPCR) protocol was developed for the simultaneous detection of the seven genotypes of PPV (PPV1~PPV7). The sensitivity of the mPCR assay was analyzed, and the detection limit was determined to be 3×103 viral copies. The assay was highly specific in detecting one or more of the viruses in various combinations in specimens. The mPCR method was evaluated with 80 serum samples, 40 lung or lymph node samples and 40 intestine or fecal samples. When applied to these samples, the mPCR method could detect the 7 viruses simultaneously, providing rapid results regarding infection and coinfection status. In conclusion, the developed mPCR assay can be utilized as an effective and accurate diagnostic tool for rapid differential detection and epidemiological surveillance of various PPVs in numerous types of field samples.

Fig 1. Optimal Ta determination for PPV mPCR.

Agarose gel electrophoresis (2%) of standard positive controls (pGEM-PPVs). Lane M, 100 bp-plus DNA ladder; lane NC, negative control for detection of PPV1~PPV7; lanes 1~11, gradient Tas of 50.0°C, 51.9°C, 53.8°C, 56.1°C, 58.0°C, 60.0°C, 62.0°C, 63.8°C, 66.1°C, 68.0°C and 70°C, respectively.

Fig 2. Specificity of the PPV mPCR method.

Agarose gel electrophoresis (2%) of specific fragments amplified by mPCR from the proviral DNA and cDNA of Pk-15 cells, PRRS (NA), PRRS (EU), PCV2, PED/TGE/Rota-mixed (vaccine), E. coli, Salmonella enterica and JEV. Lane M, 100 bp-plus DNA ladder; lane NC, negative control; lane 1, Pk-15 cells; lane 2, PRRS (NA); lane 3, PRRS (EU); lane 4, PCV2; lane 5, PED/TGE/Rota-mixed (vaccine); lane 6, E. coli; lane 7, Salmonella enterica; lane 8, JEV; lane 9, PRV; lane 10, CSFV; lanes 11~17, pGEM-PPV1, pGEM-PPV2, pGEM-PPV3, pGEM-PPV4, pGEM-PPV5, pGEM-PPV6 and pGEM-PPV7; lane 18, mixed standard of all pGEM-PPV plasmids.

Fig 3. Sensitivity of the PPV mPCR method.

The seven pGEM-PPV single plasmids, diluted from 3×10⁹ to 3×10¹ copies/μl, and the mixed plasmids, diluted from 3×10⁸ to 3×10¹ copies/μl, were used to determine the minimum detection limit of the PPV mPCR method. (A) Sensitivity for pGEM-PPV1. (B) Sensitivity for pGEM-PPV2. (C) Sensitivity for pGEM-PPV3. (D) Sensitivity for pGEM-PPV4. (E) Sensitivity for pGEM-PPV5. (F) Sensitivity for pGEM-PPV6. (G) Sensitivity for pGEM-PPV7. (H) Sensitivity for pGEM-PPV1~pGEM-PPV7. Lane M, 100 bp-plus DNA ladder; lane NC, negative control.

Fig 4. Simple infection rates of each PPV type within different types of samples.

Percentages of PPV1- to PPV7-positive serum samples (n = 80), lung/lymph node samples (n = 40), and intestine/fecal samples (n = 40). Statistically significant differences (p < 0.05, Fisher’s exact test) between types of samples within the grouped bars for each virus are marked with superscripts on the top of bars in the chart (a, b).