Propidium Monoazide Integrated With qPCR Enables Rapid and Universal Detection of Infectious Porcine Reproductive and Respiratory Syndrome Viruses

Abstract

Infectious porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS, but noninfectious PRRSV cannot. PCR and ELISA are commonly used for PRRSV detection but they cannot discriminate PRRSV infectivity. Virus isolation is a gold standard to determine virus infectivity. However, it is time-consuming. Therefore, we developed a propidium monoazide (PMA) qPCR assay for rapid and universal detection of infectious PRRSV in this study. After comparing the inactivation efficacies of distinct disinfectants, ultraviolet (UV) light, and heat, heat at 72°C for 15 min was determined as an effective strategy for PRRSV inactivation, which was confirmed by virus isolation and immunofluorescence assay (IFA) detection. In addition, PMA pretreatment parameters were optimized, including PMA concentration (5 μM), PMA binding time (25 min), PMA binding temperature (37°C), and photolysis time (25 min). The optimal concentration of primers and probes adapted from our previous study was redetermined. The optimized PMA-qPCR assay exhibited satisfied specificity, sensitivity, and reproducibility. Furthermore, the new PMA-qPCR was applied on the detection of 270 clinical samples (including 57 environmental feces, 177 lungs, 33 lymph nodes [LN], and 3 sera) and compared with previously developed qPCR. Eighty samples were qPCR positive, while only 63 samples were PMA-qPCR positive. No virus could be isolated in the 17 qPCR-positive but PMA-qPCR-negative clinical samples; meanwhile, PRRSV could be isolated in representative PMA-qPCR-positive samples, supporting that only live PRRSV isolates in distinct samples could be detected by this PMA-qPCR assay. In conclusion, this study provides the first PMA-qPCR assay for rapid and universal detection of infectious PRRSV, offering an alternative and effective method for PRRSV diagnosis, prevention, and control.

Keywords: application; infectious virus detection; porcine reproductive and respiratory syndrome virus (PRRSV); propidium monoazide qPCR (PMA-qPCR).

Figure 1

The efficacies of PRRSV inactivation by disinfectants, UV light, and heat were evaluated. The same amount of HP-PRRSV-2 XJ17-5 isolate (200 μl 103 TCID₅₀/ml) was used for all inactivation evaluations. The ΔCts (Ct (PMA-qPCR) − Ct(qPCR)) was determined to assess inactivation efficacies. (A) PRRSV inactivation effects by PCMX. (B) PRRSV inactivation effects by bromogeramine. (C) PRRSV inactivation effects by bleach. (D) PRRSV inactivation effects by exposure to UV light for 10–30 min. (E) PRRSV inactivation effects by heat from 56°C to 80°C. (F) PRRSV inactivation effects by heat at 72°C for 5–20 min. Ct, cycle threshold; PCMX, parachlormetaxylenol; PMA, propidium monoazide; PRRSV, porcine reproductive and respiratory syndrome virus; UV, ultraviolet.

Figure 2

The inactivation efficacy was confirmed by cell culture and viral antigen detection. (A) PAMs were used for infections by live or inactivated (72°C 15 min) XJ17-5 isolates (10³ TCID₅₀/ml) that were pretreated with or without PMA and then examined by IFA at 72 hpi. (B) Marc-145 cells were used for infections by live or inactivated XJ17-5 isolates pretreating with or without PMA and then examining by IFA at 72 hpi. PRRSV-specific antigen (red signal) could be detected in live XJ17-5 with PMA pretreatment but could not be detected in inactivated XJ17-5 with PMA pretreatment. Live XJ17-5 without PMA pretreatment was set as a positive control. Inactivated XJ17-5 without PMA pretreatment was set as negative control. DAPI, 4′′,6-diamidino-2-phenylindole; IFA, immunofluorescence assay; PAMs, Pulmonary alveolar macrophages; PMA, propidium monoazide; PRRSV, porcine reproductive and respiratory syndrome virus.

Figure 3

The PMA treatment parameters were optimized. (A) The influence of 1–100 μM PMA pretreatment on PMA-qPCR amplification was determined. (B) The influence of PMA binding temperature (0–37°C) on PMA-qPCR amplification was evaluated. (C) The influence of PMA binding time (5–25 min) on PMA-qPCR amplification was assessed. (D) The influence of photolysis time (exposure to blue light for 5–25 min) on PMA-qPCR amplification was tested. PMA, propidium monoazide.

Figure 4

The influence of primers and probe concentrations on PMA-qPCR amplification was determined. When the inactivated virus (72°C for 15 min) was pretreated with PMA, viral RNA was undetectable using 1 μM probe and 2 μM primers, while the other three groups had similar amplification effects at different concentrations of probe and primers. PMA, propidium monoazide.

Figure 5

Detection spectrum of the universal PRRSV PMA-qPCR assay. All live PRRSV strains including PRRSV-1 (SD1291 isolate), CH-1a-like PRRSV-2 (SD1612-1 isolate), HP-PRRSV-2 (XJ17-5 isolate), NADC30-like PRRSV-2 (SD17-38 isolate), NADC34-like PRRSV-2 (Anheal-1 isolate), and VR-2332-like PRRSV-2 (JSYC20-05-1 isolate) were detectable while all the corresponding inactivated viruses were undetectable by this PRRSV PMA-qPCR assay. PMA, propidium monoazide; PRRSV, porcine reproductive and respiratory syndrome virus.

Figure 6

The procedures of the universal PRRSV PMA-qPCR assay. (A) The procedures of PMA-qPCR for inactivated PRRSV isolates. (B) The procedures of PMA-qPCR for live PRRSV or unknown samples. PMA, propidium monoazide; PRRSV, porcine reproductive and respiratory syndrome virus.