A Triplex Propidium Monoazide (PMA) qPCR Assay Enables Rapid Discrimination of Live Porcine Reproductive and Respiratory Syndrome Viruses

Figures

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  Figure 1. The procedures of PRRSV PMA-qPCR assay for differential detection. PMA cannot enter live PRRSV particle and do not affect PCR amplification. However, PMA can enter inactivated virus and bind to nucleic acid after photolysis with blue light, which will inhibit PCR amplification.

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  Figure 2. PRRSV inactivation efficacies by UV light, heat, and disinfectants. Same amount NADC34-like PRRSV-2 BJ1805-2 isolate (0.1 MOI) was used for all inactivation evaluations. The viral amplification efficacies were determined to assess inactivation efficacies. (A) PRRSV inactivation effects by exposure to UV light for 10–30 min. (B) PRRSV inactivation effects by heat from 56 to 100°C. (C) PRRSV inactivation effects by heat at 100°C for 0.5–2 min. (D) PRRSV inactivation effects by bleach. (E) PRRSV inactivation effects by bromogeramine. (F) PRRSV inactivation effects by PCMX. The statistical significance was set at ⁣∗p < 0.05, ⁣∗∗p < 0.01, and ⁣∗∗∗p < 0.001.

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  Figure 3. The inactivation efficacy was confirmed by virus inoculation and IFA detection. Live or inactivated (100°C 1.5 min) BJ1805-2, SD17-38, and XJ17-5 isolates (0.1 MOI) were used to infect PAMs and then examined by IFA at 72hpi.

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  Figure 4. The optimization of PMA pretreatment parameters. (A) The influence of 1–50 μM PMA pretreatment on triplex PMA-qPCR amplification was determined. (B) The influence of PMA binding temperature (0–37°C). (C) The influence of PMA binding time (5–20 min). (D) The influence of photolysis time (exposure to blue light for 5–20 min). The statistical significance was set at ⁣∗p < 0.05, ⁣∗∗p < 0.01, and ⁣∗∗∗p < 0.001.

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  Figure 5. The influences of primer and probe concentrations on triplex PMA-qPCR amplification. (A–C) The influences of primer and probe concentrations on live and inactivated NADC34-like PRRSV-2, NADC30-like PRRSV-2, and HP-PRRSV-2 amplifications. (D–F) The amplifications of live and inactivated NADC34-like PRRSV-2, NADC30-like PRRSV-2, and HP-PRRSV-2 when using 2 μM probes and 3 μM primers.

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  Figure 6. Discrimination of live and inactivated PRRSV isolates by this triplex PMA-qPCR assay. The live PRRSV samples including HP-PRRSV-2 (XJ17-5 isolate), NADC30-like PRRSV-2 (SD17-38 isolate), and NADC34-like PRRSV-2 (BJ1805-2 isolate) could be simultaneously detected while the corresponding inactivated viruses were undetectable. (A) Log image. (B) Linear image.

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  Figure 7. Specificity and sensitivity evaluations of the triplex PMA-qPCR assay. (A–C) FAM fluorescent signals were observed only when live NADC34-like BJ1805-2, NADC30-like SD17-38 and HP-PRRSV-2 XJ17-5 isolates were detected but not for other viruses. (D–F) The detection limits of this triplex PMA-qPCR assay were 6 × 101.0 (60) copies/reaction for each virus, respectively.

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  Figure 8. Geographical distribution of distinct PRRSV positive samples in China determined by triplex PMA-qPCR assay. There were 65 out of 452 clinical samples were detected positive by the triplex PMA-qPCR assay. The seven PRRSV-positive regions were shown in blue. The proportions of NADC34-like, NADC30-like, and HP-PRRSV-2 positive samples in each province were shown by a colored pie chart.