Prime q pcv2 prrsv detection kit

Before applying NGS to the Korean PRRSV strains, we compared PRRSV cDNA preparation procedures using a cell-cultured reference strain (VR-2332) [see Additional file 1]. A total of 8 cDNA preparation methods were tested (designated method 1–method 8). The virus-cultured cell supernatant (method 1 ~ method 4) and concentrated supernatant (method 5–method 8) were used to determine the effect of the PRRSV concentration method. Virus concentration/ultrafiltration was performed with 15 ml of clarified sample by centrifugation at 4000 rpm and 4 °C until all the samples were filtered utilizing a VivaSpin®20 unit with a 300,000 Da molecular weight cutoff (MWCO) (Vivascience) as previously described [49 (link)]. Viral RNA was extracted using a QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. The extracted RNA was treated with a DNase Max Kit (Qiagen) and purified with Agencourt RNA Clean XP beads (Beckman Coulter) following the manufacturer’s instructions. Purified RNA was examined with a NanoDrop spectrophotometer and a Qubit 2.0 fluorometer using a Qubit RNA BR Assay Kit. Real-time reverse transcription-polymerase chain reaction (RT–qPCR) was performed for the quantification of viral RNA using the Prime-Q PCV2 PRRSV Detection Kit (Genet Bio, Daejeon, South Korea). The prepared RNAs were stored at − 80 °C until use.

Viral RNA was extracted from 100 µL of each serum sample and 1 g of each tissue sample, using a MagMAX™ Viral RNA Isolation Kit (Ambion, Applied Biosystems, Life Technologies) and a total RNA Extraction Kit (Hybrid-RTM, GeneAll, Seoul, Republic of Korea), respectively, per the manufacturers’ instructions. Serum and lung viral loads were measured using a Prime-Q PCV2, PRRSV Detection Kit (GeNet Bio, Inc., Daejeon, Republic of Korea) with a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). A standard curve was generated from known titres of PRRSV and used to calculate the quantity of PRRSV in each sample by converting each cycle threshold value to the TCID₅₀/mL-equivalent values.
The PRRSV titres in lung tissues were measured with MARC-145 cells, using a microtitration-infectivity assay. Briefly, tissue homogenates [10% (weight/volume)] from the extracted lungs were prepared in Dulbecco’s modified Eagle’s medium with antibiotics, vortexed for 10–15 min, and centrifuged at ~ 4000 × g for 1 h at 4 ℃. After centrifugation, each collected supernatant was filtered through a sterile 0.20 μm syringe filter and incubated with MARC-145 cells to measure the viral titre. PRRSV titres were calculated at 5 to 6 dpi, based on the observed cytopathic effects, and were expressed as TCID₅₀/mL.

Lung samples submitted to JBNU-VDC are routinely tested for PCV2 and PRRSV infection corresponding to client requests using the Prime-Q PCV2 PRRSV Detection Kit (GeNet Bio Inc., Daejun, Korea) according to the manufacturer’s instructions [32 (link)].
The PCV2 real-time PCR results for lung tissue samples were used to categorize individual pigs into PCV2-negative (ct > 35), LOW-PCV2-PIG (ct > 25 and ≤ 35) or HIGH-PCV2-PIG (ct ≤ 25) groups based on real-time PCR ct values. According to the previously established criteria for PCVAD based on the PCV2 ct value [23 (link), 38 (link)], HIGH-PCV2-PIG samples were classified as PCVAD-suspected, and PCV2-negative and LOW-PCV2-PIG samples were combined and classified as non-PCVAD-suspected.
Based on the PRRSV real-time PCR results, lung tissue samples were considered to be PRRSV positive (ct ≤ 35) and PRRSV negative (ct > 35) and were categorized into the following groups: PRRSV-Neg, PRRSV1 single infection, PRRSV2 single infection, and PRRSV1&2 dual infection. For further investigation regarding the correlation between PPV and PRRSV infection, PRRSV1- and/or PRRSV2-infected pigs were all combined into the category PRRSV-Pos.