Starting September 17th, a combined (multiplex) real-time, single-tube PCR assay for the detection of both US and European-like/LV isolates will be available

COMBINED NORTH AMERICAN/EUROPEAN-LIKE /LV “MULTIPRRSV” REAL-TIME PCR ASSAY

Starting September 27, a combined (multiplex) real-time, single-tube PCR assay for the detection of both US and European-like/LV isolates will be available. This assay uses the same primers and probes as the currently used singleplex assays for US and European-like/LV assays as described in the Journal of Clinical Microbiology 42(10):4453-4461, 2004, “Detection of U.S., Lelystad, and European-like PRRSV and relative quantitation in boar semen and serum samples by real-time PCR”. Extensive testing was performed to determine that the sensitivity and specificity of the assay did not change between the singleplex to multiplex assay using both in vitro transcripts and field isolates (Table 1, Figure 1a, b). The “cut off” values for sensitivities are also equivalent to a previously described “nested PCR”. Since SDSU has performed PCR for the detection of PRRSV since 1993 and the singleplex real-time PCR assays have been performed since 2003, numerous archived and current field isolates have been detected with these primers and probes. The price of this combined (multiplex) test will be the same as for the singleplex test, so there will be a significant savings if you were previously testing for both US and EuroPRRSV/LV individually.

Table 1. Cycle Threshold (Ct) levels for 10-fold serial dilutions of US (left) and European-like (right) field isolates using singleplex vs. multiplex PRRSV PCR assays on the Cepheid^® SmartCycler and ABI 7500 Real-Time PCR Instruments

PRRSV TCID₅₀/ml	US 10³	US 10²	US 10¹	US 10⁰	US 10^-1	US 10^-2	EU 10⁴	EU 10³	EU 10²	EU 10¹	EU 10⁰	EU 10^-1
Singleplex (ABI 7500)	20.33	24.31	29.05	31.59	34.58	37.46	20.45	24.94	28.82	32.43	36.30	0
Multiplex (ABI 7500)	20.24	23.6	27.96	31.93	34.88	36.19	18.02	21.21	24.85	29.83	34.33	0

PRRSV TCID₅₀/ml	US 10³	US 10²	US 10¹	US 10⁰	US 10^-1	US 10^-2	EU 10⁴	EU 10³	EU 10²	EU 10¹	EU 10⁰	EU 10^-1
Singleplex (Smart Cycler)	22.81	25.22	29.21	32.71	35.29	0	21.96	26.15	29.4	33.27	37.19	0
Multiplex (Smart Cycler)	22.25	24.90	28.88	32.08	35.69	36.83	21.70	25.16	28.25	32.60	36.66	0

Fig. 1a. Copies/ul of the serially diluted U.S. in vitro RNA transcript Fig. 1b. Copies/ul of the serially diluted EuroPRRSV in vitro RNA transcript