detection and typing of highly pathogenic porcine reproductive and respiratory syndrome virus by multiplex real-time rt-pcr检测和输入高致病性猪繁殖与呼吸综合征病毒的多路实时rt - pcr.pdfVIP

Detection and Typing of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus by Multiplex Real-Time RT-PCR 1 1 2 2 1 1 Kerstin Wernike , Bernd Hoffmann , Malte Dauber , Elke Lange , Horst Schirrmeier , Martin Beer * 1 Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany, 2 Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany Abstract Porcine reproductive and respiratory syndrome (PRRS) causes economic losses in the pig industry worldwide, and PRRS viruses (PRRSV) are classified into the two distinct genotypes ‘‘North American (NA, type 2)’’ and ‘‘European (EU, type 1)’’. In 2006, a highly pathogenic NA strain of PRRSV (HP-PRRSV), characterized by high fever as well as high morbidity and mortality, emerged in swine farms in China. Therefore, a real-time reverse transcription polymerase chain reaction (RT-qPCR) assay specific for HP-PRRSV was developed and combined with type 1- and type 2-specific RT-qPCR systems. Furthermore, an internal control, based on a heterologous RNA, was successfully introduced. This final multiplex PRRSV RT-qPCR, detecting and typing PRRSV, had an analytical sensitivity of less than 200 copies per ml for the type 1-assay and 20 copies per ml for the type 2- and HP assays and a high diagnostic sensitivity. A panel of reference strains and field isolates was reliably detected and samples from an animal trial with a Chinese HP-PRRS strain were used for test validation. The new multiplex PRRSV RT-qPCR system allows for the first time the highly sensitive detection and rapid differentiation of PRRSV of both genotypes a