Monday, May 17, 2010

Real-time Reverse Transcription Polymerase Chain Reaction Assay Using TaqMan Probe for Detection and Quantification of Infectious Myonecrosis Virus

Thales P. D. Andrade*, Thinnarat Srisuvan, Kathy F. J. Tang, Donald V. Lightner
Department of Veterinary Science and Microbiology, University of Arizona, 1117 E. Lowell, Tucson,
Arizona 85721, USA

Infectious myonecrosis, caused by Infectious myonecrosis virus (IMNV), is animportant emerging disease of shrimp that has affected the production of cultured Litopenaeus vannamei in Northeast Brazil. In this study we report the development of a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method using TaqMan probe to detect this virus in shrimp. The real-time RT-PCR showed a strong linear correlation (r2 = 0.986) between threshold cycles (CT) and RNA quantities. The assay gave negative results for other viruses, including Yellow head virus (YHV), Taura syndrome virus (TSV), Infectious hypodermal and
hematopoietic necrosis virus (IHHNV), and White spot syndrome virus (WSSV) and the necrotizing hepatopancreatic bacterium (NHPB). This real-time RT-PCR assay can detect as few as 10 IMNV copy numbers/��l RNA, while the nested RT-PCR candetect no fewer than 1000 IMNV copy numbers/μl RNA. Specific-pathogen-free L.vannamei were used in the infectivity assay. There were one control group (Group 1) and one viral challenged group (Group 2), from which shrimp were sampled for RTPCRand histological analysis. The RNA from dead shrimp was extracted and tested for IMNV by nested and real-time RT-PCR. The shrimp in Group 1 showed 100% survival, while those in Group 2 showed a 0% survival. The first mortality in the Group 2 was observed at Day 8 post-inoculation (p.i.); and the mortalities dramatically increased after Day 40 p.i. Histological sections from Group 2 shrimp taken at intervals throughout the study exhibited acute to chronic phase lesions of IMNV infection, and consecutive tissue sections reacted to the IMNV-specific cDNA probes by in situ hybridization. The real-time RT-PCR detected the presence of IMNV in all 30 of the challenged specimens in Group 2. In contrast, the nested RTPCR detected the presence of IMNV in 23 of the 30 specimens. The real-time RTPCR
revealed that the 7 specimens not detected by nested RT-PCR contained relatively low IMNV copy numbers compared to the other 23 specimens. These results demonstrate that the real-time RT-PCR developed in this study is a sensitive

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