Thales P. D. Andrade*, Rita M. Redman, Donald V. Lightner
Department of Veterinary Science and Microbiology, University of Arizona, 1117 E. Lowell, Tucson,
Arizona 85721, USA
The potential negative effect of prolonged storage of shrimp tissues in Davidson’s AFA fixative on in situ hybridization (ISH) signal was demonstrated previously for Taura syndrome virus (TSV), which has a single-stranded RNA genome. In this study we evaluated if prolonged storage of infectious myonecrosis virus (IMNV) infected shrimp in Davidson’s AFA (Alcohol, Formaldehyde, Acetic acid) fixative will degrade its double-stranded RNA genome resulting in false negative ISH reactions. Twenty-one shrimp (3 g) specific-pathogen-free Litopenaeus
vannamei were used in this study. Three shrimp were used as negative control and 18 shrimp were inoculated with a tissue homogenate prepared from frozen IMNVinfected L. vannamei obtained from Brazil in 2003 (positive control). Shrimp were collected at Day 12 post-injection and fixed in Davidson’s AFA for five different preservation times (1, 2, 4, 7 and 10 days). After the different fixation times, the Davidson’s AFA was replaced with several changes of 70% ethanol until the pH was stable. IMNV lesions were confirmed in all positive control shrimp by routine H & E histology and ISH. Myonecrosis lesions were strongly positive by ISH at all five
preservation times evaluated. Hence, in the present report it was found that the length
of time (up to 10 days) in Davidson’s AFA did not have a deleterious effect on the
ISH reaction for IMNV.
_____________________________________________________________________
*Corresponding author. Tel.: +1 520 621 4438; fax: +1 520 621 4899.
E-mail address: thalespda@hotmail.com (T. P. D. Andrade), dvl@u.arizona.edu (D. V.
Lightner).
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
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
Subscribe to:
Posts (Atom)