DEVELOPMENT AND APPLICATION OF NOVEL QUANTITATIVE AND QUALITATIVE MOLECULAR TECHNIQUES FOR DETECTION OF INFECTIOUS MYONECROSIS VIRUS (IMNV) IN PACIFIC WHITE SHRIMP LITOPENAEUS VANNAMEI

Persistent Link:
http://hdl.handle.net/10150/195726
Title:
DEVELOPMENT AND APPLICATION OF NOVEL QUANTITATIVE AND QUALITATIVE MOLECULAR TECHNIQUES FOR DETECTION OF INFECTIOUS MYONECROSIS VIRUS (IMNV) IN PACIFIC WHITE SHRIMP LITOPENAEUS VANNAMEI
Author:
Andrade, Thales Passos de
Issue Date:
2009
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Infectious myonecrosis, caused by infectious myonecrosis virus (IMNV), is an important disease of shrimp that has adversely affected the production of cultured Litopenaeus vannamei. The studies reported here were centered on development and/or validation of alternative diagnostic methods for detection of IMNV. Hence, two manuscripts were published in the Journal of Aquaculture and one manuscript was published in the Journal of Fish Diseases. Chapter 2 describes the development of a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method using TaqMan probe. The results showed that this real-time RT-PCR assay can detect as little as 10 IMNV copies/μl RNA, while the nested RT-PCR can detect 1000 copies/μl RNA. These findings suggest that the TaqMan real-time RT-PCR is “the gold standard” for screening shrimp to protect aquaculture production systems from losses caused by IMNV, because it provides quantification, higher sensitivity and specificity, and because it is less time consuming and less prone to contamination compared to conventional gel-based RT-PCR. In Chapter 3 I evaluated if prolonged storage of infectious myonecrosis virus (IMNV) infected shrimp in Davidson’s AFA fixative can degrade its double-stranded RNA genome resulting in false negative ISH reactions. 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). 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. The Chapter 4 describes the development of a reverse transcription loop-mediated isothermal amplification and nucleic acid lateral flow (RT-LAMP-NALF) for detection of IMNV. The RT-LAMP-NALF method combines simplified nucleic acid extraction, a reverse-transcription loop-mediated isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified sequences using a generic NALF qualitative detection test strip. The RT-LAMP-NALF was found to be 100 and 10 times more sensitive than one-step RT-PCR and RT-LAMP (two primer pairs), respectively. These results clearly demonstrate that the RT-LAMP-NALF method is specific, sensitive, can shorten the time for analysis, and has potential application for IMNV diagnosis in resource-poor diagnostic settings.
Type:
text; Electronic Dissertation
Keywords:
IMNV; in situ hybridization; INFECTIOUS MYONECROSIS VIRUS; Litopenaeus vannamei; Real-time RT-PCR; RT-LAMP-NALF
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Pathobiology; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Lightner, Donald V
Committee Chair:
Lightner, Donald V

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleDEVELOPMENT AND APPLICATION OF NOVEL QUANTITATIVE AND QUALITATIVE MOLECULAR TECHNIQUES FOR DETECTION OF INFECTIOUS MYONECROSIS VIRUS (IMNV) IN PACIFIC WHITE SHRIMP LITOPENAEUS VANNAMEIen_US
dc.creatorAndrade, Thales Passos deen_US
dc.contributor.authorAndrade, Thales Passos deen_US
dc.date.issued2009en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractInfectious myonecrosis, caused by infectious myonecrosis virus (IMNV), is an important disease of shrimp that has adversely affected the production of cultured Litopenaeus vannamei. The studies reported here were centered on development and/or validation of alternative diagnostic methods for detection of IMNV. Hence, two manuscripts were published in the Journal of Aquaculture and one manuscript was published in the Journal of Fish Diseases. Chapter 2 describes the development of a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method using TaqMan probe. The results showed that this real-time RT-PCR assay can detect as little as 10 IMNV copies/μl RNA, while the nested RT-PCR can detect 1000 copies/μl RNA. These findings suggest that the TaqMan real-time RT-PCR is “the gold standard” for screening shrimp to protect aquaculture production systems from losses caused by IMNV, because it provides quantification, higher sensitivity and specificity, and because it is less time consuming and less prone to contamination compared to conventional gel-based RT-PCR. In Chapter 3 I evaluated if prolonged storage of infectious myonecrosis virus (IMNV) infected shrimp in Davidson’s AFA fixative can degrade its double-stranded RNA genome resulting in false negative ISH reactions. 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). 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. The Chapter 4 describes the development of a reverse transcription loop-mediated isothermal amplification and nucleic acid lateral flow (RT-LAMP-NALF) for detection of IMNV. The RT-LAMP-NALF method combines simplified nucleic acid extraction, a reverse-transcription loop-mediated isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified sequences using a generic NALF qualitative detection test strip. The RT-LAMP-NALF was found to be 100 and 10 times more sensitive than one-step RT-PCR and RT-LAMP (two primer pairs), respectively. These results clearly demonstrate that the RT-LAMP-NALF method is specific, sensitive, can shorten the time for analysis, and has potential application for IMNV diagnosis in resource-poor diagnostic settings.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectIMNVen_US
dc.subjectin situ hybridizationen_US
dc.subjectINFECTIOUS MYONECROSIS VIRUSen_US
dc.subjectLitopenaeus vannameien_US
dc.subjectReal-time RT-PCRen_US
dc.subjectRT-LAMP-NALFen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePathobiologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorLightner, Donald Ven_US
dc.contributor.chairLightner, Donald Ven_US
dc.contributor.committeememberReggiardo, Carlosen_US
dc.contributor.committeememberBesselsen, David G.en_US
dc.contributor.committeememberTang-Nelson, Kathy F. J.en_US
dc.contributor.committeememberFitzsimmons, Kevinen_US
dc.identifier.proquest10564en_US
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