Establishment Of PCR Detection Methods For Infectious Myonecrosis Virus And Sample Detection

Infectious myonecrosis (IMN), caused by a double-stranded RNA virus that is namedinfectious myonecrosis virus (IMNV), is an emerging disease of cultured penaeid shrimp(Litopenaeus vannamei). The disease was first reported in farmed Pacific white shrimp L.vannamei from Brazil in2002. The geographic distribution of IMNV has expanded quicklythrough the coastal areas of the Northeast of Brazil. In2006, IMNV has also been observed inL. vannamei from farms in East Java Province in Indonesia because of the illegal import ofshrimp broodstock from Brazil.Gross signs of IMNV infected shrimp include whitish discoloration in the abdominalmuscles and slow mortality persisting throughout culture (cumulative mortality reaching up to70%). IMNV has been responsible for huge economic loss in shrimp farming industry.Estimates for IMNV losses from2002to2006in Brazil exceed$100million and losses inIndonesia may exceed$1million by2010. Because of the significant economical losses dueto IMNV, IMN disease was listed by OIE in2005.Since there is currently no effective treatment for IMNV infection, preventive measuresand practices are required for control, and rapid diagnosis is the most valid strategy. In thisthesis, one step RT-PCR and a real-time PCR assay for IMNV detection were establishment.Samples of shrimp from different districts were detected. The results are as follows:1. One step RT-PCR assay for the detection of IMNV was described. Four pairs ofprimers were designed from the IMNV genome sequence AY570982(GenBank), and themost sensitive pair of primers (IMNV-F5′-G G A C C T A T C A T A C A T A G C G T T G CA-3′and IMNV-R5′-A A C C C A T A T C T A T T G T C G C T G G A T-3′) to amplify a134bp DNA fragment were selected. The sensitivity of this assay was104copies of IMNVcDNA.2. A real-time PCR assay for the detection of IMNV was described. A pair of IMNVprimers (IMNV-F5′-G A T G G C A G C A C G T C A A A A T G-3′and IMNV-R5′-C A G CT G T T C C T G G G A C T T C C T-3′) to amplify a101bp DNA fragment and a TaqManprobe(5′-C T A T T T C C T C C A C T A A T T C A G A G C-3′,89–122bp in GenBankAY570892) were developed. The primers and TaqMan probe were specific for IMNV and didnot cross react with White spot syndrome virus (WSSV), Hepatopancreatic parvovirus (HPV), Monodon baculovirus (MBV), Infectious hypodermal and haematopoietic virus (IHHNV) andspecific pathogen free (SPF) shrimp DNA. A plasmid (pIMNV) containing the target IMNVsequence was constructed and used for determination of the sensitivity of the real-time PCR.This real-time PCR could detect as low as a single copy of IMNV cDNA.3.About631L. vannamei samples were collected from different districts, such asShandong, Fujian, Guangdong, Jiangsu, and Zhejiang. The experiment detected theseL.vannamei samples with the one step RT-PCR and all the samples gave the negative results.The result indicated that IMNV did not currently occur in these districts.