Sequences diagnostic for shrimp pathogens

#The invention relates to the field of diagnostic testing. More specifically, new primers have been developed for use in detection of the Infectious Hypodermal and Hematopoietic Necrosis Virus pathogen of shrimp.

#Commercial shrimp farms suffer extensive losses due to the effects of a number of common pathogens. Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) is one of the most serious viral pathogens of farmed penaeid shrimp. It is widely distributed in many countries and has a large range of hosts. For example, IHHNV causes mass mortality among the Western blue shrimp (Penaeus stylirostris) and severe deformation in the Pacific white shrimp (Penaeus vannamei). IHHNV is a small, single stranded DNA-containing parvovirus, the complete genome of which has been sequenced (Bonami et al., J. Gen. Virology 71 (Pt 11):657-2664 (1990); GenBank AF218266).

#IHHNV is extremely detrimental to the shrimp farming industry, being responsible for catastrophic epidemics worldwide. Detection of IHHNV in hatchery broodstock and in post-larvae allows infected shrimp to be eliminated before entry into a commercial production system. Consequently, a variety of methods have been developed for the detection of IHHNV in shrimp, including nucleic acid-based methods and immunological methods (Lightner et al., Aquaculture 164(1):201-220 (1998)). Nucleic acid amplification methods such as polymerase chain reaction (PCR) and isothermal amplification are of particular interest because they are simple, rapid, and sensitive. PCR methods for the detection of IHHNV, which are based on amplifying different diagnostic regions of the genome, have been described (see for example Nunan et al., Mar. Biotechnol. 2(4):319-328 (2000); Yue et al., J. AOAC International 89(1):240-244 (2006); Tang et al. Dis. Aquat. Org. 44(2):79-85 (2001); Hu et al., CN 1410549; and Dhar et al. J. Clin. Microbiol. 39(8):2835-2845 (2001)). Additionally, a loop mediated isothermal method for the detection of IHHNV is described by Sun et al. (J. Virol. Methods 131(1):41-46 (2006)).

#The various embodiments of the invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.

#FIG. 1A shows the melting curve for the IHHNV4 product and the actin internal sample control product formed by simultaneous PCR amplification of the IHHNV virus DNA and actin DNA, as described in Example 10. The melting temperature (Tm) values of the IHHNV and actin products are indicated on their corresponding melting curves.

#FIG. 1B shows the results of the agarose gel electrophoresis separation of samples containing the IHHNV4 product and the actin internal sample control product formed by simultaneous PCR amplification of the IHHNV virus DNA and actin DNA, as described in Example 10. The quantity of IHHNV and shrimp DNA is shown above each lane; “M” is a 100-bp DNA ladder.

#The following sequences conform with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

#SEQ ID NOs:1-8 are the nucleotide sequences of IHHNV diagnostic primers useful for detection of IHHNV.

#SEQ ID NOs:9-12 are the nucleotide sequences of synthetic IHHNV templates described in the General Methods Section of the Examples. These sequences are also the nucleotide sequences of amplification products obtained using pairs of IHHNV diagnostic primers disclosed herein.