title: Field-Usable Lateral Flow Immunoassay for the Rapid Detection of White Spot Syndrome Virus (WSSV).
abstract: BACKGROUND
  White spot disease (WSD), a major threat to sustainable aquaculture worldwide, is caused by White spot syndrome virus (WSSV). The diagnosis of WSD relies heavily on molecular detection of the virus by one-step PCR. These procedures are neither field-usable nor rapid enough considering the speed at which the virus spreads. Thus, development of a rapid, reliable and field-usable diagnostic method for the detection of WSSV infection is imperative to prevent huge economic losses.
  
  
  RESULTS
  Here, we report on the development of a lateral flow immunoassay (LFIA) employing gold nanoparticles conjugated to a polyclonal antibody against VP28 (envelope protein of WSSV). The LFIA detected WSSV in ~20 min and showed no cross-reactivity with other shrimp viruses, viz. Monodon Baculovirus (MBV), Hepatopancreatic parvovirus (HPV) and Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV). The limit of detection (LOD) of the assay, as determined by real-time PCR, was 103 copies of WSSV. In a time course infectivity experiment, ~104 WSSV particles were injected in Litopenaeus vannamei. The LFIA could rapidly (~ 20 min) detect the virus in different tissues after 3 h (hemolymph), 6 h (gill tissue) and 12 h (head soft tissue, eye stalk, and pleopod) of infection. Based on these findings, a validation study was performed using 75 field samples collected from different geographical locations in India. The LFIA results obtained were compared with the conventional "gold standard test", viz. one-step PCR. The analysis of results in 2x2 matrix indicated very high sensitivity (100%) and specificity (96.77%) of LFIA. Similarly, Cohen's kappa coefficient of 0.983 suggested "very good agreement" between the developed LFIA and the conventional one-step PCR.
  
  
  CONCLUSIONS
  The LFIA developed for the rapid detection of WSSV has an excellent potential for use in the field and could prove to be a boon to the aquaculture industry.
authors.affiliation: Nanobioscience Group, Agharkar Research Institute, Pune, India.
authors.forename: Prabir Kumar
authors.initials: PK
authors.lastname: Kulabhusan
authors.affiliation: Nanobioscience Group, Agharkar Research Institute, Pune, India.
authors.forename: Jyutika M
authors.initials: JM
authors.lastname: Rajwade
authors.affiliation: OIE Reference Laboratory for WTD, C. Abdul Hakeem College, Melvisharam, Tamilnadu, India.
authors.forename: Vimal
authors.initials: V
authors.lastname: Sugumar
authors.affiliation: OIE Reference Laboratory for WTD, C. Abdul Hakeem College, Melvisharam, Tamilnadu, India.
authors.forename: Gani
authors.initials: G
authors.lastname: Taju
authors.affiliation: OIE Reference Laboratory for WTD, C. Abdul Hakeem College, Melvisharam, Tamilnadu, India.
authors.forename: A S
authors.initials: AS
authors.lastname: Sahul Hameed
authors.affiliation: Nanobioscience Group, Agharkar Research Institute, Pune, India.
authors.forename: Kishore M
authors.identifier: http://orcid.org/0000-0001-5602-7518
authors.initials: KM
authors.lastname: Paknikar
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D024202:Electrophoretic Mobility Shift Assay; D005880:Gills / Q000821:virology; D006046:Gold / Q000737:chemistry; D007118:Immunoassay / Q000379:methods*; D053768:Metal Nanoparticles / Q000737:chemistry; D033561:Penaeidae / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction; D012015:Reference Standards; D015203:Reproducibility of Results; D012212:Rheology; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D046848:White spot syndrome virus 1 / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D000914:Antibodies, Viral; D006046:Gold