Shrimp hepatopancreatic parvovirus detection by combining loop-mediated isothermal amplification with a lateral flow dipstick.

ABSTRACT

Present methods such as traditional PCR, PCR-ELISA, real-time PCR and histopathology for detection of shrimp hepatopancreatic parvovirus (PmDNV) entail various disadvantages including high cost, long assay time or use of toxic substances. Loop-mediated isothermal amplification (LAMP) of target nucleotide sequences under inexpensive isothermal conditions combined with amplicon detection by chromatographic lateral flow dipsticks (LFD) allowed simpler detection within 75 min. Biotinylated LAMP amplicons from the targeted portion of the PmDNA capsid protein gene were produced under isothermal conditions at 63 degrees C for 1h and then hybridized at 63 degrees C for 5 min with an FITC-labeled probe (optimized at 20 pmol) that was specific for the LAMP amplicons (i.e., outside the primer region). The FITC-labeled, biotinylated LAMP product picked up gold-labeled, anti-FITC near the LFD origin and the whole, triple-labeled complex was captured by an immobilized biotin-binding protein to yield a red nano-gold stripe at the LFD test line. With a DNA template derived from PmDNV-infected shrimp, the LAMP-LFD detection limit was 1 ng while that for one-step PCR-electrophoresis was 10 ng. Comparative sensitivity for one nested-PCR-electrophoresis method was 1 ng but for another 0.1 ng. The LAMP-LFD method gave negative test results with DNA extracts from normal shrimp and from shrimp infected with other DNA viruses including monodon baculovirus (MBV), white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV).