A PCR-ELISA Method for Direct Detection of the Oyster Pathogen Haplosporidium nelsoni

Abstract

: A rapid method, utilizing both polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), was developed for detection of oyster MSX disease. The technique included using Haplosporidium nelsoni pathogen-specific PCR primers (based on ribosomal RNA genes), a Chelex resin (for rapid DNA extraction from oyster mantle tissues), and cloned H. nelsoni rRNA plasmid DNA (for use as a capture probe). Digoxigenin was incorporated into the pathogen-specific PCR products, which were captured by the coated probe in a fast hybridization reaction and then detected by ELISA. The sensitivity of PCR amplification on cloned plasmid DNA was 10 fg for detection by stained agarose gel, and increased to 0.01 fg for ELISA. Positive signals were observed in infected oysters using the PCR-ELISA technique. This method may be applicable to early detection of infection.

Molecular diagnostics, field validation, and phylogenetic analysis of Quahog Parasite Unknown (QPX), a pathogen of the hard clam Mercenaria mercenaria

“…In recent years tremendous progress has been made in the development of molecular diagnostic techniques. Numerous PCR assays and oligonucleotide probes are now available for a variety of bivalve mollusc disease agents including Haplosporidium nelsoni (Fong et al 1993, Stokes & Burreson 1995, Stokes et al 1995a, Ko et al 1999, Day et al 2000, H. costale (Ko et al 1995 Discussions regarding the value of these novel molecular diagnostic tools generally cite high sensitivity and specificity, and the capability of producing a rapid and cost-effective diagnosis. However, before such benefits can be realized, it is important to conduct thorough field validations against traditional standard diagnostic techniques (Hiney & Smith 1998).…”

“…Numerous PCR assays and oligonucleotide probes are now available for a variety of bivalve mollusc disease agents including Haplosporidium nelsoni (Fong et al 1993, Stokes & Burreson 1995, Stokes et al 1995a, Ko et al 1999, Day et al 2000, H. costale (Ko et al 1995, Stokes & Burreson 2001, Minchinia teredinis (Stokes et al 1995b), Mikrocytos roughleyi (Adlard & Lester 1995), Marteilia sydneyi (Anderson et al 1995, Kleeman & Adlard 2000, Marteilia refringens (Le Roux et al 1999), Perkinsus marinus (Marsh et al 1995, Yarnall et al 2000, Reece et al 2001, and Bonamia ostreae (Carnegie et al 2000, Cochennec et al 2000. Discussions regarding the value of these novel molecular diagnostic tools generally cite high sensitivity and specificity, and the capability of producing a rapid and cost-effective diagnosis.…”

An optimized DNA extraction and multiplex PCR for the detection of Fasciola sp. in lymnaeid snails

“…Twenty years ago, procedures based on the use of Chelex ® chelating resin were developed for extracting DNA from forensic-type samples (Walsh et al, 1991). Later, this resin was used to extract the DNA from oyster (Ko et al, 1999) but never from lymnaeid snails. The easiness and relatively low cost of the basic PCR protocol make it interesting to investigate the epidemiology of liver fluke infections (Caron et al, 2008) particularly during large epidemiological surveys dealing with big samples (>1000 snails).…”

A simple and reliable method for DNA extraction from bivalve mantle

“…Presumably, this was due to significant amounts of protein and other cell materials present in the mantle tissue, which might inhibit PCR amplification. In a diagnostic study for detection of oyster parasites, a similar attempt was made to extract DNA from adult oyster mantles with the standard Chelex-100 resin method (Ko et al 1999). They selected the mantle tissue and Chelex method because it was described to be easy for collection of the tissue after opening the shells, providing sufficient DNA for analysis contained in a small tissue portion, and simple and economical as compared with other conventional DNA extraction methods.…”