Development of specific PCR assays for the detection of Cryptocaryon irritans

Abstract

Cryptocaryon irritans is one of the most important protozoan pathogens of marine fish, causing the "white spot" disease and posing a significant problem to marine aquaculture. In the present study, a C. irritans-specific reverse primer (S15) was designed based on the published sequence of the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) of C. irritans and used together with the conserved forward primer P1 to develop a specific polymerase chain reaction (PCR) assay for direct, rapid, and specific detection of C. irritans. The specificity of these primers was tested with both closely and distantly related ciliates (Pseudokeroronpsis rubra, Pseudokeroronpsis carnae, Euplotes sp. 1, Ichthyophthirius multifiliis, Pseudourostyla cristata, and Paramecium caudaium), and only C. irritans was detected and no product was amplified from any other ciliates examined in this study using the specific primer set P1-S15. The specific PCR assay was able to detect as low as 45 pg of C. irritans DNA and a nested PCR assay using two primer sets (P1/NC2, P1/S15) increased the sensitivity, allowing the detection of a single C. irritans. The species-specific PCR assays should provide useful tools for the diagnosis, prevention, and molecular epidemiological investigations of C. irritans infection in marine fish.

Quantitative PCR assay for the detection of the parasitic ciliate Cryptocaryon irritans

“…We were not able to obtain any amplicons from the seawater samples without adding theronts in a specific PCR assay [9] with the P1/S15 primer set for C. irritans (data not shown). The rRNA gene copy number estimated with the qPCR assay had a significantly positive linear relationship with the number of theront cells added (p \ 0.001, Fig.…”

“…Although the life cycle and symptoms of I. multifiliis are very similar to those of C. irritans, they are taxonomically quite different [7]. Chen et al [9] reported that they had designed C. irritans-specific primers for a conventional PCR assay and that the assay should be a useful tool for the diagnosis and prevention of as well as epidemiological investigations of C. irritans infections. However, the assay could not detect C. irritans quantitatively because the assay needs nested PCR and the target region is too long to perform a qPCR assay.…”

“…A C1000 Thermal Cycler (Bio-Rad, Richmond, CA, USA) was used for PCR cycling. The primer set comprised C. irritans-specific P1 and S15 [8,9], and the final concentration of each primer was 200 nM. An initial 94°C denaturing step lasting for 5 min was followed by 35 cycles (30 s at 94°C, 30 s at 53°C, and 30 s at 74°C) and a final 74°C extension step for 5 min with KOD-Dash PCR polymerase (Toyobo Co. Ltd., Osaka, Japan).…”

Elicited cross-protection and specific antibodies in Mozambique tilapia (Oreochromis mossambicus) against two different immobilization serotypes of Cryptocaryon irritans isolated in Hawaii

“…DNA extraction from newly hatched theronts was performed with the Qiagen DNeasy kit (Qiagen, California, USA) following the manufacturer's protocol. The polymerase chain reaction (PCR) was used to amplify the rDNA fragment corresponding to the 3 0 portion of the 18S, the ITS-1, the 5.8S, the ITS-2, and the 5 0 portion of the 28S using primers P1 (forward 5 0 -GTTCCCCTTGAACGAGGAATTC-3 0 ) [6,23] and NC2 (reverse 5 0 -TTAGTTTCTTTTCCTCCGCT-3 0 ) [6,26]. PCR amplification was carried out in a final volume of 50 ml containing: 100 ng of template, 2.5 U Taq-Polymerase (Bioline, Massachusetts, USA); 0.3 ml 100 mM of each primer; 10 Â PCR buffer; 4 ml of 10 mM dNTPs in a thermocycler iCyclerTM (BioRad).…”

Sequence homogeneity of internal transcribed spacer rDNA in Mikrocytos mackini and detection of Mikrocytos sp. in a new location

“…Small et al 2007) and finfish (e.g. Chen et al 2008). The specific aims of the present study were to characterize genetic diversity within M. mackini and to survey for the presence of other genetically divergent Mikrocytos sp.…”