sybr-green-i and Protozoan-Infections--Animal

A new molecular diagnostic assay was developed for detection of Tetracapsuloides bryosalmonae the causative agent of proliferative kidney disease (PKD) in salmonid fish using a loop-mediated isothermal amplification method (LAMP). The PKD-LAMP assay amplifies the T. bryosalmonae DNA extracted from infected kidney, under constant temperature of 65 degrees C within 1 h. The required equipment for DNA amplification is only a water bath. The amplification products were detected visually by using SYBR green I dye, which turns green in the presence of amplified products and remains orange in its absence, and by electrophoresis without any difference in the sensitivity of both methods. The developed PKD-LAMP assay demonstrated an exceptionally higher sensitivity than the conventional PCR. PKD-LAMP assay was found to be 100-fold more sensitive than the PCR assay. The developed assay is simple, rapid, cost-effective, specific and highly sensitive. The assay is also characterized by its field applicability, as it does not require the use of sophisticated equipment or skilled personnel.

Topics: Animals; Benzothiazoles; Diamines; DNA, Protozoan; Eukaryota; Fish Diseases; Kidney; Kidney Diseases; Liver; Nucleic Acid Amplification Techniques; Organic Chemicals; Protozoan Infections, Animal; Quinolines; Salmonidae; Sensitivity and Specificity; Spleen

A loop-mediated isothermal amplification assay was developed for the rapid detection of Myxobolus cerebralis in both fish and oligochaete hosts. The assay was optimized to amplify parasitic DNA by incubation with Bst DNA polymerase and a set of six specially constructed primers at 65 degrees C for 60 min. The amplification products were detected visually using SYBR Green I dye which gave identical results to gel electrophoresis analysis. Parasite DNA was detected from infected oligochaetes, and from the anal fin, caudal fin, dorsal fin and operculum of clinically infected fish. This 'Myxo-LAMP' assay has a detection limit similar to that of a polymerase chain reaction assay (10(-6)), but is more rapid and only requires a water bath for amplification and is therefore practical for simple and rapid diagnosis of infected tissue.

Topics: Animals; Base Sequence; Benzothiazoles; Diamines; DNA Primers; Electrophoresis, Agar Gel; Eukaryota; Fish Diseases; Fishes; Molecular Diagnostic Techniques; Molecular Sequence Data; Nucleic Acid Amplification Techniques; Oligochaeta; Organic Chemicals; Protozoan Infections, Animal; Quinolines; RNA, Ribosomal, 18S; Sequence Analysis, DNA