Dataset Information

Development and validation of real-time PCR for rapid detection of Mecistocirrus digitatus.

ABSTRACT: Hematophagous activity of Mecistocirrus digitatus, which causes substantial blood and weight loss in large ruminants, is an emerging challenge due to the economic loss it brings to the livestock industry. Infected animals are treated with anthelmintic drugs, based on the identification of helminth species and the severity of infection; however, traditional methods such as microscopic identification and the counting of eggs for diagnosis and determination of level of infection are laborious, cumbersome and unreliable. To facilitate the detection of this parasite, a SYBR green-based real-time PCR was standardized and validated for the detection of M. digitatus infection in cattle and buffaloes. Oligonucleotides were designed to amplify partial Internal Transcribed Spacer (ITS)-1 sequence of M. digitatus. The specificity of the primers was confirmed by non-amplification of DNA extracted from other commonly occurring gastrointestinal nematodes in ruminants. Plasmids were ligated with partial ITS-1 sequence of M. digitatus, serially diluted (hundred fold) and used as standards in the real-time PCR assay. The quantification cycle (Cq) values were plotted against the standard DNA concentration to produce a standard curve. The assay was sensitive enough to detect one plasmid containing the M. digitatus DNA. Clinical application of this assay was validated by testing the DNA extracted from the faeces of naturally infected cattle (n?=?40) and buffaloes (n?=?25). The results were compared with our standard curve to calculate the quantity of M. digitatus in each faecal sample. The Cq value of the assay depicted a strong linear relationship with faecal DNA content, with a regression coefficient of 0.984 and efficiency of 99%. This assay has noteworthy advantages over the conventional methods of diagnosis because it is more specific, sensitive and reliable.

SUBMITTER: Subhadra S

PROVIDER: S-EPMC3639944 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Development and validation of real-time PCR for rapid detection of Mecistocirrus digitatus.

Subhadra Subhra S Karthik Mohanraj M Raman Muthusamy M

PloS one 20130430 4

Hematophagous activity of Mecistocirrus digitatus, which causes substantial blood and weight loss in large ruminants, is an emerging challenge due to the economic loss it brings to the livestock industry. Infected animals are treated with anthelmintic drugs, based on the identification of helminth species and the severity of infection; however, traditional methods such as microscopic identification and the counting of eggs for diagnosis and determination of level of infection are laborious, cumb ...[more]

PMID: 23646171

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Project description:Reliable species identification methods are important for industrial environmental monitoring programs. Probe based real-time quantitative polymerase chain reaction (qPCR) provides an accurate, cost-effective and high-throughput method for species identification. Here we present the development and validation of species-specific primers and probes for the cytochrome c oxidase (COI) gene for the identification of eight ecologically and economically important freshwater fish species: lake whitefish (Coregonus clupeaformis), yellow perch (Perca flavescens), rainbow smelt (Osmerus mordax), brook trout (Salvelinus fontinalis), smallmouth bass (Micropterus dolomieu), round whitefish (Prosopium cylindraceum), spottail shiner (Notropis hudsonius) and deepwater sculpin (Myoxocephalus thompsonii). In order to identify novel primer-probe sets with maximum species-specificity, two separate primer-probe design criteria were employed. Highest ranked primer-probe sets from both methods were assayed to identify sequences that demonstrated highest specificity. Specificity was determined using control species from same genus and non-target species from different genus. Selected primer-probe sets were optimized for annealing temperature and primer-probe concentrations to identify minimum reagent parameters. The selected primer-probe sets were highly sensitive, with DNA concentrations as low as 1 ng adequate for positive species identification. A decoder algorithm was developed based on the cumulative qPCR results that allowed for full automation of species identification. Blinded experiments revealed that the combination of the species-specific primer/probes sets with the automated species decoder resulted in target species identification with 100% accuracy. We also conducted a cost/time comparison analysis between the qPCR assays established in this study with other species identification methods. The qPCR technique was the most cost-effective and least time consuming method of species identification. In summary, probe-based multiplex qPCR assays provide a rapid and accurate method for freshwater fish species identification, and the methodology established in this study can be utilized for various other species identification initiatives.

Dataset Information

Development and validation of real-time PCR for rapid detection of Mecistocirrus digitatus.

Publications

Development and validation of real-time PCR for rapid detection of Mecistocirrus digitatus.

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