Development and Validation of Real-Time PCR for Rapid Detection of Mecistocirrus digitatus
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Date
2013-04
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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.
Description
TNV_PLOS_2013_8(4)1-6
Keywords
Veterinary Science