Real-time PCR for quantifying Haemonchus contortus eggs and potential limiting factors

Abstract

The purpose of this study was to evaluate the practicality of using real-time PCR for quantifying feces-derived trichostrongyle eggs. Haemonchus contortus eggs were used to evaluate fecal contaminants, time after egg embryonation, and the presence of competing and non-competing DNAs as factors that might interfere with generating reproducible results during simplex and multiplex quantitative real-time PCR (QPCR). Real-time PCR results showed linear quantifiable amplification with DNA from five to 75 eggs. However, threshold cycle (C (T)) values obtained by amplification of DNA from egg numbers between 75 and 1,000 did not differ significantly. Inhibitors of QPCR were effectively removed during DNA extraction as exemplified by the absence of any improvement in C (T) values with bovine serum albumin or phytase treatments. Changes from egg embryonation could only be detected during the first 6 h. Noncompetitive DNA did not appear to impact amplification; however, in a multiplex reaction a competing trichostrongyle such as Cooperia oncophora can hinder amplification of H. contortus DNA, when present at tenfold greater amounts. This study demonstrates the usefulness of QPCR for amplification and quantification of trichostrongyle eggs, and identifies potential limitations, which may be addressed through multiplex assays or the addition of a standard: exogenous DNA target.

Which McMaster egg counting technique is the most reliable?

“…Advances in molecular technology offer the potential for reliable and sensitive methods, which are fast and reproducible. Some authors (Harmon et al 2007;Humbert and Cabaret 1995;Learmount et al 2009;Schnieder et al 1999;Roeber et al 2011;Zarlenga et al 2001) developed different polymerase chain reaction (PCR) tools (multiplex PCR, real-time PCR, RAPD) for discriminating GI nematode eggs from faeces at both the genus and species levels. Yet, although these methods have considerable advantages, they are only semi-quantitative and have problems with extracting DNA directly from faeces resulting in low DNA yield (Bott et al 2009;Learmount et al 2009).…”

Methods for Quantification of Soil-Transmitted Helminths in Environmental Media: Current Techniques and Recent Advances

“…Removal of inhibitors can be accomplished in part by using centrifugal flotation to recover STH ova [81], though even after flotation, adding anti-inhibition agents has been found to increase the sensitivity of genetic assays for STH [82]. To determine whether inhibitors are affecting reactions in a genetic assay, internal control targets can be used (e.g., [83]).…”

Advances in the diagnosis of key gastrointestinal nematode infections of livestock, with an emphasis on small ruminants

“…However, these assays were used for the identification of larval or adult nematodes only, which limited their utility for routine diagnostic applications. Harmon et al (2007) evaluated the use of RT-PCR to quantify eggs of H. contortus from sheep feces and examined various aspects, such as the influence of fecal inhibitors on PCR, the effects of competing and non-competing DNA in multiplex reactions and the impact of embryonic development within the egg on the PCR result. The assay developed showed linear quantifiable amplification of DNA obtained from egg quantities ranging from five to 75 eggs, whereas DNA from higher egg numbers of 75-1000 eggs did not show significant differences in Ct, limiting the quantitative capacity of the assay to a narrow detection range (Harmon et al, 2007).…”