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Laboratory assessment of sensitive molecular tools for detection of low levels of Echinococcus multilocularis-eggs in fox (Vulpes vulpes) faeces.

Øines Ø, Isaksson M, Hagström Å, Tavornpanich S, Davidson RK - Parasit Vectors (2014)

Bottom Line: Results also indicate that replicates of the PCR-reactions improve detection sensitivity when egg numbers are low.The results indicate that the use of real-time PCR combined with targeted DNA extraction, improves the sensitivity of E. multilocularis detection in faecal samples containing low numbers of E. multilocularis eggs.Results also indicate the importance of replicates of the PCR-reactions when pathogen levels are low.

View Article: PubMed Central - HTML - PubMed

Affiliation: Norwegian Veterinary Institute, Post boks 750 Sentrum, 0106, Oslo, Norway. oivind.oines@vetinst.no.

ABSTRACT

Background: In endemic areas with very low infection prevalence, the frequency and intensity of Echinococcus multilocularis can be extremely low. This necessitates efficient, specific and sensitive molecular tools. We wanted to compare the existing molecular tools, used in the Norwegian national surveillance programme, and compare these with new techniques for detection of this zoonotic pathogen in fox faeces. Here we present the results of screening samples containing a known level of E. multilocularis eggs with two highly sensitive DNA isolation and extraction methods combined with one conventional PCR and three real-time PCR methods for detection.

Methods: We performed a comparison of two extraction protocols; one based on sieving of faecal material and one using targeted DNA sampling. Four methods of molecular detection were tested on E. multilocularis-egg spiked fox faeces.

Results: There were significant differences between the multiplex PCR/egg sieving DNA extraction methods compared to the new DNA fishing method and the three real-time PCR assays. Results also indicate that replicates of the PCR-reactions improve detection sensitivity when egg numbers are low.

Conclusions: The results indicate that the use of real-time PCR combined with targeted DNA extraction, improves the sensitivity of E. multilocularis detection in faecal samples containing low numbers of E. multilocularis eggs. Results also indicate the importance of replicates of the PCR-reactions when pathogen levels are low.

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Related in: MedlinePlus

Comparing the density distribution of egg numbers from samples from batches α2- egg sieving and β2 DNA-fishing. Kolmogorov-Smirnov (KS) goodness of fit test was used to determine if egg distributions between the two batches, α2 and β2, were similar.
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Figure 2: Comparing the density distribution of egg numbers from samples from batches α2- egg sieving and β2 DNA-fishing. Kolmogorov-Smirnov (KS) goodness of fit test was used to determine if egg distributions between the two batches, α2 and β2, were similar.

Mentions: A pool of fox faeces from 29 animals, which had been confirmed as E. multilocularis negative during the Norwegian 2010/2011 surveillance programme, was homogenised using vigorous stirring for five minutes. Larger debris such as hair, bones, feathers and plastic were removed from the faecal pool. One hundred and forty four 15 ml falcon tubes were filled with 3 ± 0.05 g of the homogenised fox faeces and labelled 1 to 144 (Additional file 1: Table S2). The tubes were divided into two batches (α2/β2) (Figure 1). A known number of E. multilocularis eggs were added to randomly selected numbered-tubes. Fifteen tubes in each batch had 1-4 eggs added, fifteen had 5-15 eggs added; another 15 tubes had 16-50 eggs added; 51-150 eggs were added to 14 tubes; twelve tubes had no eggs added, whilst a single tube in each batch respectively contained a high number of eggs (600-1000). Density plots for the distribution of the number of eggs per tube for the α2 and β2 batches are presented in Figure 2. The Kolmogorov-Smirnov (KS) goodness of fit test was used to determine if the egg distributions between the two batches were similar [24].


Laboratory assessment of sensitive molecular tools for detection of low levels of Echinococcus multilocularis-eggs in fox (Vulpes vulpes) faeces.

Øines Ø, Isaksson M, Hagström Å, Tavornpanich S, Davidson RK - Parasit Vectors (2014)

Comparing the density distribution of egg numbers from samples from batches α2- egg sieving and β2 DNA-fishing. Kolmogorov-Smirnov (KS) goodness of fit test was used to determine if egg distributions between the two batches, α2 and β2, were similar.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4060867&req=5

Figure 2: Comparing the density distribution of egg numbers from samples from batches α2- egg sieving and β2 DNA-fishing. Kolmogorov-Smirnov (KS) goodness of fit test was used to determine if egg distributions between the two batches, α2 and β2, were similar.
Mentions: A pool of fox faeces from 29 animals, which had been confirmed as E. multilocularis negative during the Norwegian 2010/2011 surveillance programme, was homogenised using vigorous stirring for five minutes. Larger debris such as hair, bones, feathers and plastic were removed from the faecal pool. One hundred and forty four 15 ml falcon tubes were filled with 3 ± 0.05 g of the homogenised fox faeces and labelled 1 to 144 (Additional file 1: Table S2). The tubes were divided into two batches (α2/β2) (Figure 1). A known number of E. multilocularis eggs were added to randomly selected numbered-tubes. Fifteen tubes in each batch had 1-4 eggs added, fifteen had 5-15 eggs added; another 15 tubes had 16-50 eggs added; 51-150 eggs were added to 14 tubes; twelve tubes had no eggs added, whilst a single tube in each batch respectively contained a high number of eggs (600-1000). Density plots for the distribution of the number of eggs per tube for the α2 and β2 batches are presented in Figure 2. The Kolmogorov-Smirnov (KS) goodness of fit test was used to determine if the egg distributions between the two batches were similar [24].

Bottom Line: Results also indicate that replicates of the PCR-reactions improve detection sensitivity when egg numbers are low.The results indicate that the use of real-time PCR combined with targeted DNA extraction, improves the sensitivity of E. multilocularis detection in faecal samples containing low numbers of E. multilocularis eggs.Results also indicate the importance of replicates of the PCR-reactions when pathogen levels are low.

View Article: PubMed Central - HTML - PubMed

Affiliation: Norwegian Veterinary Institute, Post boks 750 Sentrum, 0106, Oslo, Norway. oivind.oines@vetinst.no.

ABSTRACT

Background: In endemic areas with very low infection prevalence, the frequency and intensity of Echinococcus multilocularis can be extremely low. This necessitates efficient, specific and sensitive molecular tools. We wanted to compare the existing molecular tools, used in the Norwegian national surveillance programme, and compare these with new techniques for detection of this zoonotic pathogen in fox faeces. Here we present the results of screening samples containing a known level of E. multilocularis eggs with two highly sensitive DNA isolation and extraction methods combined with one conventional PCR and three real-time PCR methods for detection.

Methods: We performed a comparison of two extraction protocols; one based on sieving of faecal material and one using targeted DNA sampling. Four methods of molecular detection were tested on E. multilocularis-egg spiked fox faeces.

Results: There were significant differences between the multiplex PCR/egg sieving DNA extraction methods compared to the new DNA fishing method and the three real-time PCR assays. Results also indicate that replicates of the PCR-reactions improve detection sensitivity when egg numbers are low.

Conclusions: The results indicate that the use of real-time PCR combined with targeted DNA extraction, improves the sensitivity of E. multilocularis detection in faecal samples containing low numbers of E. multilocularis eggs. Results also indicate the importance of replicates of the PCR-reactions when pathogen levels are low.

Show MeSH
Related in: MedlinePlus