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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

A flow diagram outlining the experimental design of samples containing fox faeces with a known number of Echinococcus multilocularis eggs (α2 and β2). 144 samples were divided into two batches for DNA extraction at two different laboratories. Each of the laboratories then carried out two of the four PCR methods on the DNA extractions done at their laboratory (solid line) as well as on DNA extractions sent from the other laboratory (dotted line).
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Figure 1: A flow diagram outlining the experimental design of samples containing fox faeces with a known number of Echinococcus multilocularis eggs (α2 and β2). 144 samples were divided into two batches for DNA extraction at two different laboratories. Each of the laboratories then carried out two of the four PCR methods on the DNA extractions done at their laboratory (solid line) as well as on DNA extractions sent from the other laboratory (dotted line).

Mentions: In this study we wanted to compare different DNA isolation methods and molecular detection methods which were available in our laboratories and try to identify the best combination for use in future surveillance programs (Figure 1). The primary aim of this study was to compare the performance of two sample preparation protocols; the egg-isolation and DNA extraction used in Davidson et al.[2] ‘sieving’, with a new DNA isolation procedure ‘DNA-fishing’ using fox faeces spiked with eggs from E. multilocularis. A secondary aim was to compare a selection of real-time PCR methods for E. multilocularis detection. Comparison of the PCR systems were performed in a blinded trial using two batches of DNA samples prepared from spiked fox faecal samples containing a known number of E. multilocularis eggs. The use of spiked material was a cost-effective alternative to the use of real-life endemic samples identified by necropsy and sedimentation and counting technique. Laboratory spiking of pooled fox faeces allows almost identical samples to be analysed in parallel. The ability to select samples with very low levels of eggs may also help address the sensitivity and reproducibility of different methods more precisely. In comparison the exact egg numbers would be unknown in samples from naturally infected foxes. Results from these experiments helped with the selection of optimised methods for a surveillance programme in an E. multilocularis ‘free’ country, such as Norway.


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)

A flow diagram outlining the experimental design of samples containing fox faeces with a known number of Echinococcus multilocularis eggs (α2 and β2). 144 samples were divided into two batches for DNA extraction at two different laboratories. Each of the laboratories then carried out two of the four PCR methods on the DNA extractions done at their laboratory (solid line) as well as on DNA extractions sent from the other laboratory (dotted line).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: A flow diagram outlining the experimental design of samples containing fox faeces with a known number of Echinococcus multilocularis eggs (α2 and β2). 144 samples were divided into two batches for DNA extraction at two different laboratories. Each of the laboratories then carried out two of the four PCR methods on the DNA extractions done at their laboratory (solid line) as well as on DNA extractions sent from the other laboratory (dotted line).
Mentions: In this study we wanted to compare different DNA isolation methods and molecular detection methods which were available in our laboratories and try to identify the best combination for use in future surveillance programs (Figure 1). The primary aim of this study was to compare the performance of two sample preparation protocols; the egg-isolation and DNA extraction used in Davidson et al.[2] ‘sieving’, with a new DNA isolation procedure ‘DNA-fishing’ using fox faeces spiked with eggs from E. multilocularis. A secondary aim was to compare a selection of real-time PCR methods for E. multilocularis detection. Comparison of the PCR systems were performed in a blinded trial using two batches of DNA samples prepared from spiked fox faecal samples containing a known number of E. multilocularis eggs. The use of spiked material was a cost-effective alternative to the use of real-life endemic samples identified by necropsy and sedimentation and counting technique. Laboratory spiking of pooled fox faeces allows almost identical samples to be analysed in parallel. The ability to select samples with very low levels of eggs may also help address the sensitivity and reproducibility of different methods more precisely. In comparison the exact egg numbers would be unknown in samples from naturally infected foxes. Results from these experiments helped with the selection of optimised methods for a surveillance programme in an E. multilocularis ‘free’ country, such as Norway.

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