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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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Graph showing the percentage of E. multilocularis egg spiked faecal samples that were detected by each of four PCR methods (Taq-PCR blue diamond; EVA-PCR green square; mPCR red triangle; and CO1rtPCR purple circle) after using two different DNA extraction methods – egg sieving (dotted line, N = 59) and DNA-fishing (solid line, N = 59), once the negative and the high positive control samples (groups E, n = 12, and F, n = 1) were excluded from the data set. The 95% confidence interval is given for the detection level.
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Figure 5: Graph showing the percentage of E. multilocularis egg spiked faecal samples that were detected by each of four PCR methods (Taq-PCR blue diamond; EVA-PCR green square; mPCR red triangle; and CO1rtPCR purple circle) after using two different DNA extraction methods – egg sieving (dotted line, N = 59) and DNA-fishing (solid line, N = 59), once the negative and the high positive control samples (groups E, n = 12, and F, n = 1) were excluded from the data set. The 95% confidence interval is given for the detection level.

Mentions: Looking at the results in Figure 3, it is apparent that the samples containing 15 eggs or less were less frequently detected as positive compared to the samples that were spiked with higher numbers of eggs. Figure 5 shows the percentage of samples correctly identified as containing eggs (and the 95% confidence interval) by DNA extraction method (fishing or sieving) and detection (Taq PCR, EVA PCR, mPCR, CO1rtPCR). From this figure, it is clear that the samples examined using DNA-fishing did relatively better than those investigated using egg sieving DNA extraction. All the tests were able to detect E. multilocularis DNA in the spiked samples, with the exception of mPCR (Figure 5). The mPCR combined with DNA-fishing was only able to detect the high positive control but none of the other samples. The overall performance recorded for the two DNA extraction protocols and the four detection methods are indicated by the plot of the ROC (Figure 6). The y-axis represents test sensitivity and x-axis represents test specificity. In general, the test sensitivity increases as test specificity decreases and the higher the area under the curve the better test accuracy. By observing the curves, the three real-time PCR methods using DNA-fishing, performed better than methods using egg sieving. Although this was not the case for mPCR, as the template from the DNA-fishing seemed not to work properly with this assay. In the β2 batch, the EVA-PCR yielded the highest sensitivity followed by Taq-PCR and CO1rtPCR, respectively, for the same level of test specificity.


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)

Graph showing the percentage of E. multilocularis egg spiked faecal samples that were detected by each of four PCR methods (Taq-PCR blue diamond; EVA-PCR green square; mPCR red triangle; and CO1rtPCR purple circle) after using two different DNA extraction methods – egg sieving (dotted line, N = 59) and DNA-fishing (solid line, N = 59), once the negative and the high positive control samples (groups E, n = 12, and F, n = 1) were excluded from the data set. The 95% confidence interval is given for the detection level.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Graph showing the percentage of E. multilocularis egg spiked faecal samples that were detected by each of four PCR methods (Taq-PCR blue diamond; EVA-PCR green square; mPCR red triangle; and CO1rtPCR purple circle) after using two different DNA extraction methods – egg sieving (dotted line, N = 59) and DNA-fishing (solid line, N = 59), once the negative and the high positive control samples (groups E, n = 12, and F, n = 1) were excluded from the data set. The 95% confidence interval is given for the detection level.
Mentions: Looking at the results in Figure 3, it is apparent that the samples containing 15 eggs or less were less frequently detected as positive compared to the samples that were spiked with higher numbers of eggs. Figure 5 shows the percentage of samples correctly identified as containing eggs (and the 95% confidence interval) by DNA extraction method (fishing or sieving) and detection (Taq PCR, EVA PCR, mPCR, CO1rtPCR). From this figure, it is clear that the samples examined using DNA-fishing did relatively better than those investigated using egg sieving DNA extraction. All the tests were able to detect E. multilocularis DNA in the spiked samples, with the exception of mPCR (Figure 5). The mPCR combined with DNA-fishing was only able to detect the high positive control but none of the other samples. The overall performance recorded for the two DNA extraction protocols and the four detection methods are indicated by the plot of the ROC (Figure 6). The y-axis represents test sensitivity and x-axis represents test specificity. In general, the test sensitivity increases as test specificity decreases and the higher the area under the curve the better test accuracy. By observing the curves, the three real-time PCR methods using DNA-fishing, performed better than methods using egg sieving. Although this was not the case for mPCR, as the template from the DNA-fishing seemed not to work properly with this assay. In the β2 batch, the EVA-PCR yielded the highest sensitivity followed by Taq-PCR and CO1rtPCR, respectively, for the same level of test specificity.

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