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The best practice for preparation of samples from FTA®cards for diagnosis of blood borne infections using African trypanosomes as a model system.

Ahmed HA, MacLeod ET, Hide G, Welburn SC, Picozzi K - Parasit Vectors (2011)

Bottom Line: Better apparent sensitivity was achieved when blood was lysed prior to application on the FTA cards (73.3%) although this was not significant.This did not improve with subsequent elution using Chelex®100 (73.3%) and was not significantly different from direct DNA extraction from blood in the field (68.3%).Based on these results, the degree of effort required for each of these techniques and the difficulty of DNA extraction under field conditions, we recommend that blood is transferred onto FTA cards whole followed by elution in Chelex®100 as the best approach.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Infectious Diseases, Division of Pathway Medicine, School of Biomedical Sciences, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, UK.

ABSTRACT

Background: Diagnosis of blood borne infectious diseases relies primarily on the detection of the causative agent in the blood sample. Molecular techniques offer sensitive and specific tools for this although considerable difficulties exist when using these approaches in the field environment. In large scale epidemiological studies, FTA®cards are becoming increasingly popular for the rapid collection and archiving of a large number of samples. However, there are some difficulties in the downstream processing of these cards which is essential for the accurate diagnosis of infection. Here we describe recommendations for the best practice approach for sample processing from FTA®cards for the molecular diagnosis of trypanosomiasis using PCR.

Results: A comparison of five techniques was made. Detection from directly applied whole blood was less sensitive (35.6%) than whole blood which was subsequently eluted from the cards using Chelex®100 (56.4%). Better apparent sensitivity was achieved when blood was lysed prior to application on the FTA cards (73.3%) although this was not significant. This did not improve with subsequent elution using Chelex®100 (73.3%) and was not significantly different from direct DNA extraction from blood in the field (68.3%).

Conclusions: Based on these results, the degree of effort required for each of these techniques and the difficulty of DNA extraction under field conditions, we recommend that blood is transferred onto FTA cards whole followed by elution in Chelex®100 as the best approach.

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Related in: MedlinePlus

Cumulative number of examined FTA discs containing whole and lysed blood in relation to the sensitivity of detecting the trypanosome DNA by PCR.
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Figure 1: Cumulative number of examined FTA discs containing whole and lysed blood in relation to the sensitivity of detecting the trypanosome DNA by PCR.

Mentions: Three different sample preparations were collected from 300 animals. These preparations included, whole blood applied on FTA®cards, lysed blood on FTA®cards and direct purification of DNA from blood in the field. Ten discs from each FTA®card applied blood sample were examined individually using TBR-PCR for the detection of T. brucei s.l. The sensitivity of the tests were calculated compared to the gold standard which was defined as the total number of PCR positive results from amplification of trypanosome DNA from samples applied to FTA®cards and/or from directly extracted trypanosome genomic DNA. Figure 1 shows that there is an increase in cumulative sensitivity as the number of individually examined discs was increased. The same phenomenon was observed from both whole and lysed blood samples.


The best practice for preparation of samples from FTA®cards for diagnosis of blood borne infections using African trypanosomes as a model system.

Ahmed HA, MacLeod ET, Hide G, Welburn SC, Picozzi K - Parasit Vectors (2011)

Cumulative number of examined FTA discs containing whole and lysed blood in relation to the sensitivity of detecting the trypanosome DNA by PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108913&req=5

Figure 1: Cumulative number of examined FTA discs containing whole and lysed blood in relation to the sensitivity of detecting the trypanosome DNA by PCR.
Mentions: Three different sample preparations were collected from 300 animals. These preparations included, whole blood applied on FTA®cards, lysed blood on FTA®cards and direct purification of DNA from blood in the field. Ten discs from each FTA®card applied blood sample were examined individually using TBR-PCR for the detection of T. brucei s.l. The sensitivity of the tests were calculated compared to the gold standard which was defined as the total number of PCR positive results from amplification of trypanosome DNA from samples applied to FTA®cards and/or from directly extracted trypanosome genomic DNA. Figure 1 shows that there is an increase in cumulative sensitivity as the number of individually examined discs was increased. The same phenomenon was observed from both whole and lysed blood samples.

Bottom Line: Better apparent sensitivity was achieved when blood was lysed prior to application on the FTA cards (73.3%) although this was not significant.This did not improve with subsequent elution using Chelex®100 (73.3%) and was not significantly different from direct DNA extraction from blood in the field (68.3%).Based on these results, the degree of effort required for each of these techniques and the difficulty of DNA extraction under field conditions, we recommend that blood is transferred onto FTA cards whole followed by elution in Chelex®100 as the best approach.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Infectious Diseases, Division of Pathway Medicine, School of Biomedical Sciences, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, UK.

ABSTRACT

Background: Diagnosis of blood borne infectious diseases relies primarily on the detection of the causative agent in the blood sample. Molecular techniques offer sensitive and specific tools for this although considerable difficulties exist when using these approaches in the field environment. In large scale epidemiological studies, FTA®cards are becoming increasingly popular for the rapid collection and archiving of a large number of samples. However, there are some difficulties in the downstream processing of these cards which is essential for the accurate diagnosis of infection. Here we describe recommendations for the best practice approach for sample processing from FTA®cards for the molecular diagnosis of trypanosomiasis using PCR.

Results: A comparison of five techniques was made. Detection from directly applied whole blood was less sensitive (35.6%) than whole blood which was subsequently eluted from the cards using Chelex®100 (56.4%). Better apparent sensitivity was achieved when blood was lysed prior to application on the FTA cards (73.3%) although this was not significant. This did not improve with subsequent elution using Chelex®100 (73.3%) and was not significantly different from direct DNA extraction from blood in the field (68.3%).

Conclusions: Based on these results, the degree of effort required for each of these techniques and the difficulty of DNA extraction under field conditions, we recommend that blood is transferred onto FTA cards whole followed by elution in Chelex®100 as the best approach.

Show MeSH
Related in: MedlinePlus