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Detection of capripoxvirus DNA using a novel loop-mediated isothermal amplification assay.

Murray L, Edwards L, Tuppurainen ES, Bachanek-Bankowska K, Oura CA, Mioulet V, King DP - BMC Vet. Res. (2013)

Bottom Line: Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants.This LAMP assay successfully detected DNA prepared from representative CaPV isolates (SPPV, GTPV and LSDV), and did not cross-react with DNA extracted from other mammalian poxviruses.This study provides a simple and rapid approach to detect CaPV DNA that may have utility for use in the field, or in non-specialised laboratories where expensive equipment is not available.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Pirbright Institute, Pirbright, Surrey, UK.

ABSTRACT

Background: Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants. They are double stranded DNA viruses of the genus Capripoxvirus, (subfamily Chordopoxvirinae) within the family Poxviridae. The aim of this study was to develop a Loop-mediated isothermal AMPlification (LAMP) assay for the detection of Capripoxvirus (CaPV) DNA.

Results: A single LAMP assay targeting a conserved region of the CaPV P32 gene was selected from 3 pilot LAMP assays and optimised by adding loop primers to accelerate the reaction time. This LAMP assay successfully detected DNA prepared from representative CaPV isolates (SPPV, GTPV and LSDV), and did not cross-react with DNA extracted from other mammalian poxviruses. The analytical sensitivity of the LAMP assay was determined to be at least 163 DNA copies/μl which is equivalent to the performance reported for diagnostic real-time PCR currently used for the detection of CaPV. LAMP reactions were monitored with an intercalating dye using a real-time PCR machine, or by agarose-gel electrophoresis. Furthermore, dual labelled LAMP products (generated using internal LAMP primers that were conjugated with either biotin or fluorescein) could be readily visualised using a lateral-flow device.

Conclusions: This study provides a simple and rapid approach to detect CaPV DNA that may have utility for use in the field, or in non-specialised laboratories where expensive equipment is not available.

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Evaluation of candidate LAMP assays. A: Agarose gel (2%) showing the characteristic laddering pattern generated by LAMP for the detection of GTPV isolate Vietnam Ninh Tuan 05 by three different LAMP primer sets: EP32A (P32 gene); Epol (RNA polymerase RPO30 subunit) and ETop (DNA topoisomerase I). Results for negative controls (−) are shown. MW: molecular weight ladder (100 bp). B: Corresponding increase in fluorescence generated for EP32A (○: circle); Epol (△: triangle) and ETop (◊: diamond) LAMP assays using a real-time PCR machine. Representative signal for a negative control (□) well is shown.
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Figure 1: Evaluation of candidate LAMP assays. A: Agarose gel (2%) showing the characteristic laddering pattern generated by LAMP for the detection of GTPV isolate Vietnam Ninh Tuan 05 by three different LAMP primer sets: EP32A (P32 gene); Epol (RNA polymerase RPO30 subunit) and ETop (DNA topoisomerase I). Results for negative controls (−) are shown. MW: molecular weight ladder (100 bp). B: Corresponding increase in fluorescence generated for EP32A (○: circle); Epol (△: triangle) and ETop (◊: diamond) LAMP assays using a real-time PCR machine. Representative signal for a negative control (□) well is shown.

Mentions: In initial experiments, all three candidate LAMP assays that targeted the RNA polymerase subunit RPO30, DNA topoisomerase I and the P32 regions generated characteristic laddering patterns after agarose-gel electrophoresis (Figure 1A) and an increase in fluorescence in a real-time PCR machine (Figure 1B). However, these experiments indicated that the P32 LAMP assay was the most rapid and reliable assay, and therefore this assay was selected for further optimisation and evaluation. The optimum reagent concentrations in the LAMP reactions mixes were 1× Thermopol buffer (New England Biolabs, Hitchin, UK), 3 μM internal primers, 0.6 μM external primers, 1 mM MgSO4 (New England Biolabs, Hitchin, UK), 0.3 mM dNTPs (Sigma-Aldrich, Dorset, UK), 1 M betaine (Sigma-Aldrich, Dorset, UK), 16 U of Bst DNA polymerase (Large fragment: (New England Biolabs, Hitchin, UK), along with the addition of 2 μl of target DNA. Using the P32 primer set, loop primers were added to the reaction at an optimal concentration of 4 μM. These loop primers decreased the reaction time by nine minutes for a GTPV isolate (Vietnam Ninh Tuan 05; Figure 2). Restriction enzyme digests were performed on the P32 LAMP amplification products to confirm that the correct region of the CaPV genome had been amplified. A restriction endonuclease (BsrG I) was chosen to cut specifically a single site within the target region of the P32 LAMP assay. After incubation of 5 μl LAMP product with BsrG I at 37°C for 75 min, the digested products were visualised on a 4.0% agarose gel and could be differentiated from the characteristic laddering pattern of the LAMP reaction products confirming the sequence specificity of the DNA amplicons (data not shown).


Detection of capripoxvirus DNA using a novel loop-mediated isothermal amplification assay.

Murray L, Edwards L, Tuppurainen ES, Bachanek-Bankowska K, Oura CA, Mioulet V, King DP - BMC Vet. Res. (2013)

Evaluation of candidate LAMP assays. A: Agarose gel (2%) showing the characteristic laddering pattern generated by LAMP for the detection of GTPV isolate Vietnam Ninh Tuan 05 by three different LAMP primer sets: EP32A (P32 gene); Epol (RNA polymerase RPO30 subunit) and ETop (DNA topoisomerase I). Results for negative controls (−) are shown. MW: molecular weight ladder (100 bp). B: Corresponding increase in fluorescence generated for EP32A (○: circle); Epol (△: triangle) and ETop (◊: diamond) LAMP assays using a real-time PCR machine. Representative signal for a negative control (□) well is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3649941&req=5

Figure 1: Evaluation of candidate LAMP assays. A: Agarose gel (2%) showing the characteristic laddering pattern generated by LAMP for the detection of GTPV isolate Vietnam Ninh Tuan 05 by three different LAMP primer sets: EP32A (P32 gene); Epol (RNA polymerase RPO30 subunit) and ETop (DNA topoisomerase I). Results for negative controls (−) are shown. MW: molecular weight ladder (100 bp). B: Corresponding increase in fluorescence generated for EP32A (○: circle); Epol (△: triangle) and ETop (◊: diamond) LAMP assays using a real-time PCR machine. Representative signal for a negative control (□) well is shown.
Mentions: In initial experiments, all three candidate LAMP assays that targeted the RNA polymerase subunit RPO30, DNA topoisomerase I and the P32 regions generated characteristic laddering patterns after agarose-gel electrophoresis (Figure 1A) and an increase in fluorescence in a real-time PCR machine (Figure 1B). However, these experiments indicated that the P32 LAMP assay was the most rapid and reliable assay, and therefore this assay was selected for further optimisation and evaluation. The optimum reagent concentrations in the LAMP reactions mixes were 1× Thermopol buffer (New England Biolabs, Hitchin, UK), 3 μM internal primers, 0.6 μM external primers, 1 mM MgSO4 (New England Biolabs, Hitchin, UK), 0.3 mM dNTPs (Sigma-Aldrich, Dorset, UK), 1 M betaine (Sigma-Aldrich, Dorset, UK), 16 U of Bst DNA polymerase (Large fragment: (New England Biolabs, Hitchin, UK), along with the addition of 2 μl of target DNA. Using the P32 primer set, loop primers were added to the reaction at an optimal concentration of 4 μM. These loop primers decreased the reaction time by nine minutes for a GTPV isolate (Vietnam Ninh Tuan 05; Figure 2). Restriction enzyme digests were performed on the P32 LAMP amplification products to confirm that the correct region of the CaPV genome had been amplified. A restriction endonuclease (BsrG I) was chosen to cut specifically a single site within the target region of the P32 LAMP assay. After incubation of 5 μl LAMP product with BsrG I at 37°C for 75 min, the digested products were visualised on a 4.0% agarose gel and could be differentiated from the characteristic laddering pattern of the LAMP reaction products confirming the sequence specificity of the DNA amplicons (data not shown).

Bottom Line: Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants.This LAMP assay successfully detected DNA prepared from representative CaPV isolates (SPPV, GTPV and LSDV), and did not cross-react with DNA extracted from other mammalian poxviruses.This study provides a simple and rapid approach to detect CaPV DNA that may have utility for use in the field, or in non-specialised laboratories where expensive equipment is not available.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Pirbright Institute, Pirbright, Surrey, UK.

ABSTRACT

Background: Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants. They are double stranded DNA viruses of the genus Capripoxvirus, (subfamily Chordopoxvirinae) within the family Poxviridae. The aim of this study was to develop a Loop-mediated isothermal AMPlification (LAMP) assay for the detection of Capripoxvirus (CaPV) DNA.

Results: A single LAMP assay targeting a conserved region of the CaPV P32 gene was selected from 3 pilot LAMP assays and optimised by adding loop primers to accelerate the reaction time. This LAMP assay successfully detected DNA prepared from representative CaPV isolates (SPPV, GTPV and LSDV), and did not cross-react with DNA extracted from other mammalian poxviruses. The analytical sensitivity of the LAMP assay was determined to be at least 163 DNA copies/μl which is equivalent to the performance reported for diagnostic real-time PCR currently used for the detection of CaPV. LAMP reactions were monitored with an intercalating dye using a real-time PCR machine, or by agarose-gel electrophoresis. Furthermore, dual labelled LAMP products (generated using internal LAMP primers that were conjugated with either biotin or fluorescein) could be readily visualised using a lateral-flow device.

Conclusions: This study provides a simple and rapid approach to detect CaPV DNA that may have utility for use in the field, or in non-specialised laboratories where expensive equipment is not available.

Show MeSH
Related in: MedlinePlus