Development of a PCR diagnostic test for the rapid

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Development of a PCR diagnostic test for the rapid Powered By Docstoc
					Development of a PCR diagnostic test for the rapid               using the MoBio Clean-Up Kit (MoBio). DNA dilutions
detection   of    the  nematophagous        fungus               equivalent to 487, 363, 242, 121, 60 and 12
Duddingtonia flagrans.                                           chlamydospores were obtained and subjected to PCR
P. Kelly1, 2 B. Good1, R. Fitzpatrick1, J. P. Hanrahan1,
T. de Waal2                                                      Results and Discussion
                                                                 D. flagrans specific primers DFITS1F and DFITS2R
 Teagasc, Animal Production Research Centre,                     were constructed from the ITS regions as shown in
Athenry, Co. Galway. 2School of Agriculture Food                 Figure 1.
Science and Veterinary Medicine, UCD, Belfield D4.
Introduction                                                                    18S rRNA                    5S rRNA                          28S rRNA
                                                                                                   ITS1                       ITS2
The nematophagous fungus, Duddingtonia flagrans is a
potential biocontrol agent against nematode parasites of
ruminants (Larsen, 2006). Improved methods for the                5’-CTTCGTCCGGTCTTGACCG- 3’                                         5’-TTCGGGTTCAAA ACCAGCGTT– 3’
accurate and sensitive detection of D. flagrans would                      DFITS1F                                                           DFITS2R
                                                                                                           296 bp
significantly aid in the evaluation of this fungus as a
potential biocontrol agent in the field. Advances in
molecular diagnostics are now being used. For                    Figure 1. Schematic representation of fungal ribosomal
example, primers that target the internal transcribed            genes containing the target areas used in the design of
spacer (ITS) regions combined with the polymerase                D. flagrans species specific primers.
chain reaction (PCR) technology are increasingly being
used to develop rapid, accurate and sensitive species            D. flagrans species specific primers were found to be
specific diagnostic tests for a broad range of organisms.        highly specific when tested against 15 different fungal
In this study, we describe the development of a species          species (Fig. 2A). A single PCR product of the expected
specific PCR diagnostic test for the rapid detection of          size 296 bp was exclusively amplified from D. flagrans
D. flagrans.                                                     demonstrating the specificity of the PCR assay. The
                                                                 control PCR reaction using universal fungal primers
Materials and Methods                                            was positive for all fungal isolates (Fig. 2B). The PCR
The 16 endoparasitic fungal species as described in our          assay was also positive when tested on four different D.
previous study (Kelly et al., 2006) were used. Fungal            flagrans isolates. Soil samples containing the equivalent
genomic DNA was isolated by mechanical disruption                of 12 chlamydospores were sufficient for reliable
and enzymatic digestion. Briefly, approximately 500 mg           amplification of the D. flagrans specific PCR product.
of liquid nitrogen frozen fungal mycelium was
homogenized in 3 ml of PBS (pH 7.0) containing 3 mg                              A   1     2   3   4   5    6   7     8   9    10    11 12   13 14   15   16 17

of lysing enzyme (Sigma) and incubated at 37°C for 3 h.
This was followed by a 1 h incubation at 65°C in                       296 bp

extraction buffer (50 mM Tris HCl, 100 mM EDTA,
100 mM NaCl2, 1 % SDS, pH 8.0) containing 3 mg of                                B   1     2   3   4   5    6   7     8   9    10    11 12   13 14   15   16 17
proteinase K. Genomic DNA was extracted with phenol
and chloroform and precipitated using 0.6 volumes of                   320 bp

isopropanol. The quantity and quality of the DNA was
determined by absorbance at 260 and 280 nm. Primer
design was based on internal transcribed spacer (ITS)
DNA sequences obtained from GenBank/EMBL
databases. The ITS1 and ITS2 sequences of D. flagrans            Figure 2. Specificity of the PCR assay with genomic DNA
                                                                 isolated from 16 different fungal species. Panel ‘A’ represents
were aligned with ITS1 and ITS2 sequences derived                PCR assay using D. flagrans species specific primers. Panel
from the closest related fungal species using Clustal W.         ‘B’ represents control PCR assay using fungal universal
From this alignment two D. flagrans species specific             primers. Lane 1. 100 bp marker, 2. D. flagrans, 3. Arthrobotrys
primers were designed, DFITS1 and DFITS2 (Figure 1).             oligospora, 4. Harpsosporium lilliputanum, 5. Harpsosporium
The optimum PCR reaction conditions for the detection            helicoides, 6. Haptocillium sphaerosporum, 7. Paecilomyces lilacinus,
                                                                 8. Haptocillium balanoides, 9. Verticillium sphaerosporum,
of D. flagrans consisted of 50 ng of genomic DNA, 1 x            10. Drechmeria coniospora, 11. Pochonia. chlamydosporium,
PCR buffer, 0.2 µM dNTPs, 4mM MgCl2, 100 ng of                   12. Fusarium coeruleum, 13. Verticilllium coccospora, 14. Fusarium
each primer and 2.5 U of Taq DNA polymerase in a                 incarnatum, 15. Verticillium chlamydosporium, 16. Pyricularia
final reaction volume of 50 µl. Optimised PCR                    higginsii, 17. Fusarium oxysporum.
amplification conditions consisted of 34 cycles of 95 °C         Conclusions
for 30 s, 66.6 °C for 30 s, and 72 °C for 30 s. PCR              The PCR diagnostic test developed in this study
products were separated on 1.5% EtBr stained gels and            provides a rapid (< 8 h), accurate and sensitive test for
visualized using a Molecular Imager FX (Bio-Rad). The            D. flagrans which will enhance the evaluation of this
identity of the PCR product was confirmed by DNA                 fungus as a nematode biocontrol agent in the field.
sequencing. The primers used for universal fungal
amplification were UR (5’TCCTCCGCTTATTGATAGC-3’)                 References
and     UF     (5’-GTGAATCATCGAATCTTTGAA-3’).        The         Kelly, P., Good, B., Hanrahan, J.P., De Waal DT., &
sensitivity of the PCR assay was determined using a soil         Mulcahy, G. (2005). Proc. Agricultural Research
sampled spiked with 106 D. flagrans chlamydospores.              Forum p27.
Total DNA was extracted from this sample using the               Larsen M. (2006) Journal of Animal Science. 84: 133-
Ultra Clean Mega Kit (MoBio) and further purified                139.