©1993 Oxford University Press
Nucleic Acids Research, 1993, Vol. 21, No. 10
2525-2526
Rapid sequencing of rDNA from single worms and eggs of
parasitic helminths
Robin B.Gasser, Neil B.Chilton, Herve Hoste1 and Ian Beveridge
The University of Melbourne, Department of Veterinary Science, Princes Highway, Werribee,
Victoria 3030, Australia and 1INRA-CR Tours, Nouzilly F37380, France
Received March 19, 1993; Accepted April 7, 1993
The development of highly sensitive diagnostic techniques for
the accurate identification of individual eggs of parasite species
of medical and veterinary importance is central to the control
of the diseases they cause. Sequencing of ribosomal genes
provides a powerful molecular tool for species-level diagnosis
and phylogenetic studies (1-3), and is usually based on either
of the two original techniques (4, 5). Direct PCR cycle sequencing
(6, 7) is an attractive approach, because it is rapid, labour
effective, and can be used to generate template from minute
quantities of material (8, 9). This is especially important since
often only limited specimens are available. This method also
enables DNA template to be sequenced at high temperatures, thus
reducing artifacts due to local secondary structure. Sequencing
techniques rely on pure template DNA. Unfortunately, it is often
difficult to isolate sufficient and pure DNA template from some
parasitic helminths, because of their tough cuticle (10) and a
'white flocculate' substance found to co-precipitate with DNA
during isolation (11, 12), which inhibits subsequent enzymatic
reactions. In this paper, we describe a DNA isolation method
which overcomes these problems and a PCR cycle sequencing
technique which is sufficiently sensitive to sequence rDNA from
single nematode eggs.
1. Isolation and purification of rDNA
Worms (single worms or ~ 200 /tl packed vol.) were suspended
in 250-500 /tl 20 mM Tris-HCl, pH 8.0, 100 mM EDTA,
1% sodium dodecyl sulfate (SDS) containing 500 fig/ml
Proteinase K (Boehringer), homogenized with a polytron (Omni
1000, FSE) for 1 min (slow speed) and incubated for 10 min
at 37 °C. The use of the polytron homogenizer is much more rapid
and effective for the extraction of DNA from helminths than
simple digestion with Proteinase K, particularly for adult and
larval nematodes which have a tough cuticle (10) compared to
other helminths, such as cestodes and trematodes. Although
homogenization would appear to result in shearing of the DNA,
slight shearing is seen only in 1 % of cases (n = 100) and is
negligible for use in PCR. In the remainder of cases, a single
high molecular weight band was seen on agarose gels (Figure
1). After homogenization and incubation the suspension was
centrifuged (10,000 g) for 3 min. The supernatant was transferred
to a fresh tube and extracted once with phenol/
chloroform/isoamyl alcohol (25/24/1). The aqueous phase was
precipitated with 2 x vol. absolute ethanol (BDH, ACS grade),
immediately centrifuged for 2 min, and the DNA suspended in
100 /A H2O. If the DNA precipitated with a 'cotton wool effect',
no further purification was required. However, in many instances,
there is the co-precipitation of a 'white flocculate substance',
probably polysaccharides (11, 12), which usually inhibits PCR
or causes amplification of non-specific products (unpublished).
Prepa-Gene™ (Biorad) used according to the manufacturer's
protocol was found to be effective in removing this substance
(Figure 1) and is considerably less time consuming than caesium
-SINGLE EGGS
WORMS
PCR
300 bp
300 bp
(B)
QIAGEN COLUMN
PCR CYCLE SEQUENCING
(C)
(E)
Figure 1. Rapid PCR cycle sequencing of the second internal transcribed spacer
region (TTS-2) of Trichostrongylussm.
rDNA isolated from single or multiple
worms, purified using Prepa-Gene , is checked by agarose gel dectrophoresis
(A). PCR of rDNA from three individual worms using NCI and NC2 primers
(B) and a representative example of partial sequence of ITS-2 using -"P-labelled
NCI ( Q . PCR carried out directly on five individual eggs (without DNA isolation)
using NCI and NC2 (D) and a representative example of partial sequence of
ITS-2 using 33P-labelled NC2 (E). No DNA controls ( - ) ; positive DNA control
(+)•
2526 Nucleic Acids Research, 1993, Vol. 21, No. 10
chloride-gradient ultracentrifugation. Although the yields of DNA
purified in our experiments by Prepa-Gene™ proved to be
lower ( ~ 6 0 - 7 0 % ) than those claimed by the manufacturers
(80—90%), they are adequate and sufficiently clean for effective
PCR.
2. Amplification of ribosomal sequence and purification of
PCR products
The second internal transcribed spacer region (TTS-2) (3) was
amplified by PCR. Conserved 20-mer oligonucleotide primers
NCl:5'-ACGTCTGGTTCAGGGTTGTT-3'; NC2:5'-TTAGTTTCTTTTCCTCCGCT-3' were derived from the 5.8S and
28S sequences of the free-living nematode, Caenorhabditis
elegans. PCR mix (final concentration: 10 mM Tris-HCl, pH
8.4/50 mM KC1/2.5 mM MgCl2/250 /tM each of dATP, dCTP, dGTP, dTTP, 1 /tM of each primer and 1 unit Taq
polymerase, Perkin Elmer Cetus) was preprepared and stored
in 440 /tl aliquots at -20°C. Worm DNA in 6 /tl (0.5-10 ng)
was preheated to 95°C for 2 min and added to 44 jtl PCR mix.
DNA from single eggs was amplified directly without extraction.
Eggs isolated from female worms of Trichostrongylus
retortaeformis were washed extensively in H2O. Under a
microscope, single eggs were pipetted (Gilson P20) in 6 yl H2O
into a 0.5 ml Eppendorf tube. This tube was ultrasonicated (30
sec, 12 /tm) in a waterbath, subjected to 3 freeze (-70°C)/boil
(95 °C) cycles and centrifuged. Forty-four /tl PCR mix was then
added to the tube, spun and subjected to PCR using the same
conditions as described above. Each PCR tube was preheated
to 95°C for 2 min, spun and subjected to PCR (DNA
Thermocycler 480, Perkin Elmer Cetus): 95°C, 1 min
(denaturation); 55°C, 1 min (annealing); 72°C, 1 min (extension)
for 30 cycles. Fifteen /tl of each PCR product was checked on
a 3% TBE (89 mM Tris-HCl/borate, 2 mM EDTA, pH 8.3)
agarose gel (Figure 1). Then, primers were removed from PCR
products by purification on spun columns (QIAGEN, Diagen)
according to the manufacturer's protocol A (desalting and
concentration with QIAEX), except that the final elution in TE
(10 mm Tris-HCl, 1 mM EDTA, pH 7.4) was performed two
times for 30 min in 20 /tl. Ten /tl of this purified PCR product
(25-50 ng) was used directly in the sequencing reaction.
3. Sequencing
PCR cycle sequencing was performed (BRL kit, cat no. 8196SB)
using NCI and NC2 endlabelled with 7-ATP 33P (DuPont, cat
no. NEG 302H). As with the PCR mix (above), labelled primers
(5 /tl) and prereaction mix (26 /tl) were aliquoted prior to use
and stored at - 2 0 ° C . The QIAGEN purified PCR product was
thawed, heated to 95°C for 5 min and 10 /tl was added to the
prereaction mix. The rest of the procedure was as described by
the manufacturer. PCR was performed at: 95°C, 40 sec
(denaturation); 55°C, 40 sec (annealing); 72°C, 40 sec
(extension) for 25 cycles. Labelled PCR products were separated
for 2 hr or 4 h on 60 cm 5% polyacrylamide gels by
electrophoresis on a Base Runner rig (IBI) at 60W constant
(50°Q. Gels were dried onto Whatman 3MM, autoradiographed
onto Curix RP2 for 24 h to 5 days and developed automatically.
Usually, gels could be read accurately after 2 days. Resolution
of bands on sequencing gels was better with 33P than 32P (not
shown), which is in accordance with a recent report (7). With
this sequencing technique, we were able to resolve up to 500
bases using a 2 h and 4 h electrophoretic run. Sequences could
be read 15—25 bases from the end of either primer. Figure 1
shows partial sequence of the ITS-2 region of single worms
(0.15x0.5 mm in size) and individual eggs (~ 30x80/tin in size)
of Trichostrongylus spp. The complete ITS-2 sequences of worms
and eggs of Trichostrongylus retortaeformis were found to be
identical (Hoste et al., in preparation). To determine the accuracy
of the system, the ITS-2 of C. elegans was also sequenced and
found to be identical to that described previously by other workers
(GenBank, code CERDNA).
Our sequencing technique is simple, robust, rapid to perform
(2 days from isolating DNA to deducing the sequence) and
sensitive (can determine the sequence of individual eggs).
Although the technique has been applied to economically
important parasitic nematodes of the genus Trichostrongylus, it
has been used successfully for a wide range of other nematode
and cestode helminths from a variety of hosts, demonstrating its
general applicability. This technique is used routinely in our
laboratory for molecular phylogenetic and systematic studies on
parasites.
ACKNOWLEDGEMENTS
Thanks to Dr Warwick Grant (CSIRO) for Caenorhabditis
elegans DNA and Dr Ross Andrews for comments on the
manuscript. Financial support from the Australian Research
Council, Ian Potter Foundation, ANZ Executors and Trustees,
Department of Industry, Technology and Commerce and
Australian Academy of Science is gratefully acknowledged. Dr
Hervd Hoste, a visiting scientist at the University of Melbourne,
is a grateful recipient of a Fellowship from The French Ministry
of Research and Space Technology.
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