Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
- WO 93/~316~ PCr/USg2/06477
METHOD OF ISOL~TION OF DNA
BACKGROUND
Increasingly, DNA-based diagnostic methods are
coming into use for the diagnosis of infectious and
parasîtic diseases. ~any:of these techniques involve DNA
amplification procedures, such as the polymerase chain
reaction procadure (P~R~, as described in United States
~, Pat~nts:Nos. 4,683,195, 4J683~202~ 4,800,159, and
4,965,188 to Mullis et al. However, such DNA-based
amplification procedures require that DNA be isolated in
: ~: a stable, biologica~lly active form from blood or othPr
biological fluids; optimaI application o~ such
amplifica~:ion~procedures further requires that the DN~ to
,
be amplified be:present in the form of linear fragments
o~f less than~about~2 kb in~: length . MoreoYer ~ because
here is often a considerable lapse of time betw~en the
: < ~ ~
15~ oollection of a blood specime~ from a~patient and the
testing of DN~ isolated from:a blood:sampl , by DNA
amplification procedures,~there is a need for a method of
storing DNA stably for periods of time in blood. There :~
further exists~a need~::for the conversion of th~ DNA in
. .
20 ~ these~ ~biological samples, which may be in the form of
:~ :: closed circular catenanes or other~interlink~d forms,
into line~rized double-stranded DNA;fragments suitable ~:~
for amplification. ~This method must be rapid, easy to
. .
:: : ~
'
WO93/031S7 2 ~ P~T/US92/~,
perform, and capable of releasing the DNA efficiently
from cells or other structures and cleaving it into
fra~ments suitable for amplification.
The need for a method of isolation and storage
of DN~ is particularly acute in connection with the
diagnosis of parasitic diseases by DNA amplification
procedure~. Many of these diseases, including Chagas'
: : disease, leishmania~is, and malaria, occur primarily in
trvpical areas in which medi~al faciliti~s are relatively
~, primitive and a~substantial period of time may elapse
between collection of the sample from a patient suspected
o~in~ection and its tèsting. ~Recent1y, considerable
:
work has been directed towards diagnosis of these
15 ~:~diseases ~y PCR, as described in Avila et al. ~ ol.
Biochem _Parasitol. 42o175 11990) ) ~ Sturm et al. ~Mol.
Biochem. Paraæitol. 33:205 (1989)), Rodgers et al. (~
Parasitol. 71:267;(1990)) an~ Moser et al. (J. Clin. :~
Microbio~l. 27:l477 (1989))~.~ Additionallyr Ashall et al.
20 ~(J.Clin. Microbio1.~26:576 :~1988)) desoribe the det~ction
: ;of Trypanosoma cruzi~in ma~mmalian blood by a DNA-DNA
hybridizati~n~procedure~emp1Oying rad~iolabeled total
p~rasite D~A under conditions tha~ favor hybridization of ~.
repetitive DNA sequ:ences,
: 25 ~ ~
The use of high EDTA concentrations to isolate
:~ : DNA has-been described in~Simpson & Bsrliner, "Isolation
of the Kinetoplast DNA of Leishmania tarentolae in the
: ~
: ~:
WO93/03167 PCT/US92/0~77
3 ~1150~
form of a Network," J. Protozool. 2l:382-393 (1974)L
Zolg et al. (Am. J. TroP. Med. HYq. 3~:33 (1~88)~
describe a method for storage of blood samples to be
tested for the presence of the parasite Plasmodium
falciparum using specific DNA probes, in high salt
lysat~s. The lysates are prepared using water to lyse
the erythrocytes; a detergent/EDTA mix to lyse the
parasites, followed by the addition of cesium
tri~luoroacetate. Disadvantages of this procedure
include the necessity to lyse the erythrocytes ~irst, the
~-- reguirement for use of a detergent, and the expense of
the cesium ~alt. In addition, this system may inhibit
enzymes such as exonucleases added to he lysate to
: cleave DNA. . :
:
: 15
The need~for:a method to isolate and prevent
degradation of DNA in b1Ood samples from patients
~: suspected of infection with parasites or other infectious
agents during storage, and to prepare the DNA for
amplification and/or hybridization procedures so that
: ~ diagnosis of infection can be accomplished, has gone
~ .
unmet.
Accordingly, ~he present invention provides a
rapid and efficient method of DNA isolation, storage, and
eleavage that meets these needs and pr~vides DNA suitable
:~
WO~3/~3167 ~CT/US9~/0~;
21~ ` 4
for amplification by a sequence-specific method, such as
PCR. In general, the method of the present invention for
isolating biologic~lly active DNA from a bio~ogical
sample ha~ing DNA-containing structures comprises the
steps of:
(l) contacting a biological sample containing
DNA-containing structures with a lysis and storage buffer
comprising a non-amphipathic chaotropic salt su~ficient
to lyse DNA-containing structures in the sample and a
chelating agent to preserve the DNA from degradation to
form a mixture of the biological sample and the ly is and
storage buffer;
: (2) incubating the mixture formed in step (a)
with a metal-containing chemical nuclease that cleaves
15 the DNA to f orm DNA f ragments; and
(3) purifying the DNA fragments. .
The DNA in the DNA-containing structures can be
present in the form of catenated closed circl~s~ It can
be i~olated ~rom a parasite. In particular, the DNA can
be isolated from a kinetoplastid parasite such ~s ~
TrY~anosoma cru~i. Alternatively, the DNA can be
associated with a virus such as herpes simplex virus :~
~HSV). RNA can al50 be isolated from RNA-contalning
structures, and DNA can be produced from the RNA using
reverse transcriptase for amplification. The biological
sampl~ can be a fluid sample such as mammalian blood,
including human blood. :~
::
WO93/03167 PCT/VS92/0~77
2~ ~
The non-amphipathic chaotropic salt is
preferably a guanidinium salt, such as guanidinium
chloride or guanidinium thiocyanate. More preferably,
the guanidinium salt is guanidinium chloride. Most
preferably, the guanidinium chloride is present in at
least about 3 molar concentration in the mixture of the
biological sample and lysis and storage buffer.
The chelating agent is preferably
ethylenedia~ine- tetraacetic acid (EDTA). Most
prefPrably, the EDTA is present in at least about 0.1
molar concentration in the mixture of the biological
sample and lysis and storage buffer.
The general method can further comprise the
steps of:
(4) amplifying the DNA by a sequence-specific
: method employing at least two primers that hybridize to
,
~:: the DNA fragments; and
2~0~ (5) detecting disease assoc-'ated with th~
presence of the DNA in an organism by identifying the
DNA. ~ ~
The primers employed in ~he sequence-specific
~ 25 method can comprise~:~at least two primers that hybridize
: to a genetic marker of the organism from which the
.
biologi~al sample was isolated for identification of the
genetic marker:, :
WO93/03167 2 1 1 S ~ 6 1 PCT/US92/0~'
The metal-~ontaining chemical nuclease can be
selected from l,10-phenanthroline-copper complex,
derivatives of ferrous EDTA, metalloporphyrins, or
octahedral mekal complexes o~ 4,7-diphenyl-1,10-
phenanthroline. Preferably, the metal-containing
chemical nuclease is the l,10-phenanthroline-copper
complex.
An applicatisn of the general method is a
method for detecting a disease caused by a kinetoplastid
~- parasite. This method comprises the steps of:
(1~ contacting a biological sample ~rom a
patient suspected of~having a disease caus~d by a
kinetoplastid parasite, with a lysis and storage buffer
,
~comprising a non-amphipathic chaotropic salt su~ficient
to lyse cells containing catenated closed circular
: k~netoplast DNA in the sample and a chelating agent to
preser~e the DNA in the cells from degradation to form a
: ~ :
mixture of the biological sample and the lysis and
storage buffer;
~: ~ (2) incubating the mixture obtai~ed in st~p ~1)
with a metal-containing chemical nuclease to linearize ~.
catenated closed circular kinetoplast DNA to form
kinetoplast DNA fragments;
; 25 (3) purifying the DNA fragments to form
purified kinetoplast DNA fragments suitable for
amplificati~n;
.
. WO 93/03167 PCrlUS92~U6477
7 21150~
(4) amplifying the purified kinetoplast DNA
fragments by a sequence-speclfic method employing at
least two primers capable of hybridizing to the
linearized DNA to form amplified kinetoplast D~A; and
~5~ detecting di~ease by identifying the
prese~ce of the amplified kinetoplast DNA corresponding
to the primers and thus having sequences specific ~or
kinetoplast DNA of the parasite.
When this method is used to detect Chagas'
~' disease, caused by T. cruzî, the primers hybridize to
~onserved regions in T. cruzi kinetoplast minicircular
DNA.
A preferr~d~method according to the present
invention for the dete tion o~ ~ disease cau~ed by
~EY3L comprise5:
~:~ (1) mixing a mammalian blood sample suspRcted
of containing kinetopIastid DNA from T. cruzi with about
~; a o an equal volume of lysis and storage buffer comprisi~g 6
M guanidinium chloride and 0.2 M EDTA, pH 8.0, to form a
: ~ixture and storing the mixture at room temperature for
at least 24 hours;
(2~ incubating the mixture obtained in step (l)
with the metal-containing chemical nuclease l,lO-
phenanthroline-copper complex to cleave the kinetoplast
DNA to form linearized kinetoplast DNA;
W093/03167 2 I 1 ~ 6 ~ PCT/VS92/0~,
(3) purifying the linearized kinetoplast DNA to
form purified linearized kinetoplast DNA by:
(a) deproteinization with a phenol-
chloroform mixture;
(b) ethanol precipitation with a glycogen
carrier; and
(G) fi~ tration through a
microconcentrator;
(4) amplifying the purified linearized
kinetoplast DNA by a sequence-specific method employing
.- at l~ast two primers capable of hybridizing to conserved
regi~ns of the catenated closed circular kinetoplast DNA
of ~ cruzi; and
(5) detecting disease caused b~ a~L by
identifying the presence of amplified DNA corresponding
to the primers and thus having se~u~nc~ specific for
catenated closed circular kinetoplast DNA of To cruzi by
hybridization to the primers. Typically~ th~ linearized
DNA compris~s fragments of from about 1 kb to 2bout 1. 5
20 kb in length. ~:
The i~olation and storage method step~ of the
method of the present invention ca~ be practiced
.~ .
separately from cleavage of the DN~. A method o~
isolating and storing DNA from a cell-containing
biological sample according to the pres~nt invention
comprises the steps of: ~
W093/03t67 ~ PCT/US92/0~77
21~.S~ 1
(1) contacting a biologiral sample containing
DNA present in cells with a lysis and storage buffer in
order to li~erate the DNA from the cells, the lysis and
storage buffer comprising:
(a) a concentration of a non-amphipathic
chaotropic salt sufficient to lyse cells in the
:biological sample when the ly~is buffer i~ contacted with
the biological sample; and
. ~b) a concentration of a chelating agent
: lO su~ficient to preserve the DNA from degradation when the
lysis~ buffer i& contacted~with the biological sample; and
(2) storing the liberated DNA in the mixture
at a temperature of below about 650C.
: 15 ~ Preferably, ~he storage temperatur~ is below
about~4C in order to preserve the DNA sample for at
: least about one year.
: BRIEF DESCRIPTION_OF THE DRAWINGS
2~ .
Figure l~shows the results of te~ting the
~ stability of:plasmid~ DNA as described in Example 1,
: infra.
Figure 2~shows the results of cleaYage of
: isola~ed Trypanosoma cruzi kinetoplast DNA by the 1,lO-
: phenanthroline-oopper~ chemical nuclease (OP-Cu2~) as
described in Example 2, infra.
. L . t~ ~ ~
WO93/03167 PCT/~S92~0~'.
Figure 3 shows the frequency of single strand
nicks in oP-cu2~ linearized minicircle DNA as described in
Example 2, infra.
Figure 4 shows results of a sensiti~ity
titration of OP CuZ~-clea~ed DNA, as described in Example
4, infra.
Figure 5 shows results from a blood sample o~ a
chronic chagasic patient analyzed for the presence of
.~ cruzi minicircle sequences, as described in Example 5,
infra.
Figure 6 shows the results of PCR amplification
of T. cruzi DNA isolated from triatomid bug feces as
described in ~xample 5l ~n~
DESCRIPTION
~':
The present inve~tion is a ra~id and e~icient
method of storing, isolating~ and purifying biologically
a~ive DNA from a biological sample having DNA-con~aining
structures. The DNA-containing structures include cells
and vixal particles. The method i~ particularly useful:: 25 for isolating parasitic DNA that is present in the form
of catenated closed circles, but the method is not
limited to use on such DNA. The method allows ~or
storage of the DNA in a s~ate suitable for ~ubsequent
'.
WO93/03167 ~ PCT/US9~/0~77
ll 21 1~3~61
controlled degradation by a chemical nuclease in order to
cleave the DNA to fragments of a size suitable for
subsequent amp~ification. The DNA can then be rapidly
purified to render it suitable for use in an
amplification method such as PCR.
,
In general, the method comprises:
(1) contacting a biological sample containing
DNA-containing structures with a lysis and stor~ge buffer
0 comprising a non-amphipathic chaotropic salt sufficient
to lyse DNA-con~aining:structures in the sample and a
chelating agent to preserve the DNA ~rom degradation and
: prevent coagulation of the blood to ~orm a mixture of the
biological sample and the lysi~ and storage buff~r; -
(2~ incuba~ing~he mixture formed in step (1)
with a metal-containing:chemical nuclease that cleaves
th~ DNA to DNA fragments, and
(3) purifying the DNA fra~mentsD
The method is particularly adapted for, but is
: not limited to,~detection o~ p~ra5itic diseases caused by
parasites having catenated clo~ed circular kin~toplast
: DNA, such as Trypanosoma cruzi, the pr~tozoan causing
Chagas' disease; (Sturm et al., Mol. Bio~hem! Parasitol.
33:205 (1989l;~Avila et al., MOl! Biochem. Parasitol.
4~:175 (l990);:Moser et alOt J. Clin. Microbiol. 27:1487
(1989); Ashall et al., J. Clin. Microbiol. 26:576
(1988)); Leishmania species, the protozoa causing
.
5 ~
~ W093J03167 PCT/US92/0~'.
12
leishmaniasis (Rodgers et al., Exp. Parasitol. 71:267
(1990)), and Pla5modium falciparum, P. vivax, and P.
malariae, the protozoa causing human malaria (Zolg et
al., ~m. J. Trop. Med. Hv~. 39:33 (1988)). In addition,
it can be used for the detection of other infectious
diseases and infectious agents such as the provirus of
human immunodeficiency virus (HIV), cytomegalovirus
(CMV), hepatitis B virus, herpesvirus, Epstein~Barr
virus, and Toxoplasma gondii.
I. I50LATION OF DNA
A. The Biological Sample ::
,
The method is suitable ~or use on any
~:: : biologica1 sample containing DNA. The method i~
: particularly adapte~ for storage and isolation of DNA
: ~ ,
~:~ fr~m mammalian blood,~including human blood, but the
: : method is suitab1e~for Use on other biological samples,
~20: :such as:insect feces, biopsy tissue, u~ine, ~put~m, and
:lymphatic fluid. The method can be used on any volume of
fluid:sample, from microliters to liters, as needed.
:
~. The Lysis and Storaqe Buffer
: 25
The biological sample is mixed with the lysis
and storage buffer o~ the invention. The lysis and
storage buf~er comprises: (l) a oonFentration of a non- :
, WO93/03167 PCT/US9~/0~77
13 211~fi~
amphipathic chaotropic salt sufficient to ly~e cells in
the sample and (2) a concentration of a chelating agent
sufficient to prPserve the DNA from degradation. A non-
amphipathic chaotropic salt is a salt, other than a
detergent ha~ing distinct po~ar and non-polar moieties,
~hat disrupts non-covalent bonds, such as hydrogen bonds,
salt links, hydrophobic interactions, and van der Waals
interactions, that are primarily responsible for the
maintenance of secondary, tertiary, and quaternary
structure in proteins and nucleic acids.
The non-amphipathic chaotropic salt is
preferably guanidinium chloride or a chemically relat~d
salt ~uch as guanidinium thiocyanate; other chaotropic
salts such as lithium bromide, potassium thiocyanate, or
pota,sium iodine are also usable. Most preferably, the
guanidinium chloride i: pre~ent in at least 3 molar
co~centration in the mixture of the b~ological sample and
the lysis and storage bu~Efer.
The chelating agent is preferably EDTA,
:: although other chelating agents , such as sodium citrate ,
can also be used. Most preferably, the EDTA is present
in at least 0.1 molar concentration in the mixture of the
:25 biological sample and the lysis and storage bu~f~r.
Typically, the biological samplQ is mixed with
about an equal volume of the lysis and storage buffer
WO93/03167 2 ~ PCT/U~92/0~'
14
containing about 6 molar guanidinium chloride and about
0.2 molar EDTA, pH ~Ø
~ en the lysis and storage buffer is mixed with
th~ biological sample, the DNA-containing structure ,
such as cells or viral particles, present in the
biological sample are lysed essentially instantaneously
and the DNA is libe~ated from the DNA~containing
structures. The DNA is~stable in the mixture of the
biological sample and lysis and storage buf f er and can be
~-: stored at a storage temperature below about 65~C. A~ a
storage temperature of 37C, DNA stored in a mixture of
lysis and storage buffer and sample remains intact Xor at
:
;~ ~ least a month. At 4C or -20C, the DNA iæ stable for at
15 ~ least one year. The DNA can be stored at this stage ~or
subsequent cleavage:with the chemical nuclease and
p.urification according~to the remainder of the process of
the:present invention,:or may be used in other
: :te~chniques,:such as:nick translation to produce
20 ~ hybri~dization probes.
: When the biological sample is blood, it is
preferred to store ~he~mixture of blood and lys~s and
storage buffer for an initial period of about ~ hours at
room temperature (i.e., about 20-25C), followed by
subseguent storage at~4C. It is not necessary to use
~reshly drawn blood;; blood stored for periods of up to ~.
about l week can be used. In one version of the
WO93J03167 PCT/VS92/0~77
2 1 ~
procedure, blood is drawn into tubes containing
anticoagu1ants such as citrate and/or heparin and then
used after a period of storage of about 24 hours.
~. The Chemical Nucleas~
The DNA in the lysis and storage buffer is
fragmented by incubation of the biological sample-lysis
and storage buffer mixture with a metal-containing
ch~mical nuclease capable of nicking DNA by oxidative
~, attack on the deoxyribose moiety (Sigman & Chen, Annu.
Rev. Biochem. 59:207 (l990)). The chemical nuclease can
~e a 1,19-phenanthroIine-copper complex ~sigmant
Bio~chemistry 29:9097 (1990)); a derivative of ferrous
:~15 EDTA such as mekhidiumpropyl-EDTA-iron (Hertzberg
Devan, J. Am Chem. Soc. 104:313 (1982); Schu1tz et al.,
J. Am.~Chem. Soc. 104:6861 (1982~; Tu11ius & D~mbroski,
Proc. Nat1. Acad. Sci. U.S.A. 83:5469 (1986));
meta11Oporphyri~s~(Ward et~al., Biochemistry 25:6875
20~: (1986); Lee Doan:et al., Biochemis5ry 25:6376 (1986);
roves & Farrell, J. Am. Chem. So~. 111:4998 (1g893) or
: metal-containing octahedral complexes of 4,7-dipheny1-
: ~ 1,10-phenant~hroline such~as tri~ (4,7-dipheny1-1,10
phenanthro1ine~ ruthenium (Barton, Science 233:727
;25~ (1986~). Preferably, ~the chemical nuclease is a l,lO-
phenanthro1ine-copper~complex,~because it is not
:
: : ~ensitive to inhibition ~y buffer components. In
addition, the 1,10-phenanthro1ine-copper complex is
:
':~
WO93/03167 PCT/US92/0
~ . 16
inexpensive and its reaction can be efficiently quenched
by chelating agents in order ~o control the Qxtent of
cleavage. Other chemical nucleases, such as ferrou~-
E~TA, methidiumpropyl EDTA-iron, metalloporphyrins, and
metal-containing octahedral complexes of 4,7-diphenyl-
1,10-phenanthroline, may be useful pro~ided they are not
substantially inhibited by the buffer.
The phsnanthroline-copper chemical nuclease
reagent introduces random single strand nicks into duplex
DNA in the presence of peroxide (Sigman & Chen/ Annu.
Rev. Bioch m. 59:20~ (1990)). Because, on the average,
one double strand cleavage occurs after ten random single
strand breaks are introduced into a DNA m~lecule, this
nuclease can be used to digest catenated kinetoplast DNA
to linearize minicircles.
The che~ical nuclease reaction u~ing the 1,10-
phenanthroline-copper oomplex i5 typically perPormed as
20~ f~llows: to one volu~e of the mixture of the biological
ample and lysis and storage buffer containing 3 molar
guanidinium chloride and 0.1 molar EDTA are added 0.1
volume each of 1 molar MgCl2, 200 mlllimolar CuSO4, 20
millimolar 1,10-phenanthroline and 7.5% H2O2 (freshly
di~uted frvm 30% stock soluti~n). The reaction is
initiated by addition o~ 0.1 volume of 3-
mercaptopropionic acid and digestion of DNA is allowed to
proceed for 30 minut s at 37 C. The reaction is stopped
:.~
~ WO93/03167 ~ PCT/US92/0~77
~7 2I 1~6~
by addition of 0.1 volume of 1.5 molar 2,9-dimethyl-1,10-
phenanthroline. Reactions with other chemical nucl~ases
are carried out as specified in the literature describing
them. The cleavage process cleaves DNA to fra~ment~ that
can be used in an amplif ication process. These ~ragments
are linearized and are:typically of about 1 to about 1.5
kb length.
. The l,10-ph~nanthroline-copper complex is
: 10 believed to generate a highly reactive oxidative species
and to react with the DNA through the formation of a
reversible complex between the phenanthroline-copper
reagent and the DNA. The reaction produce~ the following
: stable products: 5'-phosphorylated-termini, 3'-
phosphorylated-termini~, free b~ses and 5-
methylenefuranone,~aq well as minor amounts of 3'-
phosphoglycolate termini. The predominant reaction
volves initial oxidative attack at the C-l hydrogen of
the deoxyribose by~the DNA-bound coordination complex.
:; : 20~ :~xidative reaction~is initiated within th~ minor grooYe
of~thc DNA and the~reagent exhibits preferential
reactlvity for DNA in;the B ~orm of the helix relative to
~: DNA in the A form of the helix. The reaction i.~ not
~: ~ specific for the~nucleotide at the site o~ scission, b~t
: 25 its rate does depend on local sequence. mhe most
important influe~ce on the intensity of cutting by the
-
:~ ~henanthroline-copper complex at any s~quence po-Qition is ::
the neighboring 5i-nucleotide. Because the reaction can
WO 93/03167 ~ ?~ PCI'/US92/0647
18
be terminated at any time by the addition of a chelating
agent with high affinity for copper, such as 2,9-
dimethyl-1,10-phenanthroline, 2,9-dimethyl-4,7-
phenanthroline, or EDTA, compensations can be made for
any variations in reactivity due to local sequence.
Preferably, the chelating agent used to quench the
reaction is 2,9-dimethyl-1,10-phenanthroline. Thus, the
cleavage method described herein is essentially
independ~nt of the DNA sequence, and the si~e of single-
stranded D~A fragments produced by cleavage u~ing thenuclease may be e~fectively controlled by regulating th~
time of digestion using the chelating agent.
In particular, cleavage with the 1,10-
phenanthroline-copper compl~x of T. cruzi kinetoplast
DNA, which is originally pr~sent in the form of closed
circular catenated minicircl~s, results in individu~l
linear DNA molecules o~ about 1. 4 kb in length.
2 ~ D . DNA Purification
.
~: The DNA is preferabIy purified to remove
substances that can interfere with subsequent primer- :
based DNA amplification. Purification typically
includ s: (1) deproteinization; (2) precipitatio~ of the
DNA; and (3~ filtration. The product of purification is
suitable for amplification by PCR or anQther primer-based
amplification step.
WO~3/03167 PCT/U~92/0~7~
19 -2~ lS~ 1
The deproteinization of the sample is typically
performed by extraction with phenol or a 1:1 mixture of
phenol and chlorof 9rm, which denatures proteins and
separates protein~ from nucleic acid. Preferably, the
deproteinization is performed with extraction with a 1:1
mixture of phenol and chloroform.
Precipitation of the DNA is typically conductQd
using ethanol in the presence o~ a glycogen carrier and
is preferably carried out in the presence of about 80
g/ml glycogen and 003 molar sodium acetate at room
temperature.
The final purification step can be performed by
filtration through a microconcentrator. A suitable
microconcentrator is a Centricon-100 microco~centrator
manufactured by Amicon (Beverly, Massachusetts)~
~ Subsequent to purification, the isolated and
: ~ 20: purified DNA can be amplified in a system, as describad
~below, or can be used for other purposes for which highly
purified DNA fragments are used, such as the generation
of hybridization probes or incorporation into cloning
vectors.
:
~ 25
: :
WO93/03167 2 3 1~ O & 1 PCr/US92/0~7
II. AMPLIFICATION OF ISOLATED DNA
The purified and fragmented DNA can be
amplifi~d by a sequence-specific method employing at
least two oligonucleotide primers that hybridize to the
DNA fragments. Such methods include, but are not limited
to, the polymerase chain reaction (PCR method) of Mullis
et al. as described in United States Patents 4,683,195,
4,683j202, 4,~00,15g, and 4,965,188 to Mullis et al., all
of which are herein incorporated in their entirety by
.- this re~erence. Preferably, the PCR method is performed
using a thermostable DNA polymerase such as the Thermus
~5L~ al~ polymerase Taq I des~ribed in U.S. Patent
Number 4,889~818 to Gelfand et al., and incorporated
1:5 herein by ~his reference. Furth~r d~tails on the PCR
method are given in PCR Protocols (M.A~ Innis et al.,
eds., Academic Press (1989)), incorporated herein by this
: reference.
:20 Other sequence-speci~ic nucleic acid
: amplification systems ~mploy ng primers are also known, ~;
such as the transcrip~ion-based amplification sy~tem of
Gingeras et al. described in European Patent Application
No. 368~06, and the similar system of Davey and Malek
described in European Patent Application No. 329822, both
incorporated herein by this reference. These sy~tems
make use of an alternating cycle of amplification as DNA
and RNA employing a primer incorporatin~ a promoter, a
WO 93/03167 PCr/US92/06477
21 2
DNA-dependent RNA polymerase such as bacteriophage T7 RNA
pol~merase, and a RNA-dependent DNA polymerase or reverse
transcriptase.
In any of these methods, the product of the
amplification is a discrete linear D~A ~ra~ment or series
of discrete DNA fragments that can be separated by
electrophoresis on an agarose gel and identi~ied by
hybridization methods such as t'Southern blot'l
hybridization Pollowing trans~er of the DNA to
nitrocellulo~e filters or other filtérs. Useful
hybridization procedures are described in E.M. Sutter,
'~De~ection of 5pecific Sequenc2s Among DNA Fragments
Separated by Gel El2ctr~phoresis,~' J. Mol. Biol. 98: 503
(1975), incorporated herein by this reference. The
hybridization is typioally carried out with radioactively
labeled DNA oligonucleotide probes; this ~erves to
identify ~pecific sequences originally present in the DNA
: used for amplification~ :If the probes are comprised of ::~
: 20 DNA associated with~a disea e-causing agent, ~uch as a
.
pathog~nic bacterium, a DNA virus, or th~ DNA provirus o~ ~ ~
.
a RNA :virus, the hybxidization proc2dure can be used to
identify DNA sequences associated with the disease~
causing agent in the original sample and thus the
presence of the diseas~
: ''' '
wo g3,03l67 2 1 ~ :~ i) 6 i PCT/US92/0~/
III. APPLICATIONS OF THE METHOD
A. Dia~nosis Of Parasitic Diseases
The DNA storage, cleavage, puri~ication, and
amplification method o~ the present invention is use~ul
for the de~ection of parasitic diseases, particularly
diseases caused by kinetoplastid trypanosome~.
These diseases include Chagas' disease, a major
.~ health problem in Latin America caused by ~l~n~ Q~
cruzi. Current ~erological and xenodiagnostic methods
~or the diagnosis o~ Chagas J disease are unreliable,
especially in th~ case o~ chronic Chagas' disea~e in
which the ~arasites are detectable within the blood only
with difficulty, if at all, because of the low
concentration of parasite~ within ~he blood.
: ~ _gEg~i kinetoplasts, which are DNA-containing
structurss in the mitochondria of the c211s, contain DNA
in the form o~ catenated minicircles, that, wh~n clea~ed
by the chemical nucleas~ and purified according to the
method of the present invention, can be efEiciently
amplified by the PCR process or an~ther s~quenc~ specific
primer-based amplification process. Oligonucleotide
primers specific to conserved regions in T. cruzi
kinetoplast minici~cles (kDNA) are used (Degrave et al.,
Mol._Biochem. parasitol. 27:63-70 (19883; Sturm et al.,
~- WOg3/03167 PCT/US~2/0~77
23 21:~SQ&~
Mol. Biochem. Parasitol. 33:205 (1989); Avila et al.,
Mol. Biol. Parasitol. 42:175 ~19~0)). These primers may
be synthesized and are also commercially available from
AMAC, Inc. (a ~ivision of Genset, loca~ed in We~tbrook,
ME)-
This results in a set of amplified DNA
fra ~ ents that are species-dependent an~ strain-
: independent and can be used to detect the presence oflO ~cruzi DNA in the blood o~ patients, as well as in
biological samples obtained from in~ected insect ve~tors
or infected mammals such as mice.
:In general, DNA isolated and amplified a~
15 described herein may be used to detect di~ease associated ~:
;with the presence of the DN~ using standard me~hod~;, such
as by DNA hybridization using radioactively labeled
probes to bind~to~specifio sequences in the DNA (Sturm et
al:.,~Mol. Biochem.~ ParasitQl 33:205 gl989) and A~ila et
20~ al.~Mol. Biochem.~Parasitol. 42::175 ~l990)).
B. Other~Appli~ations and Advanta~es
:,; .
: The method of the present invention is not
::
limited to storage, puri:fication or detection of
parasitic DNA or to minicircular DNA. Becau~e the
: ~ :
chemical nuclease:that is employed typiaally cleaYe~ DNA
into:linear f~agments of from a~out 1 kb to about 1.5 kb
:
WOg3/03167 2~ ~3 ~ 6 1 PCT/US~2/~
24
in length, an optimal size for amplification with primers
of less than lO00 bases as required for PCR, the lysis,
storage, cleavage, and purification process of the
present invention can be used to detect DNA specific ~or
any biological entity of interest from any biological
sample, including blood, urine, sputum, or lymphatic
fluid. The DNA detected can be from any etiological
agent~ including the DNA of parasiti~ protozoans,
bacteria, or viruse such as herpes viruses,
cytomegalovirus (CMV), hepatitis B virus, herpes viru ,
Epstein-Barr virus, or Toxoplasma gondii. Cellular ~NA
can also be detected, including mammalian or human DNA,
and retroviral DNA ~uch as the DNA of human
immunodeficiency uirus (HIV) associated with AIDS. The
amplification allows the detection of extr~mely small
quantities o~ DNA specific to a etiological agent, and
thus can be used to detect the presence of the disease
,
: caused by the agent earlier tha~ conventional serological
: diagnostic procedures.
~ 2~0
: When whole blood is used for the isolation of
DNA according to the method of the present in~ntion,
genetic markers of the host species (animal or human) can
be assayed in the same multiplex PCR assay in addition to
genetic markers of parasites or other etiological agents
of diseases. For example, in the case of humans, the
genstic markers assayed can include a family of immune
response genes such as the major histocompatibility l~cus
W093/03167 PCT/US92/0~77
~1~ 5a ~'~
(MHC) genes which have been shown to be correlated with
resistance to various diseases and susceptibility to
autoimmune diseases. Other appropriate genetic loci for
assay in multiplex PCR include the human homologues of
those genetic markers found in animal models to confer
resistance to parasitic diseases such as those caused by
Leis~mania or TrYpanosoma cruzi. Any desired gen~tic
locus, such as onoogenes and anti-oncogenes or genes
involved in genetic;diseases can be targeted for
~: 10 selective amplification during this assay, using the
..
~ appropriate selection of primers and the i~olated DNA.
.
RNA can also be isolated from RNA-containing
~ : structures such~as cells or viruses and stored in a ~ .:
:~ 15~ mixture~of the sample and the lysis and storage buffer.
The RNA can then~bè~tran~sor~ibed by reverse transcriptase
;:tQ;~generate a RNA-VNA~hybrid. The ~NA in the hybrid i~ :
then degraded~by~the enzyme ribonuclease H specific for
;the:~RNA~strand of~a~double-stranded RNA-DNA hybrid, and
;20~ the~resulting DNA~strand~made double-s~randed by
hybridization of~a~ itable primer and elongation of ~he
: primer~by~DNA;polymerase~.
: :An additional advantage of the method sf the
;:25~ ~iDvention is that~the~;use~of the lysis and-storage buffer
and chemical nuclease:~of :the invention can contribute to
reduction of the infectivity of endogenous inf~ctious
organisms such as~viruses or bacteria present in the
: ~
: ~ :
W093/03167 2 ¦ ¦ 5 ~ 1 PCT/US92/0
26
biological sample, because the infectivity is
substantially destroyed by lysis of the bacterial cells
or virus particles and also by cleavage of the viral
nucleic acid. Treatm~nt o~ membrane enveloped viruses
with the l,lO-phenanthroline-copper chemical nuclea~e
destroys their infecti~ity (Lembech et al., Fed. Proc.
44 ~07~ (~985).
The invention is illustrated by the following
examples. The examples are for illustrativ~ purposes
~~ only and are not t~ be construed as limiting ths scope of
the invention in any manner.
; ~ Exam le l
:
Storaqe Of DNA In Guanidinium Chloride-EDTA Buffer
,
Human intravenous blood was freshly drawn and
~ collected into tubes containing an equal volume oX 2 X GE
: lysis and storage~bu$fer (6 M guanidinium chloride, 0.2
EDT~, pH 8 . 0) ~ The resulting GEB
(gu~nidinium/EDT~1b~ood) lysate was stored at 25C for at
least 24 hours and subsequently at 4~C for up to 6
months.
: 25 : To test the stability of DNA in GEB lysates,
: plasmid DNA~ pGEM 7Z (Promega, Madison, Wisconsin), was
added to 2 tubes of GEB lysates. Each tube was stored at
either 37C or 65C. Equivalent aliquots from each tube
.
: WO93/~3167 PCT/US92/~477
27 21~ ~Bl
were taken at different time intervals for up to ~our
weeks. The ali~uots were extracted ~nce with
phenol/hloroform (1:1, v/v) and ethanol precipitated.
The isolated DNA was electrophoresed in a 1% agaro~e gel
to determine the percentage of nicked or linearized
plasmid DNA, which can be detected by its relatively
rapid mobility through the gel in contrast to intact
catenanes, which remain at the top of the gel~ :
The results are shown in Flgure l; the
electrophoretic pattern:resulting from incubation at 37C
is designated (lA~ and the pattern resulting from
incubation ~t 65C is designated (lB). The control lane
~; is unincubated plasmid DNA in 10 mM Tri~-HCl, pH 8 . 0, 1 , .
: ~: 15 mM EDTA (TE). Figure 1 shows that the DNA remains intact
~at 37C for at least a:month, with no apparent nicking or
de~radation. At 65C, the DNA is nicked aft~r a two-week
; incubation, ~as indicated:by the disappearance of the
closed circular DNA band and the increase in the nicked
20: ~circular~band. Even~-t 65C, at least 50~ of the D~A
remaine~ in the nicked circular or linear form after one
:week~incu~ation. At~4C or -20C, DNA wa~ shown to be
stable in GEB lysate~for ~at least one year. These
resul~s clearl~ demonstrate that the guanidinium
chloride-EDTA reagent is a suitable medium for the lysis
; : o~ cells contained~:in blood and the preservation of DNA
at room temperature ~or a substantial period of time. :
::
~: '
WO93/03167PCT/VS92/0~, ~` `
2 1 ~ ................................ 28
Example_2
~leavaqe of Kinetoplast DNA
Using I~Q=Rh9~ky~1ine-Copper Complex
:::
5To 0.5 ml GEB lysate containing kinetoplast
DNA, 0.05 ml o~ each o~ ~he ~ollowing solutions was
added: 1 M MgCl2, 200 mM CUSO4, 20 mM 1,10-phenanthroline,
and 7.5~ H202 (diluted fresh from 30% stock). The
reaction was initiat~d by addition of o.05 ml of 5~ mM 3-
mercaptopropionic acid. Digestion o$ DNA was allowed to
.~ proceed for 30 minutes or 60 minutes at 37C. The
reaction was stopped by addition of 0.05 ml of 1.5 M 2,9-
; dimethyl-l,lO-phenanthroline. For GEB lysates containing
_9~YZi kinetoplast DNA digestion was carried out for 60
~minutes at 37C, and aliquots were removed ~ry 10
.
minutes~ The reaction was quenched by addition o~ 2~9-
dimethyl-l,}0-phenanthrolinQ. ~he aliguotc w~re
deproteinized with phenol/chloroform (l:1) and
; ~ precipitated~with ethanol. The DNA was denatur2d in
`20 glyoxal/DMS0 and loaded onto a 1% agarose gel and
electrophoresed as described by McMaster and Carmichael
(Proc. Natl_ Acad~. ~Sci. U.S.A. 74:4835 ~1977)). Th~ g~l
was blotted and the DNA transferred to a Nytran filter
(Schuell and Schuster, Xeene, New Hampshire). Total ~.
25~ ruzi kinetoplast DNA:was nick-translated with 32P-ATP and
used as a hybridization probe~ Nick translation and
hybridization conditions were as d~scribed in Simpson et :~
'
W~93/03167 PCT/US92/0~77
29
al., Nucl. Acids Res. 13:9577 (1985)), incorporate~
reference herein.
The extent of cleavage of purified T. cruzi
kinetoplast DN~ in Tris-EDTA buffer (10 mM Tris-HCl, pH
8.0, 1 mM EDTA) by the phenanthroline-copper chemical
nucl~ase reagent was monitored by agarose gel
electrophoresis on a 1% agarose gel as a ~unction of
digestio~ time (Figure 2). In Figure 2, the M lane shows
Hind III fragments of bactexiophage ~ DNA and Hae III
,, .
fragments of bacteriophage ~X174 RF DNA as size marker~.
The disappearance of the kDNA from the well and the
appearance of ~he 1~4-kb linear minicircle band as a
function of digestion time is seen. After 19 minutes of
lS incubation, the DNA was completely cleaved to
mi~icircles. ~
Undig sted catenated kinetoplast DNA remained
in the wcll because,~::as:~a~result of its large ize, it
~; 20 was unable to enter the ~ gelO ~fter on~ minut~ of
~; digestion, no catenatcd kinetoplast DNA could be detected
in thc well and the released linearized minicira~e DNA
migrated as a 1.4 kb band (Figure 2). Continued
digestion led to the~appearance o~ a lower molecular :~
weight smear resulting~from multiple cleavag~s per
minicircle molecule. However, the reaction could be
terminated:at a~y point by the addition of a copper
W013~0~1~76 1 PCI/US92io64'
chelating agent, such as 2, 9-dimethyl-1, 10-
phenanthroline.
If the duplex minicircle DNA fragments released
from the catenated network contain excessive single
strand nicks, the DNA will not be ~n adeguate ~ubstrate
for PCR amplification. Upon denaturation, the size of
the single-stranded fragment would have to be at least
equal to the distanoe between the two P R primer~ in
order to obtain successful amplification. As described
.~ above, the DNA was cleaved with the phenanthroline-copper
chemical nuclease in GEB lysate for increasing periods of
~ time and the cleaved DN~ was electrophoresed in a
:~ d~aturing glyoxal gel~to det~rmine the size distribution
~5 o~ single-strandsd fragments. The gel was blotted onto a
nylon membrane and hybridized with 32p labeled T. cruzi
: : ki~etoplast DNA (kDNA):. At time 0, a f PW decatenated
, :~
minicircles undergoing~replication can be seen as a 1.4-
kb band in the ~e~ After cleavage, the minicircles were
:~ 20 ~ released from the kDNA:networks as double stranded
::: : :
: linearized molecules~ By denaturing the linearized
minicircles, it can be~seen that the minicircles were
increasingly nicked as a ~unction of digestion time.
Figure 3 shows that with increasing incubation time there
25 was a decrease in the~size of the single-stranded ~ :-
~ fragments caused by~nicking. However, after 30 minutes,
.
: 90% of the minicircle~ragments were larger than 310 ~ :
bases. After 60 minutes, 50% of the fragments were
- -,
~.
.
W093/03t67 PCT/~Sg2/0~77
31 2~
larger than 310 bases, and 80% of the fragments were
larger than 118 bases. An incubation time of 30 minutes
was selected for routine phenanthroline-copper digestion
of blood lysates, at which time approximately 90% of the
single-stranded fragments were longer than 310 bases, and
would be appropriate amplification target molecules for
the three sets of PCR primers which yield products of 83
bp, 122 bp, and 330 ~p respectively (Sturm et al.,
supr~)~ A control experiment showed the kinetopla~t DNA
digested with the phenanthroline-copper reagent in GEB
.~ lysate under standard conditions for 30 minutes was a
suitable template fur PCR amplification.
xample 3
Isolation Of~Cleaved Minicircle DNA
After GEB~lysates of T. cruzi were digested
with the phenanthroline-copper reagent (OP-CuZ+) for 30
minute. and the reaction guenched as described above, 500
~Ll aliquots containing ~deGreasing numbers of minicircle
molecules (calculated from the DNA concelltration) were
;~ ~ removed. Each ~00 ~ aliquot was ex~racted once with 100 ~;
~1 phenol/chloroform (1:1). The aqueous phase was - :
transferred to an~Eppendorf tube containing 40 ~g of
glycog n (2~1 of 20 mgjml stock solution~ and 50 ~1 of 3 - ;
M NaOAc. One mi~ iter of ethanol was added and the DNA
was precipitated at room temperature by spinning the i ~
tltbes in a microcentrifuge for 20 minut~s. ~: :
' :
WO93/03167 PCT/US92/0~'
21 1 S ~ 6 - The pellet was resuspended in 1 ml of water and
transferred to a Centricon-100 microconcentrator (Amicon3
containing 1 ml of water. The microconcentrator unit was
centrifuged at 1000 x g in a clinical centrifuge ~or 10
minutes. The retentate was washed a second time with 2
ml of water. After the second 10 minute centrifugation
100 ~l of concentrated retentate was collected a~
described by the manufacture of the microconcentrator.
The retentate was used for PCR ampli~ication as described
below.
Example 4
Poly~ r~ ~a~e Chain Reaction Amplification
Of ~leaved Minicircle D~
70 ~l o~ the retentate material was amplified in a
100 ~l PCR reaction. $he reaction conditions were as
follows: 10 mM~Tris-HCl, pH 8.3, 50 mM KCl, 5 m~ ~gC12,
0.1 mgjml BSA, 3 units of Taq DNA polymerase (Perkin
:20 ~: Elmer Cetus) and ~00 picomoIes of each primer. The
prImers used were three sets of primers specific ~o the
four conserv~d regions ~in T~ cruzi minicircles yielding
the 330 bp minicircle variable and conserved r~gion DNA
~ragment and the 83 bp and 122 bp conserved r~gion -:
fragments respectively,:as described by Sturm et al.,
Mol. Biochem. Parasit~l. 33:205 (1989) and Avila et al.,
Mol. Biochem. Parasitol. 42:175 ~1990), both of which are
incorporated by reference herein. The combination of the
~ WOg3/03167 - PCT/U~92/0~77
33 21.~5~6~;
26 base primer S33A and the 26 base primer S34A re~ulted
in the generation of the 83 bp conserved region fragment.
The combination of S34A and the 26 base primer S67
resulted in the generation of the 122 bp conserved region
fragment, The combination of the 26 base primer S35 and
the 21 base primer S36 resulted in the generation of the
330 bp variable region fragment (Sturm et alO, sura). ..
The~e primers are commercially available as Gense~ from
Amac/ Inc., Westbrook, ~aine. The cycling profil~ was as
:10 follows: Denaturation, annealing~ and elongation were
.
: ~ done at 94C, 60C, ~and 72C, respectively; each step was
allowed to pro eed for one minute ~or a total of 30
cycles. A 15 ,ul aliquot from each reaction was analyzed
. : ,
: on agarose/Nusieve (FNC, Rockla~d, Maine~ gels.
15 : ~
: ~ To determine the sensitivity of the P~R-
:
~ minicircle DNA as~ay, the equivalent of 20 To cruzi
:
kinetoplast DNA catenated networks were added to 10 ml
GEB~lysate. The~network DNA was cleaved with
~ ~ .
~ 20 ~:phenanthroline-copper~as described in Example 2. After : :
. .
~: : c}ea~age, the:~EB lysate was diluted with kinetoplast :.
DNA-free GEB lysate~to give DNA concentrations of from 1
; to 10,000 minicircles ~or 500 ~l of GEB lysat~. Total .
~ DNA was isolated from these aliquots as described in
:
Example 3 and subjected to PCR amplification; 1/6 of each
PCR rpaGtion was loaded onto a gel. In all cases, ~he M~:
lane shows H~e III fragments of bacteriophage ~X174 RF .-~
DNA as size mar~:er-. In~some oases, ~esults are shown
'~
W093tO3167 PCT/US92/0~
21~5~ 1 34
from hybridization with a radioactively labeled probe
a~ter blotting of the gel as described in Example 2.
Figure 4A shows results from amplification of
83-bp fragments u~ing 35 PCR cycles; the products were
electrophoresed on a 2% agarose/3% Nusieve gel. The
rapidly migrating band present in all lanes r~presentæ
PCR primer~ or primer dimers.
Figure 4B shows results from amplification o~
.~ 12~ bp fragments using 30 PCR cycles; the products were
electrophoresed on a 1% agarose/3% Nusieve gel. The top
panel shows the stained gel; the low molecular weight
: : :
band present in all lanes represents PCR primer. The
~; 15 bottom panel shows the hybridization of the blot with 32p_
labeled S34A oligonucleotide internal probe (Sturm et
al., supra3.
: :
. .
: Figure 4C shows results from amplifieation ~f
:
~ 20~ ~30 bp variable and conserved region fragments wi~h 30 ~
.
PCR cycles; the products were electrophoresed on a 1%
agarose/3% Nusieve gel. The top panel shows the stained
gel. ~Th~ bottom panel shvws results from hybridization
of the blot with 32P-labeled S67 oligonucleotide internal
25 probe ~Sturm et al., supra; Avila et al., Mol. Biochem. ~-
Parasitol. 42:175 (1990)). The C1 and C2 lanes show 500
. .
~1 o~ undigested GE3 lysate sample containing 10 kDNA
. W093/03167 PCT/
networks per 30 ml processed for PCR amplification as
described for all other samples.
As shown by the electrophoresis patterns o~
Fîgure 4, at lea~t lO0 minicircles in 500 ~l ~f GEB
lysate were detected, either by ethidium bromide staining
(83 bp PCR product3 or by hybri~ization to a 32P-labeled
oligonucleotide probe (122 bp and 330 bp PCR products~.
Th~refore this method detects the equiva}ent of 1% o~ the
:~10 minicircle content of a single T. cruzi cell in 500 ~l o~
phenanthroline-copper digested GEB lysate. These results
indicat~ that the method can be used for the detection of
:~ a single TO ~ruzi cell in
: 2~ ml of blood.
, :. ' .
Example 5 ~::::
Di~qnosis_of Chagas'_Disease ~:
: ~ ,,
.:
....
A 49 year old female patient from Ecuador
~: ;: ao: showing cardiac disturbances ~ested positive for T. cruzi
in two ~ifferent serological tests - xenodiagnosis was :~
,
: :negative. The serologioal tests employed were complem~nt
fixation (Sommerwirth and Jarett, Gradwohl's_Clini~al
Laboratory;Me~hod5 and Diaanosis (C.V. Mosby, St. Louis,
1980)) and ELISA (Goldsmith ~ Heyneman, ropical Medicine
~: and Parasitoloqy (1989))~ Xenodiagnosîs was perfo~med by ::~
the following procedure (E.L. Segura, Xenodiaqnosis. In
Cha~as' Disease Vectors (Brenner and Stoka, eds., CRC
: ,:
WO93/031~7 PCT/US92/0~7
2 1 ~r ~ 36
Press, Boca Raton, Florida, 1987), v~l. II, pp. 41-45):
Uninfected triatomids of several species, including
Triatoma dimidiata, T. protracta and Rhodnius prolixus,
were allowed to feed upon the patient. Second, third,
fourth, and fifth nymphal as well as adult ~tages were
used. Approxima~ely one-third of the bugs ~ed, and
defecati~n spots were found on the patient. Microscopic
; examination of bug feces at 30 and 60 days after feeding
re~ealed no T. cruzi
A 10 ml sample of venous blood from the patient
was obtained and stored as GEB lysate. Phenanthroline-
~ copper cleavage of the lysate was performed and DNA was
: ~ isolated from two 500 ~1 aliquots and PCR amplified as
~15 described in Examples 2 through 4, supra.
Figure 5 shows a specific amplification of T.cr~zi mi~icircle s~equences from patlent blood with two
differe~t sets ~f PCR primer~. Figure 5A shows the
~: ;` 20~ amplifi~cation of an 83 bp fragment; the products are
: analyzed on~a l~:agarose/3% Nusieve gelO The negative ~ -
:
: control in lane l~is~a GEB sample from a non-chagasic ~ ~
.
donor. The negative control ~n lane 2 is a sample ~ ~
.
: l~cking kDNA~in the P~R reaction. The M lane shows DNA
~: : : : :
:~: 25~ Hae III fragments of~X174 RF DNA:as size markers. :~
:~ Figure 5B shows thP amplificat~ion of 330 ~p variable and ::
con~erved region fragments, run on a 2% agarose gel,
blotted and hybridi~zed with 32P-labeled S67
'~
: . WO93/03167 P~T/US92/0~77
37 2,'~ ,Gl
oligonucleotide internal probe (Sturm et al., supra;
A~ila et al., supra.) A positive control is lO0 fg gel-
iso-ated OP-CuZ~-cleaved kDNA; the negative controls are
the same as (A).
Blood samples ~rom four addition~l ser~logy-
positive chronic chagasic patients (three patients were
xenodiagnosis-negative and one xenodia~nosis-po~itive)
also tested positive for T. cruzi parasite~ by PCR
~: 10 amplification of minicircle DNA using the standard
procedure of Examples 1-4. These results indicate that
the method should prove useful in diagnosis of chronic ~.
Chagas' disease.
~15The DNA storage, cleavage, purification, and
.
: :ampli~ication method was extended to an insect vector and
t~ biopsy material~from infected mice. The abdominal : ~
contents of two~ imidiata and two R. Prolixus were ~;
collected and stored~in GE bu~fer. The 5ample~ were
20~ processed as descri~èd in Examples 1-4. Figure 6 show a :
specific amplification o~ kinetoplast DNA minicircle ~:
:seguences~rom the insect abdominal contents. :~
~:~ : Minicircle variable region fragments of 330 bp were : -
amplified. The~PCR~reactions were of l00 ~l total
:
25~ volume; I5 ~l was loaded on a 2% agarose gel. The M lane ~:~
~ shows DNA Hae III fragments of ~Xl74 RF DNA as size
:~ markers. Positive~ and~negative controls are l pg kDNA
;~ and~no kDNA, respectively, in the PCR reaction.
: ~
... . .. . . . . . ... . .. .
WO93/03167 PCT/US92/0~7
rJ'~ 38
Animal biopsy material was also dissolved and
stored in GE~ Heart tissue obtained from in~ected and
uninfected mice was washed with saline solution and
stored in GE buffer. The tissue was dissolved by
incubation in GE at 37C for two days with occasional
viyorous mixing. The dissolved tissue was processed as
described in Examples 1-4. Specific PCR amplification of
minicircle sequences was obsexved from the heart tissue
lysa~es of the in~ected mouse but not from uninfected
10 ~ controls~ ~hese results indicate that the method could
.
~be used for autopsy specimens to detect Chagas' disease.
: .:
AD~ANTA~ES_~ TU~ y~ Q~
:
:~ :15The method of:the present invention has a wide
,~
: variety o~ potential applications in the diagnosis of
bacterial~ parasitic, and viraI diseases. It i~
particularly useful for the collection of specim~ns from -~
patients in the field, in clinics and under other : :
csndition~ in which storage conditions may be le~s than
optimal. It:is particularly adapted to the di~gnosis of
,
parasitic diseases caused by~inetoplastid trypanosomes,
such as T. cruzi, the causative agent of Chagasf disease,
: ~ because the catenated~kinetoplast minicircle DNA
characteristic ~f this~organism is efficiently cleaved to
linear frag~ents of a~size suitable for amplification by
a sequence-specific primer-based amplification technique
such as PCR. The~method of the invention is simple to
,
. WO93/03167 ~ PCT/US92/0~77
39 2 ~ & :1
carry out, rapid, and highly effective in detecting small
quantities of DNA specific to infectious agents. In
addition, the infectivity of biological samples
containing infectious agents is reduced by application of
the method of the invention.
': ''
Although the present invention has been ~:
described in considerable detail with reference to
certain preferred versions thereof, other versions, ::
: lO m~di~ications and variations of the invention a~ set
forth above may be made without departing from the spirit
and scope of the present invention. Therefore, the
invention:is limited only by the terms of the appended
cIaims~
:
~: 15 : ~
: ~ :
:;
.
: ~ ,., :-,'
: ~ : :
:: :: ~ , ~
~: ,
;
`
::
~ , :
: , ~ :
~ , .
