Note: Descriptions are shown in the official language in which they were submitted.
) 7 l~ ~
Disclosed are no~l method3 by which ~ rapid, multisample,
non-radioactive procedure to detect pathogons, 3uch as
Plasmodium falciparum paxa~itas, in biological fluid~
insluding human blood samples i~ achieved. Th~ detection i8
based on the use o~ parasite speci~ic DNA probe~ and sandwich
hybridization ~echnique employing ~icrotitre plate~. The high
sensitivity and specificity of these assays and the ease
with which they can be performed enable one to use them for
routine analyses o~ a large number o~ blood and other
coloured tissu~ samples o~ vertebrates and invertebrates.
E~pecially, these assays can bc u~ed to detect th~ presence
of P.falciparum. The procedure described is amenable for
application in a wide variety o~ DNA detection analysis using
non radioactive DNA probes. In regard to d~tection of
P.falciparum, the invent~on also relates to novel DNA
~ra~ments and hybridisation probes based on such fragments.
The in~ent.ion provides a diagnostic kit on the basis of the
novel methods.
t
2~7 ~1~
~AC~RO~ND 0~
~alaria i~ caus~d by protozoan parasiteu b~longing to the
genus Pla~modiu~. Th2 li~ cyale o~ ~he paras~te occurs in
two phase~ - th~ a~exual pha~ ln v~rtebrates and th~ sexual
phase in mos~uito (usually o~ the genus ~nophsles)O The ~our
species of Plasmodium respon~ible f~r human malaria are
P.falciparum, P.vivax, PJmalarias and P.ovale. ~mong these,
the first two are the most common. P.~alciparum causes the
most severe form of malaria which in S0~3 instances is fatai.
Furthermore, this parasite al~o develops re3i~tance to tha
commonly used antimalarial drugs.
The current method of diagnosis of ~alaria i~ by blood smear
examination. This method is laborious and also reguires
expertise. Further, a skilled microscopi~t i8 allowed to
examine a maximu~ of sixty slides a day. Diagnosis by
serology may also be done, but becaus~ o~ the persistence of
antibodies current infections cannot be distinguished ~rom
past infections(l). Hence, the search for a new generatior. of
diagnostic tests has included the possibility of detectin~
parasite nucleic acids as indicative of the presence of the
parasite. Theoretically such a test should reguire very
little blood (5-50 ul) that can be obtained from a ~inger
prick, and should be sensitive and rapid. As few as 50
parasites in 10 ul of blood can be detected by nucleic acid
hybridization (2). Hundreds of samples can be analyzed in a
2 ~ 3 ~ 2
day w~h 80m~ initlal tralning. Th~ sen~ltiY~y oP ~h0 a~y
enablea ~ho test to b~ UBed ln blood b~n~ Por the ~creening
o~ blood to bQ u~ed ~or trans~u~lon.
Nucl~lc acid hybridizat~on could also be per~or~ed ~n lnsect
tissue samples in order to id~nti~y th~ ve~tor sp~cia~ a~ ~
carrior. Such in~ormation would help to inte~ y vector
control ~easures in order to limit the geog~aphic ~pread o~
malaria. Alternatively, chemoprophylaxi~ ~ay b~ ~doptsd ~n
suc~ areas and evaluatlon o~ thi~ strategy ~ay be accomp-
lished using nucleic acid hybridization. Tha procedure
describ~d in this patent appl~c~t$on provid~s an e~ic~ent
means of accomplishing parasit~ detection us1ng nu~leic acid
hybr$dization technique~.
The detection method described by the present invention can
be used generally to detect the presence of pathogens in
blood, tissues, samples and body fluids of humans ~s well as
of vertebrates and invertebrates in general such ~s cattles
and insects.
The said pathogens may De e.y. bacteria, viru. and parasites such
as of the Plasmo~ium genus especially P.falciparum and P.vivax.
As further examples of patho~ens can be mentioned Shigella, e.g.
Shigella flexneri, Shigella dysenteriae, Shigell~ sonnei, and
Mycobacterium tuberculosis.
~3~7i~
Althoug~ the ~pQcific exa~ple~ ln the present appllcation
relata to P.~lc~parum, it wlll be under~tood that the
detect~on method i~ gonerally applica~le as outlinsd abov~.
PRIOR ART
Nucleic acid (DNA and RNA) ba~ed hy~ridisation ~8 now being
used in a numbar of clinical diagno~i~. Initially thi~
technology utilised radioactively labelled probes. Though
the sensitivity of the diagnosis in th~ radioactive format is
satisfactory this method i5 not popular in the clinical
laboratories owing to the precaution~ and regulations
necessary in radioactive material hanaling. Hence there is
an urgent need for non ladioactive detection in th~s field o~
pathogen detection by nucleia acid hy~ridisation~ One of the
most popular method of non isotopic detection is ba~ed on the
incorporation of biotin enzymatically (3) or photochemically
into the nucleic acid probes (4). The hybrid~ that bind the
biotin labelled probes can then be easily detected with
complexes of avidin or streptavidin a~d .suitable enzy~es like
phosphatase or peroxidase. Tho~gh the above mentioned non
isotopic method looks attractive it has llot been yet
popular. A few important problems remain to be solved. The
major problems relate to the colsured background and the
state of purity of the target ~JA. Most DNA based
diagnostics are done on membrane filter~ (either
nitrocellulose or nylon). Body ~luids like blood which are
~ ~ 3'~
to be tested ~or th~ pxe3encQ o~ pathogen~ wh~n ~p~tted
directly on the me~b~ane ~llt~r to i~moblll~ ~he D~ 'e~v0
an indell~le coloured mark whlch ~aka~ the sub3eguent aolour
developm~nt after hybridi~ation ~1~08t lmpo3~iblo. T~u~ the
only alt2rnative left i~ potting pure DNA ob~aln~d ~rom the
pathogens that are present in thQ ti~SU~ or body fluid.
Since isolation of ~NA involves a procedure which includes
centrifugation and precipitation, it severely curtall~ the
feasibility of a r~pid multisampla diagnosis. For a
preferable diagnostic procedure based on nuclei~ acid
hybridisation the following conditions are essential.
E8BE~NTIAL~ OF A 1;001:~ 1)~ BA8~D N~JLTI~a~lPI~ DI~IO~IC
P~OCl~I)U~.
1. It should be based Qn non radioactive detection
2. It should use small amount of blood (a drop fro~ a
finger prick).
3. Most of the çomponents used ~n the diagnostic kit
should be stable at room temp~rature.
4. Exact micropipeting o. individual components should be
avoided.
5. Centrifugation and precipitation steps should be
avoided.
6. It should requlre minimum train~ng for successful
operation.
By the present invention a detection method is provi~ed which
fulfills all these criterLa~
23940-683
T~.e pr~sent invantion 18 ~mm~rl~d ln the ~ollowing clau~es~
1. A ~ingl~ ~trand~d DNA ~ragmsnt (~3) hav~ng th~ 3e~uenc~
gl~en he~ow :
AGGTCTT~TGACTAACrAA>CTTAAC~AACTAACTTAGGTCTTACT$T~ACTAAAC~
or it~ compli~entary strand or the corr~ponding double
stranded sequence, It i~ pre~rred to U8~ thQ ~ingle
stran~ed ~NA.
2. .~ DNA fragment as defined by clause 1 or contiguous
seg~ent th~reof which i~ at least greater than 20 basQa or
base p~irs in length.
3. ~ ~NA fragment according to clauses 1 or 2 in single
stranded form.
4. A hybridization probe comprising a D~A fragment as
define~l in clause 1 and 2.
5. A hybridization probe accor~in~ ~o clause 4 which i~
labelle~l hy a group capable of colo~lri~net.ric detecti onO The
na~ure cf t:his group is not critical for . hi~ invention.
6. R hybridi~ation probe according to clausQ 5 wherein the
.abelle~ group ~or colourimetric detection is biotin. Biotin
is a p~eferred reporter group.
7. A hybridization probe according to clause 5 wherein the
labelled group for colourimetric detection is a chro~ophoric
reporter group.
8. A method for detecting a pa~hogsn present in blood or
other body fluid comprising of the fo'lowing 8tep8:
a) Lysing a blood sample in a solution containing Guanidine
hydrochloride (GuHCl), Sodium lauryl sareos~n~ (SLS) and
Triton~ 100*
b) Denaturing the DNA present in the said blood sample
suitably by heating and performing solution hybridization in
presence of a hybridization probe which hybridizes with DNA
of t:he said pathogen.
*Trade-mark
~s~71~r~
c~ C~pturing th~ hybrids ~or~d in ~tep 8 ~b) in
~icro~ltre pl~te co~ted wit~ ~ hybridiz~tion pro~ which ha~
a nuclaotido ~equ~nce ~ap~bl~ o~ hybrldi~ing to th~ ~ame
strand of gsno~ic DNA ~bat ~he hybridzat~on prob~ U~9~ ~n
~tap n (b) binds. In a pre~erred embodi~nt, s~peaiAlly ~or
detec~icn o~ ~r~lçl~Lg~, the nualeotld~ sequence u~d ln
coatin~ the microtlter plata i~ identical to ~he ~equenc~ o~
the hybridization probe us~d in ~tep 8(b~.
d) Washing the microtitre plate with ~ solution comprising
Standard Sallne Citr~te (S~C~, Sodiu~ dodecyl Sul~ate ~SDS)
and Triton-X-100.
e) Detection of the presence of th~ hybrids by
colourimetric methods~
9. A method according to clause 8 wherein the hybridization
probe is as defined in Clause 2 and 3. In a preferred aspect
the invention is used in the detection of plasmodial species.
10. A method according to clause 8 and 9 wherein th~ ~inal
concentration Or the reagents in step 8 (a) ars as ~ollow~ :
a) Guanidine hydrochloride : Between 1.0~ - 3.OM
b) Sodiu~ lauryl sarcosin~ : Between 0.2% - 0.5~ W/v / N/v
c) Triton-X-100 : Between 0.2% - 0.5~ v/v / v/v
The above represent preferred interval~
11. A method according to clause~ 8 and 9 wherein the ~inal
concentration in ~tep ~ ~e) are a~ follow~ .
a) Standard Saline Citrate - 0.5 X - 2.5 X SSC
b) Triton-X-100 - 0.2% - 0.5% V/V
c) Sodium dodecyl S~lphat.e - 0.2% - 0.5% W/V
The above represent preferred intervals.
12. A method according to clause 8-1 wherein the lysing
solution is used both as a solubilising agent and as
hybridization solution.
2 ~ ~3(J
13. A ~othod accord~n~ to cle~ 8 to 12 wher~in 2X ~C 1
u~d to ~omoY~ ~on~p~ci~i¢ ~ybrl~.
1~. A ~sthod accQrding to ol~u~ 8 an~ 14 wh~ln ~rlton-x-
100 and 8D~ ~r~ u~o~ ~or t~ re~o~al o~ t~ ¢olour~n~
~at~ri~l orl~lnati~g ~ro~ th~ blood.
15. A ~ethod according to ~1~U~Q~ 8 to 1~ wher~ln the
pathogen i8 P.~alclpa~un.
16. A msthod sccord~ng to clau es 8 to 14 whorein the
pathogen is P.vivax.
17. A method according to clauses 8 to 14 wherein the pathogen is
Shigella.
18. A method according to clauses 8 to 14 wherein the pathogen is
Mycobacterium ~uberculosis.
19. A diagno.stic kit for the detection of a given nucleotide
sequence prescnt ir. a target polynucleotide sequence on the basis
of the method accc.rding to clauses 8 to 18.
2 ~ 3 J 'J li ~
239~0~683
In drawings which illustrate embodiments of the
invention,
Figure 1 shows the oligo f63 that was designed from
the consensus repeated sequence (21 base repeat) of P-falciparum,
Figure 2 shows the steps of DNA bases sandwiched
hybridization as follows:
A: Solution hybridization.
B: Depicts microtitre well coated with the probe f63.
C: Capture hybridization.
D: Capture hybrids after washing and ready for colour
development~
and
Figure 3 gives a key to active elements illustrated
in Figure 2 as follows:
A: biotinylated f63 DNA
B: genomic P.falciparum DNA
C: f63 DNA
- 9a
~ ~ 3 ~ 3~
~RI~ h~ Na ~8~D ~A~D~C~ $~Ig~IO~
Background:
In the non radioactivQ ~orm~t the Pinal ~ode o~ detsct~on i~
the d~velopment o~ a colour eithQr soluble or in~oluble
depending on the nature of the substrate used in the reaction
catalysed by eith~r alkaline phosphatase or horse radish
peroxidase.Therefore it is essential to remove the residual
coloured material from the target DNA as W811 as inactivating
the endogenous enzyme. This ~akes spotting blo~d directly
cnto membrane ~ilters (as i~ done in th~ radioactive
~.ybridisation format) useless ~ince the removal of residual
blood stains from the filter i8 almoAt ~possible.To
circumvent this problem we have used the ~icrotitr~ plate
format coupled with sandwich hybridisation, the basic
principle of which is described below.
It has been shown earlier that one o~ the characteristics of
t P.falciparum genome is that it contains a 21 base pair repeat
~hat is present in tandem in a lar~e region of the genome
(5-6). The fraction of the genome represented by this
repeated sequence is about 1%. Comparisons of several clones
containing this repeat sequence have indicated a consensus 21
base pair repeat sequence. Based on this consensus sequence
~0
2 i~3 t.,~ ~J ~ ~ 2
W8 hava design0d and con~tructed a 63 ~er ollgonucl~otida
probe (ds~iqnated ~63 hQrea~t~r)~ It con~lsts o~ three 21
~r~ in tandQ~ whlch ~r~ ~ax~lly r~pre~ent~d i~ th~
repeat~d 3eguence~ o~ th~ P.~alcip~ru~ DNA (~g.l). m e
preferred U8e 0~ single ~trandad DN~ a~ ~ probe an~ ~t~ ~a~d
lenqth is based on the ~ollowing reasoningc ~ingle stranded
DNA is superior to double stranded as a probe becau~e lt
hybridise3 only to the target DNA. In cass of double stranded
DNA there is a greater probability of sel~ hybridisation thu~
reducing the effective concentration of the prv~e that i8
required to bind the target DNA. This clearly establishes the
superiority of the single stranded probe in its cost
effectiveness as it is required in a much lower amo~nt for
hybridisation. 0~ the several methods that are availabie to
make single stranded DNA, oligonucleotide ~ynthesis is most
convenient.
For detection of pathogens other than P.falcipa~umc oe has
to design an optimal DNA probe which i8 repeat~d in the
pathogen DNA. This hybridization probe can th~n be used in a
similar detection protocol of sandwich hybridization which is
given below specifically for P.falciparum.
The basic protocol for the sandwich hybridisation is given
below.( Also explained pictorially in Fig.2
11
2~ 3~
23940-683
c~a~ O~C~IVB DI~ 8~ oa~
I~a3rl3~r~o~ Ill ~ B~OOD ~ ~AalD~I~ }~3RID~A~0211
Add ons drop og~ blood sampl~ (50 ul) Pro~ nger prick
in a small pla~tic: tUb2 conta~ining I.y~ing sQlutioJ2 and
bio-f63 probQ
( $) ~ix well
Phase 1
(ii) Heat in E~oiling water bath
~or two min~te~
( i i i ~ Leave at roola tempexatura
~ r fs~r a ~ini~um o~ our hours
Para~3ite DNA-
bio-f 63 probe hybr~ ~1
See Fig.2. Plate A
~i) Trans~er the mixtur~ from the
t~ :~r. Phase 1 to w~lls in
~ rotitr~ plate~ precoated
with unl akelled ~-63 probe
Phase 2 (See Fig. 2. Plate B)
~ii) Allow to s~nd at rooD~
~ temperatur~ ~or overnigh'c.
CAPTURED
HYBRI D
See Fig.2. Plate C
(i) Wash microtitre plate wells
frGm phase 2 with 2 x SSC
con ain n~ 0.2% SDS and 0.2%
Triton ~ lOO
Phase 3
(ii~ Repsat wash procedure four
times c-ach time let stand
wash }~ufler for 5 ~inutesO
(iii) Let stand in each well APB-l
solu~ion for 30 minutes at
room tenlp2rature.
(iv) Add one drop o~ APB-l
contain~ng streptavidin
Alkalir,~ phosphatase conjugate.
(v) Let stand ~or 30 minute~ at
roo~ te~p~rature.
12
.
.
.
7 ~
23940-683
I (Yi) D~3card ~olution ln th~ W8118.
Phase 4 1 ~vli) L~t ~tan~ APB-l solution
(without B~A~ in ~h w~ or
5 ~inut~a and di~c~r~ ~olution.
!~iii) Repe~t abo~e op~ration ~our
t~
Captured hybrid ~ix~ ~in~ each well w$th APB-2
ready for solution.
detection by
colorimetry ~x) ~dd enzy~e sub~trate in AP~-2
~olution.
Fig.2 Plate D
(xi) Let stand at room te~p~rature
~or atlea~t 120 minutes.
~xii) ~ead absorbancy at 410 nm in
~ microtitre plate reader.
., ~ r
~olour with absorbancy
Phase 5 ~al~es higher ~han 0.2
~ denote~ presence of
Analysis of ~ par~s.i~e
TQXt
Result~ ~ Absence of parasite
DNA sho~ld giv~ an
absor~ancy value of
less than o.l
The success of this ~ethod depends on the ~act t~t
P.falciparum DNA remains nearly undegraded duri~g the
process.In the sol~tion hybridisation step biotinylated f6~
binds to P.falciparu~ D~A and would proceed to near
completion, the rat~ of solution hybridisation being much
,
~'~3$~2
~a3ter when co~pared to ~mobill3~d target DNA. Th~
a~ficiency o~ c~ptur~ hybridi~at~on 1~ directly proportional
to tha lQngth o~ ~h~ P.~aloipaxu~ DNA. In th~ ~xtro~e li~lt
it can be ~een th~t i~ the P.~lc~paru~ DNA 18 ~ot~lly
undegraded then even a ~eagr2 0~03~ o~ captu~e hybridisation
can bring do~n all the hybrid complex.
In the ~a~a of other pathogen~, ~he e~ficiency o~ capture
hybridiz~tion will depend on the number oP time~ the probe i~
repeated in the pathogen gencme.
239~0-683
~OTOC~ ~0~ ~O~-IBO~O~Ia D~CT~0~ 0
~t.oo~ aa~
1. Preparation o~ the probQ:
The pro~e for coating ~h~ mic,rotitre plata~: The 63 ~er
oligonucleotida( f63 ) was syn~he~ised u~ing the automated
DNA synthesi~r(Appli~d Biosyste~ 340A).
LabelQd probe ~or detectlng the th~ hybrid~: Biotlnylation
of f63 was done by photobiotinylation using photobiotin
a~etate according to published procedures.
2.Coating of microtitre plates:
All the wells in the microtitrQ plate ~Dynatech, Polyvinyl
chloride) are coated with varying amounts(luq to lOng) o~ ~-
53 in 50 ul vol~m~ contalnins 0.1 ~ ~gC12~ The coatinq is
done overnight following whi.ch the ~icrotitre plate is
exposed under germicidal W lamp ~40 watts) at a di~tanc~ of
10 cms, for 5 minutes to immobilise DNA. T~Q contents o~
~he wells are discarded subsequently and the wells are washed
with 2X SSC buffer. Unoccupied sites site~ in each of the
~ells are blocked by carrying out prehybridisation in a
buffer (200 ul/well) containing 2X SSC, 5X Denhardts, 0.5%
Triton-X-100, 0.5% SDS and 5n ugJml salmon sper~ DNA. The
prehybridisation is carlied ~ut for 4-6 hours at room
temperature. The coated plates can be stored at this stage
in room temperature
*Trade-mark
3. Coll~ction o~ ~lo~d ~ample8 ~nd ~o~u~ion hybrldl~tlon:
Blood ~a~ple~ (50 ul aliquot~) ~ro col~0ct~d gro~ ~ ~inqer
prick, dlrectly into 50 ul oY a ~olutlon contalning 4
guanidine hydrochlor~de (Gu ~Cl), 0.5% ~odlu~ lauryl
sarcosine (SLS) and 0.5~ Triton-X-lOO. Thi~ ~olution ~180
contain 5 n~ of oligonucleotide probe (biotinyla~ed ~-63).
This mixture i heated ~or 5 minutes at 95 dag C and then
kept at room temperatur~ for 4-6 hour~ for the solution
hybridisation to o~cur.
4. capture hybLidisation:
After solution hybridisat~on i~ over, the contents o~ the
eppendorf tubes are transferred into the w0118 of th~
microtitre plate that are precoated with unlabelled f-63.
This sandwich hybridisation(captur~) is allowed to go ~or 24
hours. During tnis phass, hybrid~sation occur~ between the
f-63 coated onto the plate and the rest G~ the complementary
sites available in the hybrid. The hybrid is a long piece of
target DNA carrying the biotinylated f-63 i~ certain
locations leaving behind other complementary sites. ~See
Fig~ 2).
5. Colour development:
After the san~wich hybridisation is over, the contents of the
wells o~ the microtitre plate are discarded and wells are
washed with a solution containing 2x Ssc, 0.2% SDS and 0.2%
~iJ~ 3tl~,
Triton X-100, ~our tl~0~, ~ivo ~inut~ each ~t roo~
te~perature, During thla post hybridl~atlon wash, ~11 the
coloured mat2rial5 are removed leav~ng behind the san~wlch
hybrid. The well~ axe then blocX~d ~ith A.P 7.5 whlch i~ ~
~olution conta$ning 1~ NaCl, 100 ~ Tris-cl pH 7.5, 2 mM
~gC12, O.OS% Triton-X-100 and 3% BSA, for 30 mi~utes at roo~
temperature.
sandwich hybrid~ are then detected by using ~or exa~ple,
Streptavidin-alkaline phosphatase con~ugate. The Streptavidin
al~aline phosphatase con~ugate(l ug/~13 i3 added to A.P 7.5
buffer. 50 ul of t~i8 801ution (~P 7.5 buf~er containing
streptavidin alkaline phosphatase) 18 added to each well and
incubation continued ~or another 30 minute~ at room
temperature. The exce~ unbound con~ugate ~s removed by
washing four time~, five minutes each, at room te~perature
with A.P 7.5 buffer without BSA.
Finally the wells are rinsed with A.P 9~5(substrate
inc~bation buf~er containing lOOm~ Tris-Cl pH 9.5, 100 mM
NaCl and 50 mM MgC12). 50 ul of the substrate p-nitrophenyl
i phosphate is added to A.P 9.5 at a concentration of lmg/ml
and 50 ul of this solution is added to each well. The color
~evelopment is allowed to take place for 6-12 hours. The
absor~ance (at 410 n.m.) are recorded, using a suitable
plate reader( e.g. Dynatech plate reader).
The Te~t re~ult~ ar~ given ln tha ~ollowlng tabl~.
~ESULTS
(Ab~orbance at 410 m~
. .
Amount of
parasite DNA ~mount ~ ~Ç~ çoated Q~ ~ic~b~te~la
(T9/106* DNA)
, ~
1 ug 500 ng 100 ng 10 ng
500 ng over over over over
2 5 0 ng ~ n 1~ n
12 5 n-3 " n 1~ n
6 3 ng " ~ n ~
31 ng n n n n
16 ng " ~ n 1 . 52~
~.5 ng 1.511 1.242 1.373 0.92g
__________________________________________________________
1 ug û.291 0.295 0.263 0.214
Human DNA
* T9/106 represent a chloroquine resistant P. falcipar.um
clone.
Note : In human samples 50 ul of blood has about ~Onct of
parasite (P. falciparum) DNA if the infection is about l~s .
The term "over" indicates an optical density above 2 . o.
18
