AU640745B2

AU640745B2 - A novel procedure for the detection of pathogens using dna probes

Info

Publication number: AU640745B2
Application number: AU72028/91A
Authority: AU
Inventors: Kasirajan Ayyanathan; Santanu Datta
Original assignee: Astra AB
Current assignee: AstraZeneca AB
Priority date: 1990-03-30
Filing date: 1991-03-01
Publication date: 1993-09-02
Anticipated expiration: 2011-03-01
Also published as: GB2242904B; AU7202891A; IE911010A1; CA2038742A1; GB2242904A; GB9106504D0

Description

COMMONWEALTH OF AUSTRALIA 4 7 4 PATENTS ACT 1952 FORM Case: R-1072-1 AU Class: Int. Class Application Number: Lodged: Complete specification: Lodged: Accepted: Published: Priority: Related Art:

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Name of Applicant: AKTIEBOLAGET ASTRA Address of Applicant: S-151 85 Sodertalje, Sweden.

Actual Inventor/s: KASIRAJAN AYYANATHAN; and SANTANU DATTA.

Address for Service: E.F. WELLINGTON CO., Patent and Trade Mark Attorneys, 312 St. Kilda Road, Melbourne, 3004, Victoria.

Complete Specification for the invention entitled: "A NOVEL PROCEDURE FOR THE DETECTION OF PATHOGENS USING DNA PROBES" The following statement is a full description of this invention including the best method of performing it known to us.

1 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 FORM Case: R-1072-1 AU Application Number: Class: Int. Class Lodged: Complete specification: Lodged: Accepted: Published: Priority: Related Art: 00 *5 .5 Name of Applicant: AKTIEBOLAGET ASTRA •0 Address of Applicant: S-151 85 Sodertalje, Sweden.

o• Actual Inventor/s: KASIRAJAN AYYANATHAN; and SANTANU DATTA.

S* Complete Specification for the invention entitled: "A NOVEL PROCEDURE FOR THE DETECTION OF PATHOGENS USING DNA PROBES" The following statement is a full description of this invention including the best method of performing it known to us.

1 Summary 9. tM InveZntion: Disclosed are novel methods by which a rapid, multisample, non-radioactive procedure to detect pathogens, such as Plasmodium falciparum parasites, in biological fluids including human blood samples is achieved. The detection is based on the use of parasite specific DNA probes and sandwich hybridization technique employing microtitre plates. The high sensitivity and specificity of these assays and the ease with which they can be performed enables one to use them for *:o0 routine analyses of a large number of blood and other coloured tissue samples of vertebrates and invertebrates.

Especially, these assays can be used to detect the presence

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of P.falciparum. The procedure described is amenable for *e application in a wide variety of DNA detection analysis using non radioactive DNA probes. In regard to detection of P.falciparum, the invention also relates to novel DNA fragments and hybridisation probes based on such fragments.

The invention provides a diagnostic kit on the basis of the m novel methods.

BACKGROUND OF THR INVENTION Malaria is caused by protozoan parasites belonging to the genus Plasmodium. The life cycle of the parasite occurs in two phases the asexual phase in vertebrates and the sexual phase in mosquito (usually of the genus Anopheles). The four species of Plasmodium responsible for human malaria are P.falciparum, P.vivax, P.malariae and P.ovale. Among these, the first two are the most common. P.falciparum causes the most severe form of malaria which in some instances is fatal.

Furthermore, this parasite also develops resistance to the commonly used antimalarial drugs.

The current method of diagnosis of malaria is by blood smear S.examination. This method is laborious and also requires expertise. Further, a skilled microscopist is allowed to examine a maximum of sixty slides a day. Diagnosis by serology may also be done, but because of the persistence of Santibodies current infections cannot be ristinguished from past infections(l). Hence, the search for a new generation of

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i* diagnostic tests has included the possibility of detecting parasite nucleic acids as indicative of the presence of the parasite. Theoretically such a test should require very 5 little blood (5-50 ul) that can be obtained from a finger prick, and should be sensitive and rapid. As few as parasites in 10 ul of blood can be detected by nucleic acid hybridization Hundreds of samples can be analyzed in a a so soo 0g a 0 o 0000 .0 day with some initial training. The sensitivity of the assay enables the test to be used in blood banks for the screening of blood to be used for transfusion.

Nucleic acid hybridization could also be performed on insect tissue samples in order to identify the vector species as a carrier. Such information would help to intensify vector control measures in order tr limit the geographic spread of malaria. Alternatively, chemoprophylaxis may be adopted in such areas and evaluation of this strategy may be accomplished using nucleic acid hybridization. The procedure described in this patent application provides an efficient means of accomplishing parasite detection using nucleic acid hybridization techniques.

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 as well as of vertebrates and invertebrates in general such as cattles and insects.

The said pathogens may be e.g. bacteria, virus and parasites such as of the Plasmodium genus especially P.falciparum and P.vivax.

As further examples of pathogens can be mentioned Shigella, e.g.

Shigella flexneri, Shigella. dysenteriae, Shigella sonnei, and Mycobacterium tuberculosis.

Although the specific examples in the present application relate to P.falciparum, it will be understood that the detection method is generally applicable as outlined above.

PRIOR ART Nucleic acid (DNA and RNA) based hybridisation is now being used in a number of clinical diagnosis. Initially this technology utilised radioactively labelled probes. Though the sensitivity of the diagnosis in the radioactive format is satisfactory this method is not popular in the clinical laboratories owing to the precautions and regulations *0 0 necessary in radioactive material handling. Hence there is an urgent need for non radioactive detection in this field of pathogen detection by nucleic acid hybridisation. One of the *o most popular method of non isotopic detection is based on the incorporation of biotin enzymatically or photochemically into the nucleic acid probes The hybrids that bind the biotin labelled probes can then be easily detected with complexes of avidin or streptavidin and suitable enzymes like 0 phosphatase or peroxidase. Though the above mentioned non 20 isotopic method looks attractive it has not been yet 00 popular. A few important problems remain to be solved. The major problems relate to the coloured background and the state of purity of the target DNA. Most DNA based diagnostics are done on membrane filters (either nitrocellulose or nylon). Body fluids like blood which are *0 we a 2 S0

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to be tested for the presence of pathogens when spotted directly on the membrane filter to immobilise the DNA leave an indelible coloured mark which makes the subsequent colour development after hybridisation almost impossible. Thus the only alternative left is spotting pure DNA obtained from the pathogens that are present in the tissue or body fluid.

Since isolation of DNA involves a procedure which includes centrifugation and precipitation, it severely curtails the feasibility of a rapid multisample diagnosis. For a preferable diagnostic procedure based on nucleic acid hybridisation the following conditions are essential.

ESSENTIALS OF A GOOD DNA BASED MULTISAMPLE DIAGNOSTIC

PROCEDURE.

1. It should be based on non radioactive detection 2. It should use small amount of blood (a drop from a finger prick).

3. Most of the components used in the diagnostic kit should be stable at room temperature.

4. Exact micropipeting of individual components should be avoided.

Centrifugation and precipitation steps should be avoided.

6. It should require minimum training for successful operation.

By the present invention a detection method is provided which fulfills all these criteria.

The present invention is summarised in the following clauses: 1. A single stranded DNA fragment (f63) having the sequence given below:

AGGTCTTAACATGACTAACTAAGGTCTTAACTTAACTAACTTAGGTCTTACTTTAACTAAACT

or its complimentary strand or the corresponding double stranded sequence. It is preferred to use the single stranded

DNA.

2. A DNA fragment as defined by clause 1 or contiguous segment thereof which is at least greater than 20 bases or base pairs in length.

3. A DNA fragment according to clauses 1 or 2 in single stranded form.

*6 4. A hybridization probe comprising a DNA fragment as defined in clause 1 and 2.

5, A hybridization probe according to clause 4 which is S labelled by a group capable of colourimetric detection. The nature of this group is not critical for this invention.

6. A hybridization probe according to clause 5 wherein the labelled group for colourimetric detection is biotin. Biotin is a preferred reporter group.

*5 4 7. A hybridization probe according to clause 5 wherein the S" labelled group for colourimetric detection is a chromophoric reporter group.

8. A method for detecting a pathogen present in blood or other body fluid comprising of the following steps: a) Lysing a blood sample in a solution containing Guanidine hydrochloride (GuHC1), Sodium lauryl sareosine (SLS) and Triton-X-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 the said pathogen.

c) Capturing the hybrids formed in step 8 in a microtitre plate coated with a hybridization probe which has a nucleotide sequence capable of hybridizing to the same strand of genomic DNA that the hybridization probe used in step 8 binds. In a preferred embodiment, especially for detection of P.falciparum, the nucleotide sequence used in coating the microtiter plate is identical to the sequence of *1'4 the hybridization probe used in step 8(b).

d) Washing the microtitre plate with a solution comprising Standard Saline Citrate (SSC), Sodium dodecyl Sulfate (SDS) and Triton-X-100.

e) Detection of the presence of the hybrids by 2 colourimetric methods.

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W *e 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 clauses 8 and 9 wherein the final concentration of the reagents in step 8 are as follows: a) Guanidine hydrochloride Between 1.OM b) Sodium lauryl sarcosine 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 intervals.

11. A method according to clauses 8 and 9 wherein the final concentration in step 8 are as follows: a) Standard Saline Citrate 0.5 X 2.5 X SSC b) Triton-X-100 0.2% 0.5% V/V c) Sodium dodecyl Sulphate 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.

4 0 13. A method according to clauses 8 to 12 wherein 2X SSC is used to remove nonspecific hybrids.

14. A method according to clauses 8 and 14 wherein Triton- X-100 and SDS are used for the removal of the colouring material originating from the blood.

15. A method according to clauses 8 to 14 wherein the T Ob pathogen is P.falciparum.

0 I C 16. A method according to clauses 8 to 1- wherein the pathogen is P.vivax.

17. A method accordinc; to clauses 8 to 14 wherein the pa-hogen is Shigella.

1b. A method according to clauses 8 to 14 wherein the pathogei is Mycobacterium tuberculosis.

19. A diagnostic kit for the detectior of a given nucleotide sequence present in c, target polynucleotide sequence on the basis of the method according to clauses 8 to 18.

PRINCIPLE OF DNA BASED SANDWICH HYBRIDISATION Background: In the non radioactive format the final mode of detection is the development of a colour either soluble or insoluble depending on the nature of the substrate used in the reaction catalysed by either alkaline phosphatase or horse radish peroxidase.Therefore it is essential to remove the residual coloured material from the target DNA as well as inactivating the endogenous enzyme. This makes spotting blood directly 10 onto membrane filters (as is done in the radioactive O hybridisation format) useless since the removal of residual

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Sblood stains from the filter is almost impossible.To *0 circumvent this problem we have used the microtitre plate format coupled with sandwich hybridisation, the basic principle of which is described below.

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It has been shown earlier that one of the characteristics of P.falciparum genome is that it contains a 21 base pair repeat

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that is present in tandem in a large region of the genome The fraction of the genome represented by this

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'0 repeated sequence is about Comparisons of several clones containing this repeat sequence have indicated a consensus 21 base pair repeat sequence. Based on this consensus sequence we have designed and constructed a 63 mer oligonucleotide probe (designated f63 hereafter). It consists of three 21 mers in tandem which are maximally represented in the repeated sequences of the P.falciparum DNA (Fig.1). The preferred use of single stranded DNA as a probe and its said length is based on the following reasoning. Single stranded DNA is superior to double stranded as a probe because it hybridises only to the target DNA. In case of double stranded DNA there is a greater probability of self hybridisation thus reducing the effective concentration of the probe that is 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 amount for hybridisation. Of the several methods that are available to 20 make single stranded DNA, oligonucleotide synthesis is most *5* convenient.

For detection of pathogens other than P.falciparum, one has to design an optimal DNA probe which is repeated in the pathogen DNA. This hybridization probe can then be used in a '25 similar detection protocol of sandwich hybridization which is given below specifically for P.falciparum.

*0* The basic protocol for the sandwich hybridisation is given below.( Also explained pictorially in Fig.2) FLOW CHART FOR MON RADIOACTIVE DIAGNOSIS OF' P-FLCIPARUX INFECTION IN HUM BLOOD BY SANDWICS HYB&D~8SATION Add one drop of blood sample (50 ul) from a finger prick in a small plastic tube containing Lysing solution and bio-f63 probe Mix well Phase 1 (ii) Heat in Boiling water bath for two minutes (iii) Leave at room temperature for a minimum of four hours Parasite DNAbio-f63 probe hybrid See Fig.l. Plate A *0 o' 0 Transfer the mixture from the tube in Phase 1 to wells in a a microtitre plates precoated with unlabelled f-63 probe .Phase 2 (See Fig.l. Plate B) (ii) Allow to stand at room temperature for overnight.

CAPTURED

HYBRID

See Fig.l. Plate C Wash microtitre plate wells from phase 2 with 2 x SSC containing 0.2% SDS and 0.2% S. Triton x 100 Phase 3 (ii) Repeat wash procedure four times each time let stand wash buffer for 5 minutes.

(iii) Let stand in each well APB-1 solution for 30 minutes at room temperature.

(iv) Add one drop of APB-1 containing streptavidin Alkaline phosphatase conjugate.

Let stand for 30 minutes at room temperature.

Phase 4 Captured hybrid ready for detection by colorimetry Fig.l Plate D (vi) Discard solution in the wells.

(vii) Let stand APB-1 solution (without BSA) in each well for minutes and discard solution.

(viii) Repeat above operation four times.

(ix) Rinse each well with APB-2 solution.

Add enzyme substrate in APB-2 solution.

(xi) Let stand at room temperature for atleast 120 minutes.

(xii) Read absorbancy at 410 nm in a microtitre plate reader.

0A %see 0 @0 0000 00 Sr r @0 Colour with absorbancy Phase 5 values higher than 0.2 denotes presence of Analysis of parasite DNA Text Results Absence of parasite DNA should give an absorbancy value of less than 0.1 The success of this method depends on the fact that P.falciparum DNA remains nearly undegraded during the process.In the solution hybridisation step biotinylated f63 binds to P.falciparum DNA and would proceed to near completion, the rate of solution hybridisation being much

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0 *0S* 0D 0 4.* 0 *00 0 eto oo faster when compared to immobilized target DNA. The efficiency of capture hybridisation is directly propo6rtional to the length of the P.falciparun DNA. In the extreme limit it can be seen that if the P. falciparum DNA is totally undegraded then even a meagre 0. 03% of capture hybridisation can bring down all the hybrid complex.

In the case of other pathogens, the efficiency of capture hybridiz~ation will depend on the number of times the probe is repeated in the pathogen genome.

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0S a PROTOCOL FOR NON-ISOTOPIC DETECTION OF P.FALCIPARUX DNA XI BLOOD SAMPLES 1. Preparation of the probe: The probe for coating the microtitre plates: The 63 mer oligonucleotide( f63 was synthesised using the automated DNA synthesizer(Applied Biosystems 340A).

Labeled probe for detecting the the hybrids: Biotinylation of f63 was done by photobiotinylation using photobiotin acetate according to published procedures.

q 10O 2.Coating of microtitre plates: 0 All the wells in the microtitre plate (Dynatech, Polyvinyl u chloride) are coated with varying amounts(lug to long) of f- 63 in 50 ul volume containing 0.1 M MgC12. The coating is done overnight following which the microtitre plate is exposed under germicidal UV lamp (40 watts) at a distance of cms, for 5 minutes to immobilise DNA. The contents of the wells are discarded subsequently and the wells are washed with 2X SSC buffer. Unoccupied sites sites in each of the wells are blocked by carrying out prehybridisation in a .*20 buffer (200 ul/well) containing 2X SSC, 5X Denhardts, Triton-X-100, 0.5% SDS and 50 ug/ial salmon sperm DNA. The prehybridisation is carried out for 4-6 hours at room temperature. The coated plates can be stored at this stage in room temperature 3. Collection of blood samples and solution hybridisation: Blood samples (50 ul aliquots) are collected from a finger prick, directly into 50 ul of a solution containing 4M guanidine hydrochloride (Gu HC1), 0.5% sodium lauryl sarcosine (SLS) and 0.5% Triton-X-100. This solution also contains 5 ng of oligonucleotide probe (biotinylated f-63).

This mixture is heated for 5 minutes at 95 deg C and then kept at room temperature for 4-6 hours for the solution hybridisation to occur.

4. Capture hybridisation: After solution hybridisation is over, the contents of the 9 eppendorf tubes are transferred into the wells of the icrotitre plate that are precoated with unlabelled f-63.

This sandwich hybridisation(capture) is allowed to go for 24 hours. During this phase, hybridisation occurs between the f-63 coated onto the plate and the rest of the complementary sites available in the hybrid. The hybrid is a long piece of target DNA carrying the biotinylated f-63 in certain locations leaving behind other complementary sites. (See Fig: 2).

5. Colour development: After the sandwich hybridisation is over, the contents of the wells of the microtitre plate are discarded and wells are washed with a solution containing 2X SSC, 0.2% SDS and 0.2% Triton X-100, four times, five minutes each at room temperature. During this post hybridisation wash, all the coloured materials are removed leaving behind the sandwich hybrid. The wells are then blocked with A.P 7.5 which is a solution containing 1M NaCl, 100 mM Tris-cl pH 7.5, 2 mM MgC12, 0.05% Triton-X-100 and 3% BSA, for 30 minutes at room temperature.

The sandwich hybrids are then detected by using for example, Streptavidin-alkaline phosphatase conjugate. The Streptavidin alkaline phosphatase conjugate(l ug/ml) is added to A.P buffer. 50 ul of this solution (AP 7.5 buffer containing streptavidin alkaline phosphatase) is added to each well and incubation continued for another 30 minutes at room 0o0 temperature. The excess unbound conjugate is removed by washing four times, five minutes each, at room temperature with A.P 7.5 buffer without BSA.

Finally the wells are rinsed with A.P incubation buffer containing 100mM Tris-Cl pH 9.5, 100 mM NaCI and 50 mM MgC12). 50 ul of the substrate p-nitrophenyl o*o0 phosphate is added to A.P 9.5 at a concentration of Img/ml ego 0 and 50 ul of this solution is added to each well. The color development is allowed to take place for 6-12 hours. The absorbance (at 410 are recorded, using a suitable plate reader( e.g. Dynatech plate reader).

The Test results are given in the following table.

RESULTS:

Table I 80MIT IDTh (Absorbance at 410 mm) Amount of DNA Amoun M gote n microbactgria plate (T9/106* DNA) l ug 500 ng 100ng l0ong *500 ng over over over over 250 rig o .E 125 rig :'ao 63 ng 31lng 16 rig 1.524 rig 1.511 1.242 1.373 0.929 1 ug 0.291 0.295 0.263 0.214 *Human DNA *T9/106 represent a chioroquine resistant P.falciparum clone.

Note :In human samples 50 ul of blood has about Song of parasite (P.falcipaxrum) DNA if the infection is about 1%.

The term "over" indicates an optical density above FIGURE LEGENDS Fig.l shows the oligo f63 that was designed from the consensus repeated sequence (21 base repeat) of P.falcipaium.

Legend to Fig. 2 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.

Fig. 3 depticts: 0 A: biotinylated f63 DNA B: genomic P.falciparum DNA C: f63 DNA The matter contained in each of the following claims is to be read as part of the general description of the present invention.

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C C Ref erences I. seroepidimiology of Human Malaria A multicentric study. Malaria Research Centra (ICKR) (1987) 2. Barker,R.H.Jr., Suebsaens, Rooney, Alecrin,G.C., Dourado, and Wirth,D.F. (1986) Science M, 1434.

3. Langer Waldrop A.A. and Ward,D.C. (1981), Proc.Natl. Acad. Sci. USA 11, 6633.

4. Forster Mclnnes,J.L., Skingle, D.C. and Symons, R.H. (1985) Nucleic Acids Res, 1l.2, 745.

Aslund, Franzen.L., Westin, Persson.T., Wigzell.H., and Pettersson.U. (1985) J.Mol.Biol. 18, 509.

4,00006. Francis. Ayyanathan. Bhat.P., Srinivasa.H. and Padinanaban.G. (1988). Indilan J Biochem. Biophys 0 537.

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Claims

1. A single stranded DNA fragment (f63) having the sequence given below: AGGTCTTAACATGACTAACTAAGGTCTTAACTTAACTAACTTAGGTCTTACTTTAACTAAACT or its complimentary strand or the corresponding double stranded sequence.

2. A DNA fragment as defined in claim 1 or contiguous segment thereof which is at least greater than 20 bases or base pairs in length. o. 3. A DNA fragment according to claim 1 or 2 in single stranded form.

4. A hybridization probe comprising a DNA fragment as defined in claim 1 or 2. A hybridization probe according to claim 4 which is labelled by a group capable of colourimetric detection. •Oee 6. A hybridization probe according to claim 5 wherein the labelled group for colourimetric detection is biotin. 7 6. A hybridization probe according to claim 5 wherein the labelled group for colourimetric detection is biotin .0 7. A hybridization probe according to claim 5 wherein the labelled group for colourimetric detection is a chromophoric reporter group.

8. A method for detecting a pathogen present in blood or other body fluid comprising the following steps: a) Lysing a blood sample in a solution containing GuHC1, SLS and Triton-X-100; b) Denaturing the DNA present in the said blood sample and performing solution hybridization in the presence of a hybridization probe which hybridizes with DNA of the said pathogen; c) Capturing the hybrids formed in step 8(b) on a microtitre plat, coated with a hybridization probe which has a nucleotide sequence capable of hybridizing to the same strand of genomic DNA that the hybridization probe used in step 8b, binds; d) Washing the microtitre plate with a solution comprising SSC, SDS and Triton-X-100; and e) Detecting the presence of the hybrids by S colourimetric methods. 00.

9. A method according to claim 8 wherein the denaturing step 8b is carried out by heating. A method according to claim 8 or 9 wherein the hybridization probe used in coating the microtitre plate has 't the same nucleotide sequence as the hybridization probe used in step 8b.

11. A method according to claim 8 wherein the hybridization probe is as defined in claim 2 or 3. 6. 12. A method according to any one of claims 8 to 11 wherein the final concentration of the reagents in step 8 (a) are as follows: a) Guanidine hydrochloride Between 1.OM b) Sodium lauryl sarcosine Between 0.2% 0.5% W/V c) Triton-X-100 Between 0.2% 0.5% V/V

13. A method according to claim 8 or 12 wherein the final concentration of the reagents in step 8 are as follows: a) Standard Saline Citrate 0.5 X 2.5 X SSC b) Triton-X-100 0.2% 0.5% V/V c) Sodium dodecyl Sulphate 0.2% 0.5% W/V

14. A method according to any one of Claims 8 to 13 wherein the lysing solution is used both as a solubilising agent and as hybridization solution. A method according to any one of claims 8 to 14 wherein 2X SSC is used to remove nonspecific hybrids.

16. A method according to any one of claims 8 to wherein Triton-X-100 and SDS are used for the removal of the colouring material originating from the blood.

17. A method according to any one of claims 8 to 16 S wherein the pathogen is P.falciparum. S 18. A method according to any one of claims 8 to 16 wherein the pathogen is P.vivax.

19. A method according to any one of claims 8 to 16 S wherein the pathogen is Shigella.

20. A method according to any one of claims 8 to 16 wherein the pathogen is Mycobacterium tuberculosis. '21. A diagnostic kit for the detection of a given1 nucleotide sequence in a target polynucleotide sequence, comprising components which enable the method according to any one of claims 8 to 20 to be carried out. DATED this 1st day of Marc.h, 1991 AKTIEBOLAGET ASTRA, By its Patent Attorneys, E. F. WELLINGTON CO., Byt BRUCE S. WELLINGTON