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• • C—CHEMISTRY; METALLURGY
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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• 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 routine analyses of a large number of blood and other coloured tissue samples of vertebrates and invertebrates.
• these assays can be used to detect the presence of P.falciparum.
• the procedure described is amenable for application in a wide variety of DNA detection analysis using non radioactive DNA probes.
• 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 novel methods.
• Malaria is caused by protozoan parasites belonging to the genus Plasmodiu .
• 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.
• this parasite also develops resistance to the commonly used antimalarial drugs.
• the current method of diagnosis of malaria is by blood smear 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 antibodies current infections cannot be distinguished from past infections (1). Hence, the search for a new generation of 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 little blood (5-50 ⁇ l) that can be obtained from a finger prick, and should be sensitive and rapid. As few as 50 parasites in 10 ⁇ l of blood can be detected by nucleic acid hybridization (2). Hundreds of samples can be analyzed in a 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 to limit the
• the detection method described by the present invention can be used generally to detect the presence of pathogens, especially blood pathogens, in blood, tissues, samples and body fluids of humans as well as of vertebrates and invertebrates in general such as catties 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.
• pathogens can be mentioned Shigella, e.g. Shigella flexneri, Shigella dysenteriae, Shigella sonnei, and Mycobacteriu ⁇ . tuberculosis.
• 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 necessary in radioactive material handling. Pence there is an u g ent need f zr r.on radioact i ve detection in this field of pathogen detection by nucleic
• 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 and more particularly AGGTCTTAACATGACTAACTA.
• a hybridization probe comprising a DNA fragment as defined in clause 1 and 2.
• a hybridization probe according to clause 4 which is labelled by a group capable of colourimetric detection.
• the nature of this group is not critical for this invention.
• Biotin is a preferred reporter group.
• SUBSTITUTE SHEET labelled group for colourimetric detection is a ⁇ hromophoric reporter group.
• 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 hydro ⁇ hloride (GuHCl), 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 (b) 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 hybridzation probe used in step 8 (b) binds.
• the nucleotide sequence used in coating the microtiter plate is identical to the sequence of the hybridization probe used in step 8 (b).
• SSC Standard Saline Citrate
• SDS Sodium dodecyl Sulfate
• Triton-X-100 Triton-X-100.
• step 8 (a) Guanidine hydrochloride: Between 1.0M - 3.0M b) Sodium lauryl sareosine: Between 0.2% - 0.5% W/v / N/v
• SUBSTITUTESHEET c Triton-X-100: Between 0.2% - 0.5% v/v / v/v The above repie ⁇ e ⁇ t isferred intervals.
• step 8 (e) 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.
• 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 onto membrane filters (as is done in the radioactive hybridisation format) useless since the removal of residual blood stains from the filter is almost impossible. To cirumvent this problem we have used the microtitre plate format coupled with sandwich hybridisation, the basic principle of which is described below.
• P.falciparum genome contains a 21 base pair repeat that is present in tandem in a large 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 concensus 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
• Phase 2 tube in Phase 1 to wells in microtitre plates precoated with unlabelled f-63 probe See Fig. 1. Plate B
• Phase 4 ( vii) Let stand APB-1 solution (without BSA) in each well for 5 minutes and discard solution (viii) Repeat above operation four times
• the efficiency of capture hybridization will depend on the number of times the probe is repeated in the pathogen genome.
• the probe for coating the microtitre plates: The 63 er obligonucleotide (f63) was synthesised using the automated DNA synthesizer (Applied biosyste s 340A) .
• Biotinylation of f63 was done by photobiotinylation using photobiotin acetate according to published procedures.
• All the wells in the microtitre plate (Dynatech, Polyvinul chloride) are coated with varying amounts (l ⁇ g to lOng) of f63 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 10 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 in each of the wells 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 50 ug/ml 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.
• 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 sareosine (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.
• 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, 2nM MgC12, 0.05% Trition-X-100 and 3% BSA, for 30 minutes at room temperature.
• A.P 7.5 is a solution containing 1M NaCl, 100 mM Tris-cl pH 7.5, 2nM MgC12, 0.05% Trition-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 (1 ug/ml) is added to A.P 7.5 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 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.
• A.P 9.5 substrate incubation buffer containing lOOm Tri ⁇ -Cl pH 9.5, 100 mil NaCl and 50 mM MgCl2. 50 ul of the substrate
• SUBSTITUTE SHEET p-nitrophenyl 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 development is allowed to take place for 6-12 hours. The absorbance (at 410 n.m. ) are recorded, using a suitable plate reader (e.g. Dynatech plate reader) .
• a suitable plate reader e.g. Dynatech plate reader
• T9/106 represent a chloroquine resistant P.falciparum clone.
• over indicates an optical density above 2.0.
• Fig. 1 shows the oligo f63 that was designed from the consensus repeated sequence (21 base repeat) of P.falciparum.

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• 1991
  • 1991-07-31 IL IL99025A patent/IL99025A0/xx unknown
  • 1991-08-12 MX MX9100627A patent/MX9100627A/es unknown
  • 1991-08-13 BR BR919105861A patent/BR9105861A/pt not_active Application Discontinuation
  • 1991-08-13 IS IS3742A patent/IS3742A7/is unknown
  • 1991-08-13 AU AU84099/91A patent/AU8409991A/en not_active Abandoned
  • 1991-08-13 CN CN91108921A patent/CN1059910A/zh active Pending
  • 1991-08-13 EP EP91915151A patent/EP0495075A1/en not_active Withdrawn
  • 1991-08-13 WO PCT/SE1991/000533 patent/WO1992003576A1/en not_active Application Discontinuation
  • 1991-08-13 JP JP3514014A patent/JPH05501961A/ja active Pending
  • 1991-08-14 PT PT98687A patent/PT98687A/pt not_active Application Discontinuation

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