EP0135108A2
Expired / Lapsed
EP
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Nucleotide hybridization assay for protozoan parasites
Assignee: Rockefeller University
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Filing: 1984-08-07
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Granted: —
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Expires: 2004-08-07
Abstract
Abstract The present invention provides a method for detection of protozoan parasites in blood or other specimen from their mammalian hosts. The method comprises nucleic acid hybridization of repetitive nuclear DNA fragments of the parasites. Hybridization probes have been prepared for this purpose by cloning the repetitive nuclear elements that are species-specific in appropriate vectors. The sensitivity of these probes has been increased by further sub-cloning to make them capable of cascade hybridization. The assay is highly specific and sensitive for detection of disease-causing protozoan parasites of the commonly occurring Trypanosomatidae of the genus Leishmania and genus Trypanosoma, as well as for malaria-causing protozoan and other parasitic microorganism of mammals.
Inventors
- 👤 Paul Lizardi
- 👤 Nadia Nogueira
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-31 07:54
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/44; C07K14/445; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C12Q1/6888; C12Q1/6893; Y; Y02; Y02A; Y02A50/00; Y02A50/30
Claims Analysis
Total Claims: 65
Claim 1
Independent Claim
38 words
Method for detecting protozoan parasites in a biological sample from a vertebrate comprising contacting said sample with a labeled hybridization probe recognizing species-specific, repetitive nuclear DNA in said parasite and detecting said labeled probe bound to said parasite.
Claim 2
Dependent Claim
38 words
Method for detecting protozoan parasites in a biological sample from a vertebrate comprising contacting said sample with a labeled hybridization probe recognizing species-specific, repetitive nuclear DNA in said parasite and detecting said labeled probe bound to said parasite.
Claim 3
Dependent Claim
31 words
Method of Claim 1 wherein said hybridization probe comprises DNA or RNA having a nucleotide sequence corresponding to or complementary to all or a portion of said species-specific repetitive nuclear DNA.
Claim 4
Dependent Claim
14 words
Method of Claim 2 wherein said hybridization probe is DNA isolated from said parasite.
Claim 5
Dependent Claim
11 words
Method of Claim 2 wherein said hybridization probe is synthesized chemically.
Claim 6
Dependent Claim
27 words
Method of Claim 2 wherein said hybridization probe is synthesized from the component nucleotides in the presence of appropriate enzymes under conditions suitable reaction between said nucleotides.
Claim 7
Dependent Claim
35 words
Method of Claim 1 wherein said hybridization probe comprises DNA or RNA having a nucleotide sequence corresponding to or complementary to all or a portion of said species-specific repetitive nuclear DNA in an appropriate vector.
Claim 8
Dependent Claim
25 words
Method of Claim 6 wherein said vector is selected from the group consisting of F, coli plasmids, filamentous phages, lamboid bacteriphages, salmonella phages and yeast.
Claim 9
Dependent Claim
12 words
Method of Claim 7 wherein said vector contains salmonella SP6 phage promoter.
Claim 10
Dependent Claim
12 words
Method of Claim 7 wherein said E. coli plasmid is PUC 13.
Claim 11
Dependent Claim
13 words
Method of Claim 1 wherein said hybridization probe is capable of cascade hybridization.
Claim 12
Dependent Claim
30 words
Method of Claim 1 for detection of T. cruzi wherein said hybridization probe is capable of recognizing the approximately 200 base pair nuclear DNA fragment in T. cruzi nuclear genome.
Claim 13
Dependent Claim
17 words
Method of Claim 11 wherein said hybridization probe comprises said DNA or RNA in a suitable vector.
Claim 14
Dependent Claim
12 words
Method of Claim 12 wherein said vector is an E. coli plasmid.
Claim 15
Dependent Claim
10 words
Method of Claim 13 wherein said vector is PUC 13.
Claim 16
Dependent Claim
30 words
Method of Claim 1 for detection of Plasmodium falciparum wherein said pybridization probe is capable of recognizing the approximately 200 base pair nuclear DNA fragment in plasnodium falciparum nuclear genome.
Claim 17
Dependent Claim
17 words
Method of Claim 15 wherein said hybridization probe comprises said DNA or RNA in a suitable vector.
Claim 18
Dependent Claim
12 words
Method of Claim 15 wherein said vector is an E coli plasmid.
Claim 19
Dependent Claim
10 words
Method of Claim 17 wherein said vector is PUC 13.
Claim 20
Dependent Claim
20 words
Method of Claim 1 for detection of T. brucei gambiense wherein said hybridization probe is capable of recognizing Sloof DNA.
Claim 21
Dependent Claim
11 words
Hybridization probe recognizing species-specific repetitive nuclear DNA of a protozoan parasite.
Claim 22
Dependent Claim
11 words
Hybridization probe recognizing species-specific repetitive nuclear DNA of a protozoan parasite.
Claim 23
Dependent Claim
14 words
Hybridization probe of Claim 20 labelled with a radioactive element or a chromophoric group.
Claim 24
Dependent Claim
11 words
Hybridization probe of Claim 21 wherein said chromophoric group is biotin.
Claim 25
Dependent Claim
26 words
Hybridization probe of Claim 20 comprising DNA having a nucleotide sequence corresponding to all or a portion of the species-specific repetitive nuclear DNA in said parasite.
Claim 26
Dependent Claim
13 words
Hybridization probe of Claim 23 wherein said DNA is isolated from said parasite.
Claim 27
Dependent Claim
11 words
Hybridization probe of Claim 23 wherein said DNA is chemically synthesized.
Claim 28
Dependent Claim
28 words
Hybridization probe of Claim 23 wherein said DNA is synthesized from the appropriate nucleotides in the presence of appropriate enzymes under conditions suitable for reaction between said nucleotides.
Claim 29
Dependent Claim
27 words
Hybridization probe of Claim 20 comprising DNA or RNA having the complementary sequence of all or a portion of the species-specific repetitive nuclear DNA in said parasites.
Claim 30
Dependent Claim
32 words
Hybridization probe of Claim 20 comprising DNA or RNA having a nucleotide sequence corresponding to or complementary to all or a portion of said species-specific repetitive nuclear DNA in an appropriate vector.
Claim 31
Dependent Claim
27 words
Hybridization probe of Claim 28 wherein said vector is selected from the group consisting of E. coli plasmids, filamentous phages, lamboid bacteriopha- ges, salmonella phages, and yeast.
Claim 32
Dependent Claim
13 words
Hybridization probe of Claim 29 wherein said vector contains salmonella SP6 phage promoter.
Claim 33
Dependent Claim
11 words
Hybridization probe of Claim 29 wherein said plasmid is PUC 13.
Claim 34
Dependent Claim
19 words
Hybridization probe of Claim 31 wherein said DNA, complementary DNA or RNA is in tandem array in said vector.
Claim 35
Dependent Claim
18 words
Hybridization probe of Claim 28 comprising repeated segements of said DNA, complementary DNA or RNA in said vector.
Claim 36
Dependent Claim
46 words
Hybridization probe of Claim 20 for Plasmodium falciparum comprising DNA having all or a portion of the sequence of the approximate 340 or 1200 bp sequenced nuclear DNA fragments of Plasmodium falciparum DNA or RNA complementary to all or a portion of said sequenced nuclear fragments.
Claim 37
Dependent Claim
9 words
Hybridization probe of Claim 34 in an appropriate vector.
Claim 38
Dependent Claim
13 words
Hybridization probe of Claim 35 wherein said vector is an E. coli plasmid.
Claim 39
Dependent Claim
12 words
5 37. Hybridization probe of Claim 45 comprising pTCNRE and pTC-multi NRE.
Claim 40
Dependent Claim
13 words
Hybridization probe of Claim 34 wherein said nuclear DNA fragment has the sequence:
Claim 43
Dependent Claim
20 words
Hybridization probe of Claim 38 wherein said sequence exhibits mini-variation of about 5 to 20 % at each base pair.
Claim 44
Dependent Claim
48 words
Hybridization probe of Claim 20 for Plasmodium falciparum comprising DNA having all or a portion of the sequence of the approximate 340 or 1200 bp sequenced nuclear DNA fragments of Plasmodium falci p arum DNA or RNA complementary to all or a portion of said sequenced nuclear fragments.
Claim 45
Dependent Claim
9 words
Hybridization probe of Claim 40 in an appropriate vector.
Claim 46
Dependent Claim
13 words
Hybridization probe of Claim 41 wherein said vector is an E. coli plasmid.
Claim 47
Dependent Claim
9 words
Hybridization probe of Claim 41 comprising pPFR-1 and pPFR-6.
Claim 48
Dependent Claim
13 words
Hybridization probe of Claim 40 wherein said nuclear DNA fragment has the sequence:
Claim 49
Dependent Claim
13 words
Hybridization probe of Claim 40 wherein said nuclear DNA fragement has the sequence:
Claim 50
Dependent Claim
8 words
DNA having the nucleotide sequence: or fragments thereof.
Claim 51
Dependent Claim
8 words
DNA having the nucleotide sequence: or fragments thereof.
Claim 52
Dependent Claim
8 words
DNA of Claim 46 isolated from T. cruzi.
Claim 53
Dependent Claim
10 words
DNA of Claim 46 synthesized in vitro chemically or ciochemically.
Claim 54
Dependent Claim
9 words
DNA or RNA complementary to DNA of Claim 46.
Claim 55
Dependent Claim
8 words
DNA of Claim 46 in an appropriate vector.
Claim 56
Dependent Claim
8 words
DNA having the nucleotide sequence: or fragments thereof.
Claim 57
Dependent Claim
8 words
DNA having the nucleotide sequence: or fragments thereof.
Claim 58
Dependent Claim
8 words
DNA of Claim 51 isolated from Plasmodium falciparum.
Claim 59
Dependent Claim
10 words
DNA of Claim 51 synthesized in vitro chemically or biochemically.
Claim 60
Dependent Claim
9 words
DNA or RNA complementary to DNA of Claim 51.
Claim 61
Dependent Claim
8 words
DNA of Claim 51 in an appropriate vector.
Claim 62
Dependent Claim
8 words
DNA having the nucleotide sequence: or fragments thereof.
Claim 63
Dependent Claim
8 words
DNA having the nucleotide sequence: or fragments thereof.
Claim 64
Dependent Claim
8 words
DNA of Claim 56 isolated from Plasmodium falciparum.
Claim 65
Dependent Claim
10 words
DNA of Claim 46 synthesized in vitro chemically or biochemically.
Claim 66
Dependent Claim
9 words
DNA or RNA complementary to DNA of Claim 56.
Claim 67
Dependent Claim
8 words
DNA of Claim 56 in an appropriate vector.
References Cited (Backward Citations)
No citation records indexed.
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