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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
• • 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
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  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/81—Packaged device or kit

Definitions

• the present invention relates to a method and kit for the diagnosis of Schistosomiasis in human sample matter by Polymerase Chain Reaction (PCR) .
• the method is based on the detection of the parasite DNA and is especially useful in cases of low infection intensity, for which parasitological stool tests (Kato-Katz) demonstrate little sensitivity.
• Schistosomiasis is a disease caused through infection by parasites of genus Schistosoma, with Schistosoma mansoni, Schistosoma japonicum and Schistosoma haematobiom being the principal infectant species.
• the disease has been recorded for 4000 years and still constitutes an important problem for public health, affecting more than 200 million people in undeveloped countries.
• the species S . mansoni is endemic in 54 countries and territories of South America, the Caribbean, Africa and the eastern Mediterranean region.
• the method of diagnosis constitutes a primordial instrument for the programs to control the. endemic. It is highly desirable to develop techniques that are more efficient than those presently available.
• the diagnosis of the infection can be done through confirmation by microscopy of the presence of parasite eggs in the faeces or urine, depending on the species.
• the Kato- Katz method (KATZ, N., A. Chaves, J. Pellegrino, 1972, A simple device for quantitative stool thick smear technique in Schistosomiasis mansoni. Rev. Inst. Med. Trop. Sao Paulo. 14: 337-340) is the technique that offers the best conditions of efficiency, cost and operation; (WHO, 1994, The Control of schistosomiasis. Technical Report Series, 830, Geneva, 86pp.).
• the serological diagnosis constitutes an alternative to the parasitological ' test, being, after the latter, the second most widely employed.
• antibodies produced by infection with S . mansoni are detected by techniques such as the reaction of indirect immunofluorescence and ELISA (Enzyme Linked ImmunoSorbent Assay) .
• this technique has the disadvantage of not distinguishing between active infection and past infection.
• the low specificity of antibodies against S. mansoni in the absence of active infection is explained by crossed reactivity with other parasites (Correa-Oliveira R, Dusse LMS, Viana IRC, Colley DG, Carvalho OS, Gazzinelli G 1988. Human antibody response against schistosomal antigens. Am J Trop Med Hyg 38: 348-355) by the presence of unisexual infections, by contact with other cercariae, by the transfer of maternal antibodies and by the persistance of antibodies after a successful previous treatment.
• Schistosoma mansoni Another alternative for the diagnosis of Schistosoma mansoni consists in detecting antigenic substances released by the parasite, allowing to differentiate between past and active infection and eliminating the problem of the unspecificity of the antibody diagnostic.
• the search for circulating antigens presents other disadvantages, such as low sensitivity to light ' infections, the high cost, difficulty of operation and a dependency on the production of monoclonal antibodies (DE Jonge,N., A.L.T.Rabello, F.W.Krijger, P.G.Kremsner, R.S.Rocha, N.Katz e A.M. Deelder.1991.
• PCR Polymerase Chain Reaction
• Document EP 550 8'83 describes an experiment using PCR to detect, in faecal matter, the presence of the DNA of protozoan G. lamblia .
• the procedure was carried out using, as target, the sequence of the gene RNA 18S of G. lamblia .
• the applicant designed oligonucleotide primers sufficiently complementary to a region of the target sequence of the ribosomic RNA 18S of G. lamblia .
• Document WO 94/04681 is related to the development of specific oligonucleotides to be used as primers of the PCR for the detection of the DNA sequence encoding the protein of the wall of Cryptosporidium sp. , and thus as a kit for the diagnostic of parasites of this genera.
• the document WO 93/03167 which is an application of a process for the isolation, storage and cleavage of DNA capable of furnishing it in an adequate form for amplification through a specific sequencial process, such as PCR.
• the process is particularly employed ' for the detection of parasitic diseases caused by micro-organisms having concatenated closed circular kinetoplast DNA such as T. cruzi, Leishmania sp . , P. falciparum, P. vivax and P. malariae .
• RNA arbitrarily primed PCR allows the accelerated generation of expressed sequence tags.
• the primers described in the present invention were designed based on the S. mansoni sequence originally described by Hamburger et al . shown in Figure 1, (Hamburguer, J, Turetski, T, Kapeller, I & Deresiewicz, R. 1991. Highly repeated short DNA sequences in the genome of Schistosoma mansoni recognized by a species-specific probe. Molecular and Biochemical Parasitology 44: 73-80).
• the design of the primers was done after the visual inspection of the original sequence, so as to avoid the presence of complementary regions within each initiator or between both primers.
• the primers were chosen in a manner so as to amplify a short sequence. Also, all the stages of the present invention were strictly standardised in a manner as to function satisfactorily with chemically complex biological samples such as faeces and serum.
• the object of the present invention is the detection of Schistosoma sp . in biological samples by PCR.
• a first embodiment of the present invention refers to a method for the diagnosis of Schistosoma through the amplification by PCR of a DNA sequence of Schistosoma sp. .
• the method of the present invention is characterised by the stages of:
• stage (c) amplify a region of the Schistosoma DNA extracted in stage (b) 'with specific primers constructed from the original sequence described in ID SEQ n.l; (d) separate the products of the amplifications of stage (c) by electrophoresis, followed by detection using appropriate colouring methods.
• the invention is directed to a diagnostic kit to be used in the detection of Schistosoma
• a basic kit includes all the reagents necessary for carrying out the PCR technique, namely: specific primers, nucleotides and appropriate buffer solution for the amplification by PCR.
• the kit may contain the enzyme Taq polymerase in quantities sufficient for amplification, standard DNA to be used as positive control of the reaction, buffer solution of the sample to prepare the amplified material for electrophoresis and protocol and instructions manual for the user.
• FIGURE 1 Shows the DNA sequence of the repeater unit of S. mansoni . It indicates, in bold type, the localisation of the specific primers described in the present invention.
• FIGURE 2 Demonstrates the sensitivity of the PCR in the detection of the S. mansoni DNA.
• FIGURE 3 Shows the capacity of the PCR technique of the present invention to amplify the DNA of various species of Schistosoma sp. , including the three species of greater clinical-epidemiological importance in the world: S. mansoni , S. japonicum and S. heamatobium.
• FIGURE 4 Shows the specificity of the PCR of the present invention.
• FIGURE 5 Illustrates the comparison in terms of sensitivity between the PCR of the present invention and the parasitological test for the diagnosis of S. mansoni in human faeces .
• FIGURE 6 Shows the detection, by PCR, of S. mansoni DNA in faecal samples of infected patients.
• FIGURE 7 Shows the detection, by PCR, of S. mansoni DNA in samples of human serum. DETAILED DESCRIPTION OF THE INVENTION
• Primers single strand synthetic oligonucleotide, normally used in pairs in hybridisation with strands complementary to a DNA section. The inner extremities of the primer/DNA template complex are used by the DNA polymerase as points of initiation of the synthesis in a PCR.
• PCR Polymerase Chain Reaction
• An inadequate choice of primers may produce various undesirable effects, such as: impossibility of amplification, amplification at various sites, formation of primer dimers, amongst others, rendering the amplification reaction non-informative.
• the object of the present invention is accomplished by the amplification, by PCR, of a specific region of the Schistosoma sp . geno a and latter separation by electrophoresis of the products of this amplification, followed by appropriate colouring techniques that permit an adequate visualisation of the DNA in the gel including, but not limited to: colouring by silver salts, radioisotopes and enzymes combined with substrates that permit their detection, without the undesirable effects mentioned above.
• the method of the present invention may be employed for the detection of the DNA of the genera Schistosoma in any biological sample that contains cells or DNA free of the parasite having sufficient integrity to be amplified by PCR.
• Some samples such as faeces and urine, need to be submitted to some kind of treatment so that ' the membranes or envelopes that may eventually enclose the DNA in the cell of the parasite, may be ruptured, releasing the DNA in solution.
• Other samples e.g. the serum of infected people, already contains the free molecules and do not need such treatment.
• the membranes can be ruptured by the use of special chemical substances, such as detergents and chaotropic salts, or by using physical and mechanical processes, such as induced osmotic pressure and sonication. After the rupture of the cell membranes, the DNA must be isolated from the other cell molecules, which can also be done by physicochemical processes, such as precipitation by ethanol or with the use of silica matrixes.
• the DNA may be detected in the sample, after amplification by PCR.
• the DNA must be, preferentially, purified employing standard techniques for the removal of eventual substances that inhibit the amplification reaction.
• the DNA is selectively amplified by Polymerase Chain Reaction.
• PCR Polymerase Chain Reaction
• the reaction is a sequential process that requires at least two primers, small sequences of DNA complementary to the parasite DNA, that will be extended enzymatically, presenting a faithful copy of this DNA. Adding large quantities of primers, together with other necessary reagents, millions of copies of the parasite DNA are obtained.
• the primers of the PCR employed here were specially designed for this invention, based on the original highly repetitive sequence of the S . mansoni genoma as described in ID SEQ n.l and also illustrated in Figure 1.
• primers may be constructed having nucleotide sequences that are functionally equivalent in relation to ID SEQ n.2 and ID SEQ n.3. Such sequences are termed equivalent if functionally the corresponding biopolymers can perform the same role, without being identical, in view of the usage or purpose considered.
• the equivalent sequences may be the result of variability, as such, any modification in a sequence, spontaneous or induced, whether by substitution and/or deletion and/or insertion of nucleotide, and/or extension and/or shortening of the sequence at one of its extremities.
• An unnatural variability can result from genetic engineering techniques.
• Each primer of the pair is, preferentially, constructed in a manner as to be substantially complementary to a different strand of the sequence that flanks the specific sequence of the Schistosoma sp. genoma to be amplified.
• a primer from each pair is sufficiently . complementary to be able to hybridise with a part of the sequence in the sense and the other primer of each pair is also sufficiently complementary to hybridise with a different part of the same sequence in the antisense.
• the sequence of the primer not necessarily needing to mirror the exact sequence of the template, the closer the terminal 3' sequence corresponds to the exact sequence the better the link during the matching stage of the polymerase chain reaction.
• the primers may be prepared by any appropriate method known to those skilled in the art and include, for example, cloning and digestion of adequate sequences and direct chemical synthesis.
• the product from the amplification promoted by PCR is a fragment, or a series of DNA fragments of different sizes which can be separated through electrophoresis, followed by appropriate techniques of colouring which allow adequate visualisation of the DNA in gel. Due to the great specificity of PCR, the amplified DNA of the parasite can be differentiated from the other products of the amplification based on its specific size. In this manner, the presence or absence of the infection can be determined, most times, simply by visual analysis of the amplified products, therefore, by the presence or not of the fragment with a weight corresponding to that of the parasite.
• the PCR is employed to amplify and visualise a specific fragment of DNA originally described in S.
• the amplification of the specific region of the Schistosoma sp. genoma is done by PCR, with the primers specially constructed to be used in the present invention and defined in Table 1 or functionally equivalent sequences, therefore, that are capable of amplifying the sequence of S . mansoni or homologous sequences of other species of Schistosoma .
• Table 1 Primers employed in the reaction by PCR for the amplification of the highly repetitive region of the Schistosoma sp . genoma.
• the kit of the present invention includes all the reagents necessary to allow the detection of any infection caused by helminths of the genera Schistosoma .
• the kit consists of specific primers for a specific region of the Schistosoma sp. genoma as shown in Table 1 or functionally equivalent sequences and, furthermore, reagents and additives normally used in the PCR technique are also supplied, e.g. appropriate nucleotide, such as ' dGTP (desoxyguanidine-triphosphate) , dATP (desoxyadenosine- triphosphate) , dCTP (desoxycitidine-triphosphate) and dTTP (desoxytimidine-trisphosphate) ; appropriate buffer solution (e.g.
• the eggs of S. mansoni were extracted from the livers of mice previously infected with 100 cercariae, and stored in 0.9% saline solution at -20°C until being used (Pellegrino e Siqueira, 1956, Rev. Bras. Malar. 8: 589).
• 10 ⁇ l of the saline solution containing 100.000 eggs/ml was mixed with 90 ⁇ l of distilled water and the final solution submitted to 5 minutes of agitation in a mechanical mixer. This mixture, containing ruptured eggs, was used directly in the extraction of the DNA.
• 100 ⁇ l of the ruptured egg solution was diluted in 200 ⁇ l of buffer containing 10 mM of Tris-base, pH 8.0; 270 mM of EDTA, pH 8.0; 1% of Sodium Dodecyl Sulphate (SDS) ; 1% of polyvinylpolypyrrolidone (PVPP) .
• This mixture was incubated at 95°C for 20 minutes, with a rapid manual agitation after the first 10 minutes of incubation and, then centrifuged for 10 minutes at 8000 x g, at room temperature.
• the DNA contained in the skim was precipitated with ethanol, the skim was removed and the precipitate was incubated for 15 minutes at 37°C for the evaporation of the.
• the amplification reaction involved denaturation of the double-stranded parasite DNA at 95°C for 45 -seconds and primer annealing at 63°C for 30 seconds. These two steps were repeated sequentially in 35 consecutives cycles. In the first cycle, the denaturation step was prolonged for five minutes, in order to assure complete denaturation, and at the last cycle an additional step, at 72°C for 2 minutes, was included to finalise the extension of the remaining annealed primers.
• Figure 2 shows the electrophoretic pattern obtained from the amplification of the DNA of S . mansoni and, also, the maximum sensitivity obtained for the detection of this DNA.
• lane M is the molecular weight marker; in lanes 1 to 5: 20, 10, 5, 1 and 0.5 fg of DNA, respectively. The reaction by PCR was capable of detecting down to 1 fg of S. mansoni DNA.
• Figure 3 shows the electrophoretic pattern obtained after the amplification of the DNA of five other species of genus Schistosoma, amplified with the same primers and in the same PCR conditions as used for S. mansoni .
• Lane M molecular weight marker; lane 1: DNA of S . mansoni; lane 2: S.
• lane 3 S. japonicum (strain from the Phillipines)
• lane 4 S . japonicum (strain from Japan)
• lane 5 S. ma tthei
• lane 6 S. bovis
• lane 7 S . leipperi
• lane 8 negative control.
• the PCR reaction was capable of amplifying the DNA of all the species of Schistosoma sp . tested.
• Figure 4 shows the attempt to amplify the DNA of worms of other genera, also under the same conditions used for S. mansoni .
• Lane M molecular weight marker
• lane 1 positive control (DNA of S.
• Example 2 the eggs contained in the infected faeces (naturally or artificially) were ruptured in the manner, described in Example 1.
• the DNA was then extracted from the faeces by the same technique used for the extraction of DNA from pure S . mansoni eggs (Example 2) .
• Figure 5 shows the products of the amplification of the S. mansoni DNA in faeces artificially infected with the eggs of the parasite. Briefly, 100 mg of a faecal sample of a patient, containing 216 eggs/gramme, was mixed to 900 mg of negative faeces, forming a sample with an expected concentration of approximately 20 eggs/gramme. This procedure was repeated two more times originating faeces samples with 2 and 0,20 eggs/gramme, approximately.
• lane M is the molecular weight marker
• lane 1 positive control (O.lng of S. mansoni egg DNA)
• lanes 2 to 5 amplified DNA of samples containing 200, 48, 4.8 and 0.48 eggs/gramme of faeces, respectively.
• the PCR was capable of detecting the DNA of S . mansoni down to the sample containing approximately 4.8 eggs/gramme, whilst the sensitivity of the Kato-Katz method was of 48 eggs/gramme, therefore, 10 times lower.
• Figure 6 shows the result of the amplification of the S. mansoni DNA in faeces of patients with various concentrations of parasites, previously determined by the Kato-Katz method.
• Lane M molecular weight marker
• lane 1 positive control
• lanes 2 to 9 samples of human faeces containing 0.96; 0.168; 432; 600; 96.912 and 72 eggs/gramme, respectively.
• the result of the PCR was in accordance with those obtained by the parasitological test in all the samples.
• EXAMPLE 5 Detection of ,S . mansoni in the serum of patients.
• the DNA of 4 samples of serum was purified using 100 ⁇ l/sample employing the Glass-max ® DNA isolation spin cartridge system (Life Technologies) , in accordance with the instructions of the manufacturer. 2 ⁇ l of this DNA were then used in the amplification by PCR, in the same conditions described above.
• the serum from persons previously examined by the Kato-Katz method was used, having 2 positive and 2 negative samples.
• Figure 7 shows the result of this amplification.
• lane M is the molecular weight marker
• lane 1 positive control
• lanes 2 to 5 serum of persons containing 0; 96; 0 and 216 eggs/gramme of faeces respectively.

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