Nucleic acid probes specific for Plasmodium vivax and methods of using the same
BACKGROUND OF THE INVENTION AND PRIOR ACT 
     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 the 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. Further, this parasite also develops resistance to commonly used antimalarial drugs. The frequency of the cases caused by P. vivax varies between different countries but generally it is about 50-80%. The occurrence of P. malariae and P. ovale is rare. 
     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. The search for a new generation of diagnostic tests includes the possibility of detecting parasite nucleic acids as indicative of the presence of the parasite. Such a test requires very little blood (5-50 ul) that can be obtained from a finger prick, is sensitive and rapid. As few as 50 parasites in 10 ul of blood can be detected by nucleic acid hybridization (1). 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 geographic spread of malaria. Alternatively, chemoprophylaxis may be adopted in such areas and evaluation of this strategy may be accomplished using nucleic acid hybridization. 
     Double stranded DNA is of complementary nature. Under certain conditions of temperature and salt concentration, the complementary strands of DNA may be denatured or dissociated and reproducibly reassociated. The reassociation, may also occur between DNA and complementary RNA. The reassociating DNA and RNA may be tagged with detection devices (isotopic or non-isotopic) and appropriate detection methods may be employed. In the case of P. vivax, there is no report of any potential DNA probe sequences. The development of specific nucleic acid sequences to detect P. vivax would be important in order to determine the incidence of P. vivax in a geographically defined area. The term `probe` is used to denote a set of DNA sequences obtained biologically or synthetically. 
     Nucleic acid hybridization may be performed on any biological sample suspected of harbouring the parasite. Blood, for example, may be taken directly, solubilized in alkali and spotted on nitrocellulose or similar solid supports by standard methods (2). The DNA is immobilized on the supports and is brought in contact with the specific probe under appropriate conditions of temperature, ionic strength etc (3) which would favour specific reannealing of the probe and target nucleic acids. If the probe carries an isotopic tag (usually  32  P), radioautography is performed to detect positive samples from negative ones. If the probe carries a non-isotopic tag e.g. biotin (4), an enzyme system e.g. avidin-alkaline phosphatase is employed and after a cascade of events colour develops in samples that are positive for the test. 
     In yet another technique (5), blood or the tissue sample to be analyzed may be collected directly in a chaotropic agent such as 4M guanidine thiocyanate. The hybridization process is performed in solution and the mixture is then filtered through a solid support under conditions which facilitate the binding of target-probe hybrids only while excess of probe does not bind to the matrix. Radioautography or colorimetric detection may be performed on this matrix. This hybridization format is especially useful when single stranded RNA probes are used. 
    
    
     DETAILS OF THE INVENTION 
     The present invention describes the identification of specific DNA sequences for P. vivax and the nucleotide sequences of these probes for P. vivax. Based on these characterizations, a diagnostic procedure for the specific detection of P. vivax has been developed employing nucleic acid hybridization. The test is rapid, sensitive and can be used on a large scale for purposes of epidemiological surveys. 
     The present invention relates to: 
     1. The new constructs or plasmids, pARC 117, pARC 145 and pARC 1153, deposited in the form of transformed E. coli at The National Collection of Industrial Bacteria Ltd., Torry Research Station, P.O. Box 31, 135 Abbey Road, Aberdeen, AB9 8DG, Scotland on dates and under deposition numbers as given below: 
     pARC 117: NCIB 40114, E. coli RR1 M15 pARC 117, Feb. 15, 1989 
     pARC 145: NCIB 40110, E. coli RR1 M15 pARC 145, Feb. 7, 1989 
     pARC 1153: NCIB 40108, E. coli RR1 M15 pARC 1153, Feb. 7, 1989. 
     2. The DNA sequences of pARC 117, pARC 145 and pARC 1153 and substructures thereof comprising at least 20 consequtive nucleotides. 
     3. A DNA probe for detection of P. vivax, consisting of the nucleotide sequence identified below for pARC 117. 
     4. A DNA probe for detection of P. vivax, consisting of the nucleotide sequence identified as SEQ ID NO: 1 in the Sequence Listing and identified below for pARC 145. 
     5. A DNA probe for detection of P. vivax, consisting of the nucleotide sequence identified as SEQ ID NO: 2 in the Sequence Listing and identified below for pARC 1153. 
     6. A method for the detection of P. vivax in a biological sample of vertebrates or invertebrates by hybridization of a probe comprising a nucleotide sequence identified as SEQ ID NO: 3 in the Sequence Listing and as identified for either of pARC 117, pARC 145 and pARC1153, in radioactive or non-radioactive labelled form with their respective homologous sequences in the P. vivax genome, and detecting the labelled probe which is bound to the P. vivax genome. 
     The present invention is exemplified by but not limited to the diagnosis of diseases. Epidemiological screening, forensic investigations, determination of food contamination, public health surveys, preventive medicine, veterinary and agricultural applications with regard to diagnosis of infectious agents may be covered by this disclosure. 
     Identification of DNA sequences specific for P. vivax 
     Since P. vivax has not yet been cultured in vitro, genomic DNA from this parasite was obtained from the blood of an infected patient. After removing as much of the buffy coat as possible, the genomic DNA was prepared by standard methods (6), digested with the enzyme Sau 3A cloned into the Bam H1 site of the known plasmid vector, pUC 18. Species specific DNA sequences were identified by screening duplicate filters with nick-translated P. vivax DNA and nick-translated normal human DNA in a differential screening format. Three specific probes of P. vivax designated pARC 117, pARC 145 and pARC 1153 were obtained and they were subcloned into the M13 vectors and their DNA sequence determined by di-deoxy chain termination method (7). None of them had any characteristic repetitive elements within the region sequenced. A computer based homology search utilising updated databanks such as Genbank and EMBL revealed no significant homology with any published nucleotide sequences. The highest homology was obtained with pARC 117 and rat parvalbumin (about 41%). This figure is insignificant for all practical purposes. 
     The said probes for P. vivax, were specific and did not cross react with isolates of P. falciparum (FIG. 1). 
     Description of the detection procedure using the specific P. vivax probes 
     There are two main procedures for nucleic acid hydridization. In one method, the sample nucleic acid is immobilized on a solid support while the probe sequence is in solution (2). In the other method, both the sample nucleic acid and the probe sequence and are in solution (5). 
     Thus, one nucleic acid hybridization assay involves spotting of the blood or tissue sample on a solid support e.g. nitrocellulose which is then hydridized with the probe (isotopically/non-isotopically tagged) under appropriate conditions of temperature etc (3). This method is well documented and in the context of malaria diagnosis has been used by many investigators (8). However, the tissue sample has to be processed for parasite DNA extraction and then used in the dot blot assay. This is not desirable in the design of a diagnostic assay that is to be used for mass screening. In the experience of the inventors, direct spotting of infected blood on the matrix was not satisfactory. Erythrocyte membrane proteins and other plasma components in the blood, presumably proteins, and other tissues when used sterically hinder the binding of parasite DNA to the matrix. Consequently, during the hybridization process the spotted sample detaches from the matrix leading to a loss of specific signal. The problem may be overcome by prior washing of the samples to remove the interfering plasma constituents which will make the test laborious or by spotting very small amounts of the blood which would contribute to loss of sensitivity of detection. 
     The inventors have therefore, resorted to a solution hybridization format which requires no sample processing and is more rapid to perform than the filter hybridization format. The test utilises the following steps: 
     a) Tissue sample is collected directly in a solution of a chaotropic salt or a denaturing agent e.g. 25 ul of blood in 50 ul of 6M guanidine thiocyanate. If the probe has an isotopic tag, guanidine thiocyanate at a concentration of 4M can be used as the chaotropic salt. If the probe is non-isotopic (e.g. biotin) and has to be detected by an enzyme reaction, guanidine thiocyanate cannot be used since it binds to nitrocellulose, polyvinylidene difluoride (PVDF) filters and other similar matrices and denatures the enzyme conjugate subsequently used in the reaction. Guanidine thiocyanate can however, be used with non-isotopic probes provided the matrices are pretreated. 
     In the present invention, when guanidine hydrochloride is used as a solubilizing agent, it is found to be as effective as guanidine thiocyanate. It has the added advantage that it does not bind to the solid matrix and can therefore be used for non-isotopic probes. 
     b) The probe (1 ng) is added. The probe added is preferably a single stranded nucleic acid, DNA or RNA which may be obtained biologically or synthetically by methods well documented in the art. RNA is the preferred probe. 
     c) The sample is heated at 85° C. for five minutes and allowed to cool at room temperature (about 28°-35° C.) for two hours. 
     d) The sample is then diluted at least ten fold with a salt solution containing RNAseA and incubated for a further fifteen minutes. When single stranded DNA is used as the probe, unhybridized probe may be removed by hydroxylapatite chromatography. 
     e) The sample is then filtered through the matrix e.g. nitrocellulose or polyvinylidene fluoride filters and the filters are rinsed twice in salt solution containing RNAse A for fifteen minutes each time. 
     f) The appropriate detection method is then carried out. 
     Preparation of hybridization probes 
     The hybridization probes may be prepared by cloning the genomic elements in suitable vectors. These vectors may be plasmids (E. coli plasmids), filamentous phages (M13), lamboid bacteriophage, cosmids, salmonella phages and yeast. Any vectors documented in the art may be employed. 
     Hybridization probes may be labelled radioactively by nick-translation with  32  P using DNA Polymerase and [α- 32  P] dNTP. For non radioactive labelling Bio-dUTP can be used in nick-translation reaction. Short oligonucleotide probes can be end-labelled with [α- 32  P] or [α- 32  P] dNTP at their 5&#39; or 3&#39; termini respectively. 
     Single stranded DNA probes labelled with  32  P can be obtained using the M 13 vector system (9). Single stranded DNA probes can be labelled with biotin by the polymerase chain reaction in presence of Bio-dUTP, other deoxyribonucleotides and Taq DNA polymerase (10). 
     In hybridization assays utilising RNA hybridization labelled RNA, complementary to DNA is prepared. Methods for the construction of such systems are well documented in the art (11). The present invention includes also such assays. The present invention thus includes the use of double-stranded DNA, single-stranded DNA, and RNA as probes for detecting the P. vivax genome. 
     EXAMPLE 1 
     This example illustrates the method by which species specific probes for P. vivax were obtained. 
     1. Total DNA from P. vivax infected blood was isolated, digested with the enzyme Sau 3A and cloned in the Bam H1 site of plasmid vector, pUC 18. 
     2. Clones were replica spotted on two filters of nitro-cellulose. One filter was screened with nick-translated radioactive human DNA and the other filter was screened with nick-translated radioactive P. vivax DNA. 
     3. The hybridized replicas were autoradiographed to produce hybridization signals. 
     4. Clones that reacted with P. vivax DNA and not with human DNA were considered as species specific and were selected for use as hybridization probes for P. vivax. 
     5. In this way, three clones were identified. These clones are denoted pARC 117, pARC 145, and pARC 1153 respectively. The nucleotide sequences of the three clones are given and identified as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, in the Sequence Listing. The nucleotide sequence of each clone was determined by standard methods with the following result. ##STR1## 
     EXAMPLE 2 
     This example illustrates the preparation of specific hybridization probes. 
     Hybridization probes were made by labelling the insert DNA in pARC 117, pARC 145 and pARC 1153 by nick translation in the presence of E. coli DNA polymerase or T4 DNA polymerase and [α- 32  P] dNTPs yielding a specific activity of 10 7  cpm to 5×10 7  cpm ug DNA. Single stranded or synthetically derived DNA may be end labelled at the 5&#39; ends using polynucleotide kinase and [α- 32  P] ATP. They may also be labelled at the 3&#39; ends using terminal transferase and [α- 32  P] dNTPs. Biotinylated single stranded DNA may be synthesised by the polymerase chain reaction employing Taq DNA polymerase and Bio-dUTP. 
     Single or double stranded nucleic acids may be biotinylated by photoactivatable biotin. Chemical biotinylation of nucleic acid may also be done. 
     Single stranded RNA may be generated by fusing the DNA of interest to a fragment of the Salmonella phage SP6 promoter in a suitable vector. The vector may be propagated in E. coli. DNA of this vector can be transcribed in vitro using the SP6 RNA polymerase. During this process radioactive RNA of high specific activity may be obtained using [α- 32  P] UTP. Biotinylation of this RNA is also possible. 
     EXAMPLE 3 
     This example illustrates the species specificity of the three P. vivax clones described in example 1, viz., pARC 117, pARC 145 and pARC 1153. 
     Genomic DNAs of nine different isolates of P. vivax were prepared and spotted on nitrocellulose filter. The filter was probed with the respective P. vivax specific probes which were radioactive. The probes reacted only with P. vivax samples and not with P. falciparum nor human DNA (FIG. 1). 
     EXAMPLE 4 
     This example illustrates the assay for Plasmodium vivax using a solution hybridization format. 
     Infected blood (20 ul) or tissue is taken directly into 50 ul of guanidine thiocyanate containing 1 ng of pARC 117 RNA probe radiolabelled as described in Example 2. 
     The sample is heated at 85° C. for five minutes and left at room temperature for two hours. 
     The sample is then diluted ten fold in 2×SSC containing 10 ng RNAse A and incubated at room temperature for a further 15 minutes. 
     The sample is then filtered through a nitrocellulose or polyvinylidene difluoride filter matrix and the filter rinsed in 2×SSC containing RNAse A buffer twice for 15 minutes each. 
     The filter is dried and autoradiographed to film for hybridization signals to develop. 
     The assay described is easy to perform and requires very little laboratory equipment such as is likely to be present in peripheral field conditions during mass surveys. 
     EXAMPLE 5 
     This example illustrates the solution hybridization procedure employing guanidine hydrochloride instead of guanidine thiocyanate as described in Example 4. 
     4M guanidine thiocyanate as the chaotrope and non-radioactive probes that are detected by protein conjugates are incompatible. The chaotropic salt binds to nitrocellulose and PVDF filters and denatures the protein conjugates that are used for colour development. 
     Guanidine hydrochloride when used as a solubilising agent is as effective as guanidine thiocyanate. Since it does not bind to the matrix (nitrocellulose or polyvinylidene difluoride filters), it enables the use of non-radioactive probes that are detected by protein conjugates. 
     Guanidine thiocyanate may be used in conjunction with non radioactive probes as described earlier only if the filter is pretreated, e.g. with 3% BSA before the guanidine thiocyanate solution is filtered through it. 
     REFERENCES 
     1. Pollack, Y., Metzger, S., Shemer, R., Landau, D., Spira, D. T. and Golenser, J. (1985). Am. J. Trop. Med. Hyg. 34, 663. 
     2. Kafatos, F. C., Jones, C. W. and Efstratiadis, A. (1979) Nucl. Acids. Res 7, 1541. 
     3. Maniatis, T., Fritsch, E. F. and Sambrook, J., Molecular Cloning, A Laboratory Manual, Cold Spring Harbour, N.Y. 1982. 
     4. Langer, P. R., Waldorp, A. A. and Ward, D. C. (1981) Proc. Natl. Acad Sci. U.S.A. 78, 6633. 
     5. Thompson, J. and Gillespie, D. (1987) Anal. Biochem. 163, 281. 
     6. Panyim, S., Wilairat, P. and Yuthavong, Y. Application of Genetic Engineering to Research on Tropical Disease Pathogens with special reference to Plasmodia-WHO publication. 
     7. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. G. H. and Roe, B. A. (1980). J. Mol. Biol. 143, 161. 
     8. Holmberg, M., Bjorkmann, A., Franzen. L., Aslund, L., Lebbad, M., Pettersson U. and Wigzell, H. (1980), Bull, W.H.O. 64, 579. 
     9. Burke, J. F., (1984) Gene 30, 63. 
     10. Saiki, R. K., et al (1988) Science 239, 487. 
     11. Green, M. R., Maniatis, T. and Melton, D. A. (1983), Cell 32, 681. 
     FIGURE LEGEND 
     FIG. 1: Specificity of the P. vivax probes pARC 117 (A), pARC 145 (B) and pARC 1153 (C). Double stranded inserts isolated from the three clones were tested as probes. Pv1 to Pv9 represent DNA preparations from nine P. vivax infected blood samples. FCK 2 and T 9 represent P. falciparum DNA preparations. NH represent Normal human DNA. 
     
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SEQUENCE LISTING                                                          
(1) GENERAL INFORMATION:                                                  
(iii) NUMBER OF SEQUENCES: 3                                              
(2) INFORMATION FOR SEQ ID NO:1:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 87 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: both                                                    
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(A) ORGANISM: Plasmodium vivax                                            
(vii) IMMEDIATE SOURCE:                                                   
(B) CLONE: pARC 117                                                       
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                   
ATGTAAGAGCACATGAGATTTTATAAGGATTTCATTTTACTCAGGGTGAAATGAAGAAGC60            
ACTAAAAGATTTTGAGTAGAGTTTCAT 87                                            
(2) INFORMATION FOR SEQ ID NO:2:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 249 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: both                                                    
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(A) ORGANISM: Plasmodium vivax                                            
 (vii) IMMEDIATE SOURCE:                                                  
(B) CLONE: pARC 145                                                       
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                   
CCAAGTGAAGAAAGGTGGAAGGGCCAGCAGGAGAGCTGGTCACTGCATTGTCTCTCTGAG60            
GTCTGTAGGCCAGAAGCTCCCCAGGACTTAGACCCTACTAAATGGGGTAGAGAGTAAGGG120           
GCAG CCATCACTTATCACTGGCTGTCCTGAGGGTTTGGTGTACAGCATGGCTTGTGGTCA180          
GAGGCCTGTCAGCTGGGCTCCAAGAGTCCTAGTGAATGTAAACAGTGCAGACCTTTTCTG240           
GGGGGAAGG 249                                                             
(2) INFORMATION FOR SEQ ID NO:3:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 663 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: both                                                    
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(A) ORGANISM: Plasmodium vivax                                            
(vii) IMMEDIATE SOURCE:                                                   
 (B) CLONE: pARC 1153                                                     
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                   
TTTGTGTGATTTTTGTGATTTTTGATGGAAACCTGAATATTTGGGGTAATTATGTGATTA60            
GACTCAGGATTTTATTTAAATCTTCTGTTTTAGCTAGCCTCCTCTGACACTAGCTTGGCA120           
GGAACGAGGGCAG GAGAGCATTGCTGATGCATTCCTGCCTCTTTTCTTCCTTGTTACTCC180          
CAAGTGGGTGTAAAAATCCAGGTTTCCCACTGTTTCCTCCTTTAAATTAATTAATTAATT240           
TTTAATGTTGGCAAATAAAAATTATATATTGTGTATATTTATGGGGTACAACATGATAT T300          
TTGATATATGTATACATTGCAGAATGGCTAAATTAAGCTAATTAACATACATATTACCTC360           
ACATAATCAATTTTTTTGTGGTGAGAGCACCTGCAATCTACTCTTTTAGCAATTTTCAAG420           
TATATAAAACATTGTTATTAACTATGGTCACCTCAT TGTACAATATGTTTTTTGAACTTA480          
TTCCTCCTAAGTATAATTTTGTACTCTTTGACCAACATCTCCCCAGACCCCTCAATGCCC540           
ACCCTCTGGTAACCAACATTCTACTCTTTGCTTTTCAACTTTTATAGATTCCATATGAAG600           
TAGGATCATGCTG TATTTGTCTTTGTGCCTGGCTTATTTCCTTTACATACTGTTCTCTAG660          
GTG663