Method for detecting biological toxins

Abstract

Biological toxins are indirectly detected by using polymerase chain reaction to amplify unique nucleic acid sequences coding for the toxins or enzymes unique to toxin synthesis. Buffer, primers coding for the unique nucleic acid sequences and an amplifying enzyme are added to a sample suspected of containing the toxin. The mixture is then cycled thermally to exponentially amplify any of these unique nucleic acid sequences present in the sample. The amplified sequences can be detected by various means, including fluorescence. Detection of the amplified sequences is indicative of the presence of toxin in the original sample. By using more than one set of labeled primers, the method can be used to simultaneously detect several toxins in a sample.

What is claimed is:  
     
       1. A method for detecting a DNA coding for a toxin of biological origin or a DNA coding for an enzyme specific to toxin production, said toxin suspected of being present in a sample known not to contain any organisms which produce said toxin, comprising the steps of: 
       a) performing an amplification on said sample which is suspected of containing a unique DNA having a unique sequence of nucleotides coding for the toxin or for an enzyme specific to toxin production, said sample not containing any organisms which produce said toxin, by initiating a polymerase chain reaction with a pair of primers complementary to said ends of said sequence of nucleotides and a polymerase which catalyzes said polymerase chain reaction, said amplification being sufficiently great that the amount of DNA other than said unique DNA in the amplified sample is negligible in comparison to the amount of said unique DNA in the amplified sample if said unique DNA is present in the unamplified sample; and  
       b) detecting the presence of amplified unique DNA in said amplified sample.  
     
     
       2. The method as recited in claim  1 , wherein the toxin is selected from the group consisting of botulin, tetanus, ricin, cholera, diphtheria, aflatoxins, perfringens toxin, mycotoxins, shigatoxin, staphylococcal enterotoxin B, T2, seguitoxin, saxitoxin, abrin, cyanoginosin, alphatoxin, tetrodotoxin, aconotoxin, snake venom and spider venom. 
     
     
       3. The method as recited in claim  1 , further comprising the step of: 
       performing a further amplification on said amplified sample by initiating a polymerase chain reaction with a pair of primers complementary to a unique sequence of nucleotides within said amplified unique sequence of nucleotides and a polymerase which catalyzes said polymerase chain reaction, at least one member of said second pair of primers having a detection label bound thereto, thereby producing a detectable amount of double stranded, twice-amplified DNA with said detection label on at least one end thereof if said toxin is present in the unamplified sample.  
     
     
       4. The method as recited in claim  3 , wherein said detection of the presence of amplified unique DNA in said amplified sample in step (b) comprises the steps of: 
       (1) spotting said twice-amplified sample directly on a membrane, and  
       (2) detecting any labelled DNA using one of spectroscopic, photochemical, biochemical or immunochemical techniques.  
     
     
       5. The method as recited in claim  1 , wherein said detection of the presence of amplified unique DNA in said amplified sample in step (b) comprises the steps of: 
       (1) using gel electrophoresis to separate any DNAs in the sample;  
       (2) denaturing the DNAs;  
       (3) adsorbing the denatured DNAs on a nitrocellulose membrane;  
       (4) incubating the nitrocellulose membrane under renaturing conditions with a solution containing fluorescent or radio-labelled molecules able to base pair with said unique DNA; and  
       (5) detecting the position and size of the labelled, unique DNA using autoradiography or fluorometry.  
     
     
       6. The method as recited in claim  1 , wherein said detection of the presence of amplified unique DNA in said amplified sample in step (b) comprises the steps of: 
       placing any amplified DNA in a matrix of agarose through which said amplified DNA moves under the influence of an electric or magnetic field; and  
       identifying said unique DNA based on its mobility in said matrix.  
     
     
       7. The method as described in claim  3 , wherein at least one of said primers used in said further amplification includes a specific binding site for a double-stranded DNA binding protein, and wherein any of said twice-amplified DNA present includes said specific binding site at one end and a biotin molecule at its other end. 
     
     
       8. The method as recited in claim  7 , wherein said detection of the presence of amplified unique DNA in said amplified sample in step (b) comprises the step of: 
       (1) using a capture reagent on a membrane attached to an absorptive pad;  
       (2) reacting any of said twice-amplified DNA, including said double stranded DNA binding site and said biotin molecule, with a color reagent to form a complex;  
       (3) applying the complex to a membrane attached to an absorptive pad, said membrane having bound thereto a double-stranded DNA-binding protein, whereupon the mixture is captured on the membrane;  
       (4) detecting the color on the membrane.  
     
     
       9. The method as recited in claim  7 , wherein said detection of the presence of amplified unique DNA in said sample in step (b) comprises the steps of: 
       (1) using gel electrophoresis to separate any DNAs in the sample;  
       (2) denaturing the DNAs;  
       (3) adsorbing the denatured DNAs on a nitrocellulose membrane;  
       (4) incubating the nitrocellulose membrane under renaturing conditions with a solution containing fluorescent or radio-labelled molecules able to base pair with said unique DNA; and  
       (5) detecting the position and size of the labelled, unique DNA using autoradiography or fluorometry.  
     
     
       10. The method as recited in claim  3 , wherein the detection label is selected from the group consisting of biotin, fluorophores and chromophores. 
     
     
       11. A method for detecting a plurality of DNAs coding for toxins of biological origin or DNAs coding for enzymes specific to production of said toxins in a sample suspected of including at least one of said toxins and known not to contain any organisms which produce said at least one toxin, comprising the steps of: 
       a) performing an amplification on said sample which is suspected of containing a plurality of unique DNAs each having a unique sequence of nucleotides coding for a respective one of said toxins or for an enzyme specific to production of a respective one of said toxins, said sample not containing any organisms which produce said toxin, by initiating a polymerase chain reaction with a plurality of pairs of primers, each pair of said plurality of primers being complementary to said ends of a respective one of said sequences of nucleotides, and a polymerase which catalyzes said polymerase chain reaction, said amplification being sufficiently great that the amount of DNA other than said unique DNAs in the amplified sample is negligible in comparison to the amount of said unique DNA in the amplified sample if any of said unique DNAs is present in the unamplified sample; and  
       b) detecting the presence of any of said amplified unique DNAs in said amplified sample.  
     
     
       12. The method as recited in claim  11 , further comprising the step of: 
       performing a further amplification on said amplified sample by initiating a polymerase chain reaction with a plurality of pair of primers, each pair of primers being complementary to a unique sequence of nucleotides within a respective one of said unique sequences of nucleotides and a polymerase which catalyzes said polymerase chain reaction, at least one member of each of said second pairs of primers having a detection label bound thereto, thereby producing a detectable amount of double stranded, labelled, twice-amplified DNA if any one of said toxins is present in the unamplified sample.  
     
     
       13. The method as recited in claim  12 , wherein the label associated with each primer pair is unique to said primer pair, thus permitting specific identification of the particular nucleotide sequence amplified. 
     
     
       14. The method as recited in claim  12 , wherein said detection of the presence of amplified unique DNA in said amplified sample in step (b) comprises the steps of: 
       (1) spotting said twice-amplified sample directly on a membrane, and  
       (2) detecting any of said labelled DNAs present on said membrane using one of spectroscopic, photochemical, biochemical or immunochemical means.  
     
     
       15. The method as recited in claim  11 , wherein said detection of the presence of amplified unique DNA in said amplified sample in step (b) comprises the steps of: 
       (1) using gel electrophoresis to separate any DNAs in the sample;  
       (2) denaturing the DNAs;  
       (3) adsorbing the denatured DNAs on a nitrocellulose membrane;  
       (4) incubating the nitrocellulose membrane under renaturing conditions with a solution containing fluorescent or radio-labelled molecules able to base pair with said unique DNAs; and  
       (5) detecting the position and size of any of said labelled, unique DNAs present on said membrane using autoradiography or fluorometry.  
     
     
       16. The method as recited in claim  11 , wherein said detection of the presence of amplified unique DNA in said amplified sample in step (b) comprises the steps of; 
       placing any amplified DNA in a matrix of agarose through which said amplified DNA moves under the influence of an electric or magnetic field; and  
       identifying said unique DNA based on its mobility in said matrix.  
     
     
       17. The method as described in claim  12 , wherein at least one member of each of said primer pairs used in said further amplification includes a specific binding site for a double-stranded DNA binding protein, and wherein all of said twice-amplified DNAs present include said specific binding site at one end and a biotin molecule or a fluorophore at its other end. 
     
     
       18. The method as recited in claim  17 , wherein said detection of the presence of amplified unique DNA in said amplified sample in step (b) comprises the step of: 
       (1) using a capture reagent on a membrane attached to an absorptive pad;  
       (2) reacting any of said twice-amplified DNAs present, including said double stranded DNA binding sites and said biotin molecules, with a color reagent to form a complex;  
       (3) applying the complex to a membrane attached to an absorptive pad, said membrane having bound thereto a double-stranded DNA-binding protein, whereupon the complex is captured on the membrane; and  
       (4) detecting the color on the membrane.  
     
     
       19. The method as recited in claim  17 , wherein said detection of the presence of amplified unique DNA in said amplified sample in step (b) comprises the steps of: 
       (1) binding a double-stranded DNA-binding protein to a solid support;  
       (2) occupying all double-stranded DNA-binding sites of said protein on said solid support by treating said support with a color indicator which occupies said binding sites of said protein but has a significantly lower affinity for said binding sites of said protein than do any of said twice-amplified DNAs present in the sample;  
       (3) treating the solid support with the twice-amplified sample amplified sequences having a normal affinity for said binding sites whereupon any of the twice-amplified DNAs present in said twice-amplified sample displaces said indicator from said support; and  
       (4) colorimetrically or fluorometrically detecting the amount of said indicator displaced from said support.  
     
     
       20. The method as recited in claim  1 , wherein said sample is an environmental sample, water supply sample, purified commercial toxin preparation, food or beverage sample, blood sample, mucous sample, saliva sample and urine sample. 
     
     
       21. The method as recited in claim  20 , wherein said environmental sample is an air, soil, surface swipe, or water sample. 
     
     
       22. The method as recited in claim  11 , wherein said sample is an environmental sample, water supply sample, purified commercial toxin preparation, food or beverage sample, blood sample, mucous sample, saliva sample and urine sample. 
     
     
       23. The method as recited in claim  22 , wherein said environmental sample is an air, soil, surface swipe, or water sample. 
     
     
       24. The method as recited in claim  1 , wherein said method consists essentially of steps (a) and (b). 
     
     
       25. The method as recited in claim  11 , said method consists essentially of steps (a) and (b).