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Method for detecting biological toxins

a biological toxin and detection method technology, applied in the field of detecting biological toxins, can solve the problems of time-consuming and costly steps, and have not been developed a quick and efficient method for detecting the presence of biological toxins in samples

Inactive Publication Date: 2001-08-07
THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE NAVY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

It is another purpose of the present invention to detect target nucleic acid sequences using a simple and easy to use method, which detection is indicative of the presence of toxins coded by the sequences.

Problems solved by technology

These steps can be time-consuming and costly.
To date, there has not been developed a quick and efficient method for detecting the presence of a biological toxin in a sample, said detection being possible even if the toxin has been denatured or the organism responsible for producing the toxin is no longer present.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

A crude, unpurified type D Clostridium botulinum (BotD) toxin preparation, and a DNA extract of the same crude toxin preparation, were selected as samples. Each was amplified and detected separately. The nucleic acid sequence coding for this toxin is known.

Ten .mu.l of each BotD DNA sample (0.5 mg. / 0.5 ml 0.2 M NaCl, 0.05 M Na acetate, pH 6.0) was prepared as follows:

0.5 ml toxin preparation was added to an equal volume of saturated phenol. The resultant mixture was vortexed briefly, and centrifuged for 5 min. at 14,000.times.g. The supernatant was recovered and the phenol extraction step was repeated on the recovered supernatant. An equal volume of chloroform was added to the supernatant recovered from the repeated phenol extraction. The supernatant was removed and chloroform extraction repeated. The DNA was then precipitated as a pellet from supernatant ethanol. The pellet was then resuspended in 50 .mu.l distilled H.sub.2 O.

The preparation was added to the conventional PCR reagen...

example 2

A crude, unpurified type A Clostridium botulinum (BotA) toxin preparation, and a DNA extract of the same crude toxin preparation, were selected as samples. Each sample was amplified and analyzed separately. The nucleic acid sequence coding for this toxin is known.

Ten .mu.l of the preparation made according to the steps set out in Experiment 1 was added to an aqueous solution including the same conventional PCR reagents as in Example 1, except that the primer pair consisted of BotA1 and BotA3. These primers were designed by co-inventor James Campbell to match unique DNA sequences in the gene encoding type A botulism toxins, and synthesized by the Synthecell Corporation. Primer sequences are as follows:

Bot A1 5'-ATT AAT TAT AAA GAT CCT-3' (SEQ ID NO: 3)

Bot A3 5'-AAC TTC AAG TGA CTC CTC-3' (SEQ ID NO: 4)

The thus prepared reaction mixture was cycled thermally for two minutes at three successive temperatures.

1. A high "denaturing" temperature (94.degree. C.);

2. A low "annealing" temperat...

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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.

Description

BACKGROUND OF THE INVENTION1. Field of the InventionThis invention relates to detecting biological toxins and, more particularly, to a method for amplifying genes coding for the toxins and detecting the amplified genes, which detection is indicative of the presence of the toxins.2. Description of the Related ArtThe ability to detect the presence or absence of biological toxins, such as botulin (a toxin produced by the bacteria Clostrium botulinum) or aflatoxins (a toxin produced by the fungus Aspergillus favus) contaminating food, various biological warfare agents contaminating the air, and various contaminants of water or biological samples, has been a desire of scientists for ages. Such an ability would help prevent disease, incapacitation or other maladies attributable to the toxins, and would be useful in countering the use of biological weapons.Current tests for detecting toxins usually depend on chromatographic techniques, such as gas or high-pressure-liquid chromatography, ma...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/689C12Q1/686C12Q2600/142
Inventor LIGLER, FRANCES S.CAMPBELL, JAMES R.
Owner THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE NAVY
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