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US researchers take a strip off botulism

By Joe Whitworth+

20-Feb-2014
Last updated on 21-Feb-2014 at 10:31 GMT

Photo: Alice Lin. Robert Hnasko (left) and biologist Larry Stanker examine lateral flow devices that can identify botulinum toxin serotypes

Photo: Alice Lin. Robert Hnasko (left) and biologist Larry Stanker examine lateral flow devices that can identify botulinum toxin serotypes

A test strip that can detect botulism-causing toxins has been developed by US researchers.

The test detects the toxin and not the bacteria and distinguishing between them is informative as some serotypes (such as A and B) are most commonly associated with the disease in humans.  

Botulinum neurotoxins (BoNT) are produced by Clostridium botulinum and are classified into seven, antigenically distinct serotypes, designated A–G.

The tests require a liquid sample and a device to deliver the sample to the sample pad.  The use and interpretation do not require any substantial training.

How it works

Robert Hnasko, molecular biologist for Agricultural Research Service (ARS), USDA's scientific research agency, said the test was for rapid, preliminary screening in foodborne outbreaks of botulism in which the culprit food has not yet been pinpointed.

These tests function similar to the home pregnancy tests – in that a small amount of liquid sample is placed on one end of the strip and the fluid moves up the test strip by capillary or wicking action past a test window,” he said.

“Within that window a color line will appear indicating, one the test performed as expected; shown by a color control line (C). This line will always resolve to a color if the test functions normally.

“Two, the test line or line/s which will also resolve to a color if the target (ie BoNT A and/or BoNT B) is present in the sample.” 

Botulism danger

Modern industrial canning is designed to kill the spores of C. botulinum so most human cases arise from home-canned low acidic foods, meat products like sausage/ham or fermented fish.

Photo: Stephen Ausmus. The lateral flow device can provide positive identification of botulinum toxin in 20 minutes

An average of 145 cases are reported each year in the US of which 15% are foodborne.

Hnasko said that if no toxin is there a single control line (C) will appear indicating the test functioned normally and no toxin was present at the set sensitivity. 

“If either A or B toxin is present two lines will appear the control (C) line and either the (A) or (B) lines indicating which toxin was detected,” he said.

“If both A and B toxin are present in the sample three lines will appear A+B and the C.”

Test strip use

The strip which measures about a quarter-inch wide by 2.25 inches long can provide results in less than 20 minutes when used as the basis of a field-ready test kit.

It is capable of resolving BoNT/A and /B as two independent colorimetric lines on one strip, with sensitivities >10 ng/mL for purified toxins and 10–500 ng/mL in toxin fortified beverages.

The strip fits into a holder (technically a lateral flow device) like those in pregnancy test kits for at-home use.

Only a small amount of prepared sample is needed, and the results, shown on a color display, are easy to see and understand.

The strip is equipped with laboratory-built proteins, known as monoclonal antibodies, which bind exclusively to A- or B-type (serotype) botulinum toxins.

Together, these types are responsible for more than 80% of cases of foodborne botulism in the US.

Testing the device

The device could detect 5 ng/mL of purified BoNT/A and 10 ng/mL of BoNT/B in 2% and 1% milk, respectively.

In undiluted apple juice, 25 ng/mL of BoNT/A and 10 ng/mL of BoNT/B could be detected.

Using monoclonal antibodies in a lateral-flow device to detect botulinum toxins isn't new but it is believed to be the first that can concurrently detect and differentiate the A and B serotypes.

The gold standard of detection of BoNTs is the mouse bioassay, which can detect 10 pg/mL of toxin.

However, the assay requires days to complete, large numbers of animals and can only be performed at a select number of laboratories in the US.

Hnasko said the mouse bioassay is a sensitive method to detect botulism and there are numerous methods that include different types of immunoassays, ie those assays that use antibodies. 

“The test strip is not designed to replace other methods of detection but rather augment – these test strips offer rapid, simple, stable, inexpensive, disposable tests that are field deployable, such as outside the lab.” 

He added they were working with commercial partners through technology transfer to move from the laboratory to detection. 

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