FI104737B - Method for detecting toxins and kits therefore - Google Patents

Description

104737

Method for detection and packaging of toxins

The invention relates to a method of Bacillus cereus. emetic toxin and related mitochondrial and membrane toxins, especially toxins of the genus Bacillus. The invention also relates to a package useful for carrying out the process.

Bacillus cereus is known to cause clinical infections, food poisoning and toxin-induced symptoms of diarrhea and vomiting. Lethal deaths, caused by infections and emetic food poisoning, have recently been reported. B.cereus is ubiquitous in nature, but has also been isolated from environments where its presence may pose a serious health risk, such as baby food and hospital bed linen. B. cereuj strains have been shown to produce seven different toxins. Of these toxins, the emetic toxin is very likely the 15 most dangerous. However, due to the lack of suitable test methods, it is the least known.

The emetic toxin, or vomiting factor, is a peptide that is thermostable and resistant to proteolytic degradation. It is formed in the exponential stationary phase of the bacterial growth phase also at -0 0 at optimum temperatures of 25-30 ° C.

Its toxicity tests include a laborious and expensive monkey feed assay and in vivo / Yro cell culture lysing systems on HEp-2 cells or Chinese hamster ovary cells (reviewed by B.cereus, see Drobniewski, FA, Clin.Microbiol.Rev. 6 , 1993, m.p.324-338). Although very different foods such as vegetables, pasta, meat, eggs, and dairy products are combined with the emetic toxin that causes gastrointestinal disease, insufficiently cooked or reheated rice foods continue to cause a disproportionate number of cases.

x · _ Biological activities of B.cereus emetic toxin, cereulide, described by Agata, N. et al. in FEMS Microbiol.Lett. 121: 31-34 and Isobe et al. in Bioorg.Med.Chem.Lett. 5: 2855-2858, include vomiting in primates (e.g. Turnbull, PCB, et al., Am.J.Nutr. 32: 219-228) and mitochondrial swelling in HEp-2 cells (Sakurai, N. et al. , Microbiol. Immunol. 38: 337-343).

2 104737

Although the structure and mitochondrial toxicity of cereulide have been recently investigated, there has so far been no rapid detection method for toxin from bacteria, food poisoning cases or contaminated food.

The present invention relates to a sensitive, rapid and reproducible bioassay for the detection of emetic mitochondrial toxin from B.ceeus reus, which is identical in all criteria to cereulide. The method of the invention is also suitable for detecting other microbial toxins which are toxic by the same mechanism of destruction of mitochondria or membrane, such as, for example, 10 B. licheniformis toxin.

The bioassay of the invention is based on the lost motility of live spermatozoa, in particular bald spermatozoa, in the presence of said toxin.

Accordingly, the present invention relates to a method for detecting a Bacillus cereus emetic toxin and related mitochondrial or membrane toxins in a food, feed or drug sample suspected of containing said toxin, characterized by exposing boar sperm to said sample and determined.

20

Figure 1 is an electron microscope image of boar spermatozoa exposed to B. cereus F 5881 cell extract.

Figure 2 is an electron microscope image of boar spermatozoa exposed to B.cereus 25 for ATCC 14579 for comparison.

In accordance with a preferred embodiment of the invention, the spermatozoa used are live sperm cells of the boar, e.g., commercially available.

To perform the test according to the invention, the sample to be tested, typically a food sample, a feed sample or also a drug sample, is preferably extracted with a solvent to extract the toxin. Any solvent that is compatible with the toxin can be used! 3 104737 for extraction. One such solvent group is aliphatic alcohols, typically lower alcohols, such as methanol, or water, but other types of solvents, such as lipid solvents, may be used provided that they do not adversely affect the test and are not harmful to the sperm used. The amount of extractant used can be determined by one of ordinary skill in the art, but the appropriate amount for many purposes, for example to extract a food sample, is 0.5 to 2 volumes of extractant per volume of sample. After extraction, the extracts obtained are preferably dried, for example by evaporation, to obtain a sample of the toxin containing the extracted toxin in dry form.

10

For the bioassay, the extracted and dried material may be redissolved in a suitable solvent which is non-toxic to sperm cells, for example dimethylsulfoxide or methanol. A suitable ratio of a toxin-containing sample to a solvent such as DMSO is 5 to 50 mg of sample per ml of solvent. The test sample thus dissolved is then tested for sperm toxicity by contacting the sample with sperm. For testing, sperm can be diluted with a diluent, suitably at a concentration of 10-20% semen, so that the diluted semen contains 1 suitably 30-60 x 10 cells / ml. The diluent may be a commercial diluent, e.g., type MR-A; Kubus, S.A., Madrid, Spain. The amount of dissolved test sample 20 should be limited so as not to significantly alter the sperm cell environment, which may have a detrimental effect on semen and the test. A suitable amount of the reconstituted test sample, taking into account diluted semen, is 5-40 μΐ, suitably 10–20 μ ml per ml of semen. The amount of semen sample may vary but should preferably be at least 1 ml, suitably 1-5 ml, since too small a sample can have a detrimental effect on living sperm cells and the test.

'' An alternative way to carry out the method of the invention is to expose the sperm to a toxin via air contact. In this case, the sample to be tested can be extracted and evaporated as described above but does not need to be reconstituted. After 30, the spermatozoa are exposed to the toxin sample by sealing both in a closed space such as a closed container, e.g. a petri dish with a lid. The toxin sample 1 • 4 104737 and the sperm do not need to be in direct contact with one another because the toxin evaporates and affects the movement of the sperm through the air.

After the spermatozoa have been exposed to the test sample, the motility of the spermatozoa is determined. This assay may conveniently be performed 8 to 48 hours after exposure. Sperm motility is expressed as a proportion of the number of sperm cells moving and is usually expressed as a percentage. Sperm motility can be assessed subjectively using a microscope equipped with phase contrast optics. The percentage of highly mobile sperm cells can also be measured objectively by commercially available devices, such as a computer-linked sperm mobility analyzer, e.g., HTM-S type, Hamilton-Thorn Research, Danvers, Mass., USA.

As shown in the following examples and tests, the emetic seed-15 fluid toxin was directly extractable from food and measured by reduced baldness of bald spermatozoa within 24 hours, allowing the detection of toxin without the time consuming culturing and isolation of suspected B-cereus strains. The test can use commercially available semen and does not require laboratory animals. This is the first rapid and sensitive biotes-20 ti described to detect an emetic toxin from B. Cere us.

A: Sperm are transcriptionally inactive, which makes them insensitive to other types of toxins, such as those that affect the synthesis or regulation of proteins or nucleic acids. They connect external signals to cellular signals, such as motility, which are mainly controlled by ion currents. This makes spermatozoa specific and sensitive indicators of ion channel-forming toxins. Sperm motility depends only on oxidative phosphorylase • V V.

thio in the mitochondria. ATP production by substrate-level phosphorylation in boar sperm is too slow to maintain motility. Spermatozoa exposed to cereulene-30-dintoxin exhibited loss of motility and swollen, degraded mitochondria, as shown in Figure 1. However, vitality staining and measurement of cellular ATP levels revealed intact plasma membranes and ATP-γ! 5 104737 nan, which can be explained by ongoing substrate-level phosphorylation in the cytoplasm. Thus, the sperm cell was paralyzed but not deadly * damaged.

* 5 It is thus believed that cereulidin-toxin poison sperm by acting as an ion phosphorus, leading to a loss of mobility caused by blocking oxidative phosphorylation in the mitochondria, i.e. it is a mitochondrial injury.

The toxin in the sample can be isolated and identified by evaluating, in addition to motility assay, plasma membrane damage, for example by staining, and possibly determining ATP concentration and possible mitochondrial damage in exposed sperm cells as described below. Thus, for example, if, in addition to reduced mobility, the plasma membrane is damaged, the toxin is most likely-.

min membrane toxin and different from emetic toxin.

15

As shown in the following examples, 50% of the purified emetic toxin had an effective concentration of 0.5 ng toxin per ml in diluted (diluted 12%) boar semen containing 30-60 x 10 * spermatozoa / ml. This amount of toxin corresponds to the toxin produced by 10M05 CFU B.cereiw cells.

20

When testing a food sample for the presence of a toxin, the amount of sample may, of course, vary depending on the type of food being tested and the degree of suspected contamination. However, a suitable amount of food to be tested is 1-10 g which, after extraction and possible redissolution as described above, can be brought into contact with the diluted semen as described above.

- '· In the following examples, the detection limit for food that was rice was 3 g rice, which ~ •. = contained 10 ** 10 * CFU of emetic B.cereus per gram of rice.

The invention also relates to a kit for carrying out the detection method according to the invention. Such a package contains, in the same package, the equipment for the toxin. for determining the mobility of exposed sperm such as solvents for sample extraction and means for diluting semen, and possibly means for reconstituting the extracted sample, such as those mentioned above. Such solvents may, for example, be contained in different ampoules. In addition, such a kit may include means for detecting membrane damage in exposed spermatozoa, such as membrane dyes, e.g., Dapi dye, and possibly means for determining ATP content in exposed cells. Means for determining membrane damage and ATP content are known to those skilled in the art and are commercially available.

10 EXAMPLES

Extraction of the Ethical Toxin from Pure Cultures and Directly from Food (i) Cell Extracts Bacteria were grown on trypsin soybean agar plates at 28 ° C for 10 days to stabilize the growth phase and obtain mainly spore-forming and lysed cells as detected by phase contrast microscope. Colonies were scraped from agar and suspended in 100 mg / ml in a suitable solvent. The extracts were filtered (0.2 μΐη pores) and the filtrate diluted with dimethyl sulfoxide (DMSO) was tested for 20 toxins.

(ii) Methanol extraction of toxin from pure cultures 500 mg cells were harvested from trypsin soybean agar plates grown at 28 ° C for 10 days and extracted with 100 ml of 100% methanol as described by Andersson et al. (Appl.Environ.Microbiol. 63: 387-393). The evaporated residue diluted with dimethyl sulfoxide (DMSO) was tested for toxin.

m • * «• * (iii) Methanol extraction of toxin directly from food

Cooked rice (60 g, 150 mL) was experimentally infected with 10x10 6 CFU per gram of B.cereus or Bacillus mycoides strain. The rice was kept at room temperature for 2 days. The rice was examined by a phase contrast microscope and found to contain many spores and lysed cells. On the 2nd day the rice was reheated to 104737

7 I

to about 80 ° C (30 min) and incubated for another two days at room temperature.

On the fourth day, the rice was extracted with 1 volume of methanol, the extract evaporated and the residue diluted with DMSO examined for sperm toxicity.

5 Sperm Toxicity Testing

Bald semen was filtered through a gauze cloth and diluted to 12% with commercial semen diluent MR-A (Kubus, S.A. Madrid, Spain) to a density of 30x10x50x10x sperm / ml. The 10 semen diluted with MR-A were gently mixed and exposed to the cell extracts and rice extracts obtained above. The extracted and evaporated residue obtained was diluted with DMSO (5-50 mg / ml). A volume of 20 μ1: η was dosed with 2 ml of diluted boar semen. Sperm motility was subjectively evaluated on a phase contrast microscope using a heated (37 ° C) table; 15 precise percentages of highly motile sperm cells were measured objectively by sperm

with a motion analyzer (HTM-S, club 7.2; Hamilton-Thom Reasearch, Dan-

vers., Mass.). The experiment was recorded positive when> 50% reduction in sperm motility was observed in both instrumental and visual methods compared to control sperm, i.e. spermatozoa exposed to only 20 diluents (DMSO).

Plasma membrane integrity was tested with a combination of two fluorescent dyes, calcein AM and ethidium homodimer-1 (Molecular Probes, Inc., Eugene,

Oreg.) (Januskauskas et al., Acta Vet.scand. 36: 571-574). Cellular ATP content was measured by bioluminescence (Januskauskas, ibid.) Using a Bio-Orbit 1235 luminometer and ATP Biomass kit 1243-118 (Bio-Orbit, Turku, Finland). The morphology of thymic mitochondria was analyzed by transmission electron microscopy (Andersson et al., Ibid.). The osmolality of diluted boar semen was determined by an automated micro osmometer (Herman Roebeling Messtechnik, Ber-30 line, Germany). Analyzes were performed in duplicate and results are averaged.

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Score

The following Table 1 shows the motility of boar spermatozoa after exposure to cell extracts of different 5aci / ftw strains.

5

Mobility is expressed as a percentage expressing the number of fast moving spermatozoa as measured by a Hamilton-Thorn sperm analyzer. The mean difference in replicate experiments was £ 20% when comparing bacterial extracts against sperm cells from three different boars. A sperm suspension containing 30 x 10 * - 50 x 104 sperm per 10 ml was exposed to extracts of 2 mg (wet weight) of bacteria / ml diluted boar semen.

TABLE 1

Cell extract prepared in% of mobile sperm after days 1, 2, 4 and 5 12 4 5

B.cereus 4810/72 <5 <5 <5 <5 B.cereus NC 7401 <5 <5 <5 <5: Other strains B.cereus ATCC 14579T> 60> 60> 60> 50 B.mycoides ATCC 6462T > 60> 60> 60> 50 B.licheniformis DSM13T> 60> 60> 60> 50! No bacteria> 60> 60> 60> 50 ψ

From the results, it can be seen that the cell extracts from both B.cereus 4810/72 and NC 7401 inhibited sperm motility within one day at a dose of 2 mg (wet weight) bacteria / ml diluted boar semen-5. Similarly prepared cell extracts of B.cereus ATCC 14579 (strain), 9 104737 B.mycoides ATCC 6462 (strain) and B.licheniformis DSM 13 (strain) had no effect on sperm motility even after day 5 of challenge. sperm motility assessed by phase contrast microscopy was objectively verified by measuring the percentage of highly motile sperm cells using a Hamilton-Thorn sperm analyzer. frequencies as evidenced by the Transmission Electron Microscope, indicating that the filtrate contained mitochondrial toxin 10. No mitochondrial swelling was observed in spermatozoa exposed to extracts of B.cereus ATCC 14579 (Figure 2) or B.mycoides ATCC 6462.

Sperm Toxicity of B.cereus Strains of Different Origin Many strains of B.cermus of different origins were tested for sperm toxicity. '

The results are shown in Table 2. It can be seen that cell extracts from five strains, namely F- = 5881, F-47, F-4426, 4810/71 and NC 7401, quenched the movement of bald spermatozoa at very low concentrations corresponding to 50 ng (dry weight) B.cereus cells (equivalent to 104 CFU) per ml of diluted boar seed:

20 fluids. Extracts from other tested J3.cerm strains had no I

effect on sperm motility even at 10,000 fold concentrations (£ 10® v CFU / ml).

The EC? Value is reported in Table 2 as the bacterial end point dilution that produced> 50% reduction in sperm motility. Extracts were prepared as described in Table 1 and diluted to the end point by double dilution in DMSO. The mean difference between two replicate • experiments was <20%.

m i - TABLE 2 10 104737 B.cereusAzMt EC »(mg EC» (CFU Strain (s) bacterial / ml) bacterial / ml) 5____

Food Poisoning> 4> 10 * IH41385 Outbreak IH4106 F-3371 F-3453 F-528 10 Food Poisoning Outbreak 0.002-0,0005 104 - 105 4810/72 NC7401, F-47 F-4426, F-5881

Paper machine> 4> 10 * E1PCA, E60, T3 15

Living Norway Six> 4> 10 * NS61

Dairy Products> 4> 10 * K8, R15, NCFB634 20___ SMR182_

Toxicity of Selected Toxins and Chemicals to Boar Sperm Viability Parameters Toxicity thresholds for purified sperm toxin from B.cereus 4810/72 were compared to the values of selected bacterial toxins and chemicals. The results are shown in Table 3. EC? Is reported as the point-dilution of terminal dilution of boar diluted semen which produced> 50% change in vitality parameters compared to spermatozoa exposed to diluent alone. The mean difference between the two replicates was <20% for all vitality parameters. The swelling of the mitochondria is detected by a transmission electron microscope.

• «

Damage to membrane integrity is detected by vitality staining using 35 fluorescence microscopes. The concentration of sperm toxicity was determined by reversed phase high performance liquid chromatography using valinomycin (Nova-Pak Cl8 column 3.9 x 300 mm, internal diameter 4 μπι, eluent A: water with 0.1% trifluoroacetic acid (TFA) as standard); 104737 acetonitrile with 0.075% TFA; gradient from 50% A and 50% B to 100% B in 10 minutes at a flow rate of 1 mL / min; detection at 214 nm; 50% injection methanol with 0.1% TFA).

v 5 The results revealed the sperm toxicity of three materials: a toxin purified from B.cereus 4810/72, valinomycin (depsipeptide and potassium ionophore, Booth, I.

1988, pp. 377-428, IN, C Anthony (ed.) Bacterial energy transduction, Academic

Press, London, England) and Gramicidine (membrane channel linear homopeptide, potassium ionophore and protonophore, Booth, ibid.). Low concentrations (<3 ng / ml) of these materials caused loss of sperm motility, concentrations of <400 ng / ml swollen mitochondria but did not deplete cellular ATP even at high concentrations (up to 12500 ng / ml). Calcium mycine A 23187 (polyether calcium ionophore) inhibited sperm motility at 32 ng / ml and depleted sperm ATP at 125 ng / ml but did not cause 1 5 morphological damage to mitochondria even at 2000 ng / ml.

It was found that sperm cells were relatively insensitive to other bioactivities tested

for those peptides, 50% effective concentrations (EC1) for vitality parameters ranged from 100 to 50,000 ng / ml. Finally, sperm cell damage (loss of mobility 20 and swollen mitochondria) caused by B.cereus 4810/72 was similar to "those caused by valinomycin and gramicidin but had little resemblance to the other toxins tested.

w - «- _ • - TABLE 3 12 104737

Toxin or chemical EC '(ng / ml)

Mitochondrial-End-Inactivated Damage Memos * Secured Movement of Brain * ATP Concentrations

Sperm toxin purified <400> 1000 0.5> 1000 produced from B.cereus 4810/72 5 Valinomycin <400> 1000 2> 1000

Calcimycin A 23187> 2000 125 32 KO

Gramicidin (A, B, C> 3000 NO <3 NO

and D)

lonomycin> 1000 10000 1000 HO

10 Polymyxin B sulfate> 50000> 50000> 50000 KD

Surfactin> 50000> 50000> 50000 ND

Modular) ini HO> 40000> 40000 HO

Anatoxin A HD> 40000> 40000 HD

H, H-Dihexylcarbodiimide 1000 10000 100 * 1000 HD

15 2,4-Dinitrophenol> 10000> 10000 1000 * 10000 HD

The following Table 4 shows the effects of cell extracts of Bacillus licheniformis, a bacterium also associated with food poisoning, on the vitality parameters of boar sperm. Sperm cells were exposed to bacterial cell extracts for 2 to 3 days in amounts of 2 and 4 mg (wet weight) extract / ml diluted boar semen. Mobility was determined using a Hamil-ton-Thorn sperm motility analyzer, the membrane was checked for vitality; staining using a fluorescence microscope, degenerated achromomes were detected by Giemsa staining under a light microscope and the mitochondria were examined with a transmission electron microscope.

«• | i; 1 TABLE 4 13 104737

Cell Extract Prepared- Moving Cell-ATP Damaged Sperm Cells X

• strains of sperm / ig ml · '

X of bones

, Menbraani Akrosomi Hitochondrio 5 Bacillus licheniformis

From the harm mixture 553/1 <1 <0.1> 70> 50 <1 553/2 <1 <0.1> 70> 50 <1 575u / 5 <1 <0.1> 70> 50 <1 10

Of unpasteurised milk 51/97 <1 <1 52/97 <1 <1 15 54/97 <1 <1

Reference cells B. licheniformis7> 60> 3 <20 <10 <1 B.mycoides1> 60> 3 <20 <10 <1 20 B.cereusT> 60> 3 <2 ° <10 <1 B.cereus1> 60> 3 < Z0 <1 °

B.cereus Emetic «801 <1> 3 <2 ° <1 °> 5 ° 25 NC7401 <1> 3 <20 <10> 5 ° F-5881 <1> 3 <2 ° <10> S0

No bacteria ____> 3 <2 ° <1 °> 50 30

From the results, it can be seen that the cellular extracts of B. licheniformis rabbits isolated •

in milk mixture and unpasteurized milk caused sperm cell motility

the ATP content was low as well, resulting from extensive membrane damage.

35

Detection of toxin from contaminated food

# - • I

Batches of cooked rice were inoculated with different strains of Bacillus and extracted as described above, and bald sperm cell motility was measured one and two days after the '40 challenge. The blank was performed with diluent alone (DMSO).

• 4 14 104737

The bacteria were also cultured on trypsin soybean agar for 10 days at 28 ° C and 500 mg (wet weight) cells were extracted with methanol. The evaporation residue of the methanolic extract was dissolved in DMSO. Diluted boar semen was exposed to serial dilutions of the extract (in DMSO) and sperm motility was measured for one and two days.

T

5 after exposure. These results are shown in Table 5 below.

ED, which shows an end point dilution of methanol-extracted solid (dry weight) samples per milliliter of diluted boar semen, resulted in> 50% reduction in sperm motility. The mean difference between two replicates of 10 replicates was 20% with semen from different boars.

Extracts of rice contaminated with B.cereus 4810/72 or B.cereus F-5881 inhibited sperm motility in amounts equivalent to 3 g (7.5 ml) of rice.

Extracts made from rice contaminated with B.mycoides ATCC 6462T or B.cereus ATCC 15 14579T did not show any mobility paralysis up to amounts equivalent to> 60 g (150 ml) of cooked rice.

Table 4 also shows the sperm toxicity of B.cereus and B.mycoides-yppikan-20 extracted from rice. The yields of B.cereus F-5881, 4810/72, ATCC 14579T and B.mycoides ATCC 6462T were 24, 16, 9 and 13 mg (dry weight) per gram (wet weight), respectively. Corresponding EC 50 values for methanol soluble substances in boar sperm were 0.072 / zg (corresponding to 2x105 CFU strain F-5881) / ml, 0.03 μ% (corresponding to 105 CFU strain 25 4810/72) / mI, 17 μζ ( corresponding to 101 CFU strain ATCC 14579T) / ml and> 68 μζ (corresponding to> 1010 CFU strain ATCC 6462T) / ml diluted boar semen.

«<

• I

4 i TABLE 5 15 104737

Inoculum Inoculum Concentrations of Various Parameters of Equivalent Methanol Extracts Elolle den ECm (fig / ml) ----- B.cereus Conta Methanol Extracts Contaminated) Mination (CFU / ml inoculated rice ml Cg) of rice) dry samples / tg / ml) B.cereus F-5881 0.072 10'-10T 38 7.5 (3) B.cereus «810/72 0.026 101-10T 30 7.5 (3) 5 B.mycoides ATCC> 68 > 101-10 '> 650 »150

6 «62T

B.cereus ATCC 17> 10'-101 1523> 150 (60)

1 «579T

• r - • - Z1

Claims (14)

A method for detecting Bacillus cereus emetic toxin and the corresponding mitochondrial or membrane toxins in a food, feed or drug sample suspected of containing said toxin, characterized in that bile sperm is subjected to said sample and the motility of said sperm is determined. . 2. A method according to claim 1, characterized in that the sperm are diluted with diluent for seminal fluid before being subjected to the sample. 10 Method according to claim 1 or 2, characterized in that the sample is a food sample, which is a rice, pasta, meat, vegetable, egg or dairy product sample. 4. A process according to claim 3, characterized in that the sample is extracted with ice with an extractant, which is a lipid solvent, such as a lower alcohol, before the sperm is exposed to the sample. 5. A process according to claim 4, characterized in that the extractant is methanol. 20 6. A process according to claim 4 or 5, characterized in that after extraction and evaporation, the sample is redissolved in a solvent. Process according to claim 6, characterized in that the solvent is dimethylsulfoxide or methanol. Method according to claim 4 or 5, characterized in that the extracted sample is dried and the diluted seminal fluid is exposed to the dried sample VP by placing the seminal fluid and sample in a closed vessel. 30 Method according to claim 3 or 4, characterized in that 1-10 g of samples are collected for use in the detection process. 104737 10. A process according to claim 2, characterized in that the diluted semen fluid contains 30-60 x 10 6 cells / ml and that motility is determined using 5-40 µl test sample per ml diluted seed liquid. 5 11. A method according to any of the preceding claims, characterized in that the motility is determined 8-36 hours after subjecting the sample to the sperm. 12. A method according to which as claimed in the preceding claim, characterized in that a reduction of> 50% in the motility of the sperm is interpreted as evidence of the presence of harmful amounts of toxin in the sample. 13. A method according to any one of the preceding claims, characterized in that, in addition to the motility of the sperm, one or more of the following cells are determined by vitality parameters: membrane integrity, cell ATP content and mitochondrial morphology. Packaging for use in detecting Bacillus cereus emetic toxin and corresponding mitochondrial or membrane toxins in a food, feed or drug sample, by subjecting bile sperm to the sample and determining their motility, which packaging contains - agent for determining the motility of the sperm • such as at - means for extracting the sample and / or - means for diluting the semen and possibly 25 means for detecting membrane damage, e.g. membrane dye, and - means for measuring the cell ATP content of sperm, all of which are packaged in a single package. **. ··. • ·