Biology Reference
In-Depth Information
production had been rapid and that there had been marked temperature
abuse during storage or transport of the fi sh. Type E toxin was confi rmed
by toxin neutralization and the mouse bioassay and by PCR.
PCR for Detection of C. botulinum Type E Strains
Hielm et al. (1996) developed protocols for combined PCR-MPN detection
and enumeration of C. botulinum types A, B, E, and F in fi sh and sediment
samples. Spore counts of type E in sediment samples varied from 95 to 2710
per kg of sample. Rainbow trout were seeded with spores of C. botulinum
type E at 10 2 to 10 6 spores per kg of tissue in addition to inoculating fi sh
intestines. Each sample was subjected to a 5-d enrichment in TPGY broth
followed by transfer of 0.5 ml into 10 ml of TPGY broth with overnight
incubation prior to PCR. Washed vegetative cells from such enrichment
broth cultures were boiled for 10 min and 1 ml incorporated into PCR
reactions. The primers GF-1/GF-3 ( Table 5.2 ) from Franciosa et al. (1994)
amplifi ed a 760-bp sequence of the botulinum neurotoxin type E gene
( bontE ). All seeded samples were detected as positive.
Alsallami and Kotlwoski (2001) developed improved primer pairs
for detection of the BoNTB and BoNTE genes. The detection limit was
increased from 1 to 0.1 ng of DNA by increasing the annealing temperature
from 50°C to 62°C. The primers BoTE1/BoTE2 ( Table 5.2 ) amplifi ed a 307-
bp sequence of the botnE gene.
Lindström et al. (2001) developed a multiplex PCR for detection of
C. botulinum types A, B, E, and F in food and fecal material. The primer pairs
( Table 5.2 ) yielded amplicons of 782-, 205-, 389-, and 543-bp respectively.
With a two-step enrichment the detection limit in food and fecal samples
was 10 spores per 0.1g of sample material for type E.
Kimura et al. (2001) developed a Rti-PCR assay for quantifying
C. botulinum type E in modifi ed-atmosphere packaged fi sh samples (jack
mackeral). The primers BE1430F/BE1709R ( Table 5.2 ) amplifi ed a 269-bp
sequence of the botnE gene. The dual labeled probe BE1571FP ( Table 5.2 )
was labeled at the 5'-end with 6-FAM and at the 3'-end with TAMRA. The
quantifi able range was 10 2 to 10 8 CFU/g which allowed detection much
earlier than the toxin could be detected with the mouse bioassay.
Sharkey et al. (2004) developed a competitive reverse transcription
PCR assay (cRT-PCR) to quantify toxin-encoding mRNA production by
a type E strain in media with either sorbic acid or sodium nitrite. The
primers mRNA-EF/mRNA-ER ( Table 5.2 ) amplifi ed a 250-bp sequence of
the BoNTE mRNA. A 10-fold reduction in toxin mRNA production and a
25-fold reduction in the proportion of mRNA to total RNA was estimated
when either 1 mg/ml of sorbic acid or 100 mg/ml of sodium nitrite was
added to the medium at pH 7.0.
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