A review of: Sakuma, T., Kurosaki, Y., Fujinami, Y., Takizawa, T. and Yasuda, J., 2009. Rapid and simple detection of Clostridium botulinum types A and B by loop-mediated isothermal amplification. Journal of Applied Microbiology, 106(4), 1252-1259.
Botulism is a mainly food-borne intoxication disease caused by a gram-positive, spore-forming bacterium called Clostridium botulinum. The bacterium can produce seven recognised types of neurotoxin (BoNT), labelled as BoNT/A through to G. Types A, B, and E are most commonly associated with illness in humans. Type A and B account for more than 85% of food-borne botulism cases in humans. Type F is also associated, but rarely. The other types (C, D, G) cause illness in other mammals, birds and fish. The spore-forming bacterium is commonly found in soils, aquatic sediment and fish. BoNT consists of heavy and light chains - Part of the heavy chain binds to the neurological cell membrane, and the light chain is correlated with the inhibition of acetylcholine release at neuromuscular junctions. Acetylcholine is a major neurotransmitter, and once inhibited neurotransmission failure will occur, leading to serious neurological disorders and potentially death.
The aim of this study was to develop a detection method for the bacterium toxins A and B using a loop-mediated isothermal amplification (LAMP) method. Currently the typical method of detection is the mouse bioassay due to its great sensitivity and specificity. However it also comes with some problems, being very costly and time consuming, and also raising animal rights issues. PCR-based methods have also been well developed but carry the same problems of time and cost due to the need for sophisticated equipment. So this study aimed to test if LAMP methods could be as sensitive and specific at detection, while being convenient, less costly and respectively quick.
14 Clostridium botulinum strains and 17 non-Clostridium botulinum strains were used. All strains were grown on GAM agar plates for one or two days at 37°C. C. botulinum type A spores were prepared by growing the selected A strain on GAM plates at 30°C anaerobically for seven days. Type B spores were prepared using the corresponding B strain on plates at 37°C for seven days, both of which were followed by aerobic incubation. Colonies were collected from the plates, and the DNA was isolated and extracted. The BoNT gene fragment encoding the light chain of C. botulinum was amplified by PCR using the extracted DNA for strains A and B, and designed primers for each type. The fragments were then inserted into a vector and several clones were sequenced. LAMP assays were then performed at a constant temperature of 63°C for 60mins. A positive result/detection was indicated by a turbidity measurement of >0.1.
The results showed that LAMP assays for BoNT/A and BoNT/B genes were very successful in detecting only type A and type B strains, within a very short time period of only 23 minutes. This presents the LAMP assays as very rapid techniques with highly specific results for both type A and type B strains of C. botulinum. The sensitivity of LAMP assay for both strains was also determined as very high. Positive results were also achieved within 30 minutes when the amount of DNA/assay was 10pg +. The sensitivity is almost equal to results achieved by PCR-based methods in past studies for BoNT/A genes but this method is considerably quicker and very simple in comparison. The study also examined the usefulness of the LAMP method in detecting the toxins in food. Based on previous studies, honey and canned fish were tested, and detection was very successful and very quick for both spores and vegetative cells, achieving positive results in <25mins. However, in larger scale food samples (>1kg) this method would be much more timely due to cultivation before DNA preparation and so it may not really be applicable.
In conclusion, while the methodology is quite confusing and long-winded, it is actually exceptionally simple compared with any other applicable methods. The paper also does a very good job of splitting the methods into steps which makes it much easier to digest. The introduction isn’t very long and is pretty cracking on its own for a simple understanding of the study. Overall, it was shown that this method is highly specific and sensitive for the detection of C. botulinum types A and B. It is also incredibly simple in comparison to other methods. It is not costly due to no need for sophisticated equipment (positive results achieved by visibility – turbidity), and it is very quick making applying it to environmental samples quite possible.
Botulism is a mainly food-borne intoxication disease caused by a gram-positive, spore-forming bacterium called Clostridium botulinum. The bacterium can produce seven recognised types of neurotoxin (BoNT), labelled as BoNT/A through to G. Types A, B, and E are most commonly associated with illness in humans. Type A and B account for more than 85% of food-borne botulism cases in humans. Type F is also associated, but rarely. The other types (C, D, G) cause illness in other mammals, birds and fish. The spore-forming bacterium is commonly found in soils, aquatic sediment and fish. BoNT consists of heavy and light chains - Part of the heavy chain binds to the neurological cell membrane, and the light chain is correlated with the inhibition of acetylcholine release at neuromuscular junctions. Acetylcholine is a major neurotransmitter, and once inhibited neurotransmission failure will occur, leading to serious neurological disorders and potentially death.
The aim of this study was to develop a detection method for the bacterium toxins A and B using a loop-mediated isothermal amplification (LAMP) method. Currently the typical method of detection is the mouse bioassay due to its great sensitivity and specificity. However it also comes with some problems, being very costly and time consuming, and also raising animal rights issues. PCR-based methods have also been well developed but carry the same problems of time and cost due to the need for sophisticated equipment. So this study aimed to test if LAMP methods could be as sensitive and specific at detection, while being convenient, less costly and respectively quick.
14 Clostridium botulinum strains and 17 non-Clostridium botulinum strains were used. All strains were grown on GAM agar plates for one or two days at 37°C. C. botulinum type A spores were prepared by growing the selected A strain on GAM plates at 30°C anaerobically for seven days. Type B spores were prepared using the corresponding B strain on plates at 37°C for seven days, both of which were followed by aerobic incubation. Colonies were collected from the plates, and the DNA was isolated and extracted. The BoNT gene fragment encoding the light chain of C. botulinum was amplified by PCR using the extracted DNA for strains A and B, and designed primers for each type. The fragments were then inserted into a vector and several clones were sequenced. LAMP assays were then performed at a constant temperature of 63°C for 60mins. A positive result/detection was indicated by a turbidity measurement of >0.1.
The results showed that LAMP assays for BoNT/A and BoNT/B genes were very successful in detecting only type A and type B strains, within a very short time period of only 23 minutes. This presents the LAMP assays as very rapid techniques with highly specific results for both type A and type B strains of C. botulinum. The sensitivity of LAMP assay for both strains was also determined as very high. Positive results were also achieved within 30 minutes when the amount of DNA/assay was 10pg +. The sensitivity is almost equal to results achieved by PCR-based methods in past studies for BoNT/A genes but this method is considerably quicker and very simple in comparison. The study also examined the usefulness of the LAMP method in detecting the toxins in food. Based on previous studies, honey and canned fish were tested, and detection was very successful and very quick for both spores and vegetative cells, achieving positive results in <25mins. However, in larger scale food samples (>1kg) this method would be much more timely due to cultivation before DNA preparation and so it may not really be applicable.
In conclusion, while the methodology is quite confusing and long-winded, it is actually exceptionally simple compared with any other applicable methods. The paper also does a very good job of splitting the methods into steps which makes it much easier to digest. The introduction isn’t very long and is pretty cracking on its own for a simple understanding of the study. Overall, it was shown that this method is highly specific and sensitive for the detection of C. botulinum types A and B. It is also incredibly simple in comparison to other methods. It is not costly due to no need for sophisticated equipment (positive results achieved by visibility – turbidity), and it is very quick making applying it to environmental samples quite possible.
4 comments:
Hi Sami
I hope the revision is going well!
Its surprising just how quickly new technology and techniques are being developed. I heard the other day how a small genome can be sequenced on a device the size of a memory stick in about an hour, something ridiculous like that anyway.
Just one quick question about your review. How does the LAMP assay works? It sounds familiar, is it a relativley new method?
Hey,
Thats quite cool but are there many cases of botulism anymore?
Hi Lee!
The LAMP assay is indeed a relatively new technique (Notomi et al, 2000), and it is replacing many others due to reductions in cost and its simplicity. The LAMP assay in this study was performed using a Loopamp DNA Amplification Kit. Lots of these sorts of tests have kits you can buy now – even easier! There’s quite a lot of rambly info in the paper if you wanted to have a look, or it may be worth referring to the original Notomi paper.
Hope your revisions going well too!
Hi Matt!
Botulism is very rare, but the botulinum toxin is also the most lethal known toxin so it’s not to be ignored. Incidents still occur from improper canning or preservation of food, especially when done at home, and most likely within communities that still practice traditional food preparation methods. There are also cases of outbreaks within food companies as recently as 2007 (Castleberry’s Food Company). And if you choose to believe reports...A couple of people got infected with botulism at the end of last year from Lloyd Grossman korma curry sauce, leading to a recall of nearly 50,000 units. So.. it is rare, but its still pretty important to keep improving prevention methods.
But the focus is more really on how useful the technique has become, as its being increasingly used to detect many other infectious diseases now too.
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