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Abstract

Sufficient quality and quantity of extracted DNA is critical to detecting and performing genotyping of Clostridium botulinum by means of PCR-based methods. An ideal extraction method has to optimize DNA yield, minimize DNA degradation, allow multiple samples to be extracted, and be efficient in terms of cost, time, labor, and supplies. Eleven botulinum toxin–producing clostridia strains and 25 samples (10 food, 13 clinical, and 2 environmental samples) naturally contaminated with botulinum toxin–producing clostridia were used to compare 4 DNA extraction procedures: Chelex^® 100 matrix, Phenol-Cloroform-Isoamyl alcohol, NucliSENS^® magnetic extraction kit, and DNeasy^® Blood & Tissue kit. Integrity, purity, and amount of amplifiable DNA were evaluated. The results show that the DNeasy^® Blood & Tissue kit is the best extraction method evaluated because it provided the most pure, intact, and amplifiable DNA. However, Chelex^® 100 matrix seems to be suitable for PCR-based methods intended for laboratory diagnosis of suspected outbreaks of botulism, because it is faster and cheaper compared to DNeasy^® Blood & Tissue kit, and for samples in which the mean of Ct values obtained are statistically different (P>0.05) with respect to the best method, no lack of PCR amplification was shown. In addition, molecular methods for laboratory diagnosis currently are based on a microbial enrichment step prior to PCR, and so the differences in amplification seem to not influence the analytical results.

C lostridium botulinum strains are anaerobic spore-forming bacteria classified into 4 distinct metabolic groups based on their phylogenetic and physiological properties, or into 7 types based on the botulinum neurotoxin (BoNT) produced.¹ A number of BoNT subtypes have been recognized considering the variability in gene or protein sequences.² BoNTs, the etiologic agent of botulism, represent the more toxic substances known, with as little as 30 ng being potentially lethal.³ Since the 1930s, the use of BoNTs as a possible bioweapon has been hypothesized,^4,5 because BoNTs can be easily disseminated. High mortality rates should be expected if specific antitoxin treatment is not administered, and this requires special action for public health preparedness.

BoNTs are labeled as category A biological agents by the US Centers for Disease Control and Prevention,⁶ and botulism has been included in the disease surveillance systems of several countries. Clinical diagnosis requires laboratory confirmation based on microbiological and in vivo mouse bioassay investigations. To reduce the use of animals in identifying and typing C. botulinum strains, several PCR-based methods have been developed and reported in the literature.^7-17 In addition to being used for detection, molecular genotyping techniques have increasingly been used to correlate the isolates arising from clinical and food vehicles and to perform molecular epidemiology investigations. Among molecular genotyping methods, PCR-based techniques used in C. botulinum investigations include amplified fragment length polymorphism (AFLP), amplified rDNA restriction analysis (ARDRA), microarray, multilocus sequence typing (MLST), multiple locus variable-number tandem-repeat analysis (MLVA), random amplified polymorphic DNA (RAPD), and restriction fragment length polymorphism (RFLP).^18-24 High-quality DNA is a prerequisite to performing PCR-based techniques, and the selection of an appropriate DNA extraction method plays a crucial role in this regard.²⁵ An ideal extraction method should optimize DNA yield, minimize DNA degradation, allow the extraction of multiple samples, and be efficient in terms of cost, time, labor, and supplies.²⁶ In this article, we compare 4 genomic DNA extraction methods to establish the most suitable approach to obtaining high-quality and high-purity DNA for detection and genotyping by PCR-based techniques.

Material and Methods

Strains

Table 1 shows the 11 C. botulinum strains used in this study; they were from the Italian National Reference Centre for Botulism (NRCB) strains collection. All strains were cultured from frozen stocks and streaked for isolation in egg yolk agar (EYA) plates. Plates were incubated in anaerobic conditions at 37°C for 48 hours. For each strain, a well-isolated colony was transferred in 10 ml of tryptone-peptone-glucose-yeast extract (TPGY) broth and cultured in anaerobic conditions at 30°C for 24 hours. One hundred μl of each culture was subcultured in triplicate in 10 ml of TPGY and incubated under anaerobic conditions at 30°C for 24 hours. At the end of the incubation time, each culture was centrifuged at 4,000×g for 5 min at 4°C, and the pellet was washed twice in 10 ml of phosphate buffered saline (pH 7.4). After washing, each pellet was resuspended in 10 ml of TE buffer, divided into 5 aliquots of 2 ml, and submitted to DNA extraction as reported below.

Table 1.

List of strains submitted to DNA extraction and Bonferroni's multiple comparison tests applied to the Ct values obtained by real-time PCR amplification

					Methods (Ct Value)
					Chelex		Phenol-Chloroform-Isoamyl Alcohol		NucliSENS^®		DNeasy^® Blood and Tissue Kit
No.	Strain	Collection	Metabolic Group	bont type	Mean Diff.	P<0.05	Mean Diff.	P<0.05	Mean Diff.	P<0.05	Mean Diff.	P<0.05
1	C. botulinum type Bf	NRCB Cl 644	I	B	^*	^*	8.34	Yes	5.90	Yes	4.51	Yes
				F	0.30	No	2.80	Yes	1.32	Yes	^*	^*
2	C. botulinum type A(B)	NCTC 2916	I	A	2.91	Yes	3.91	Yes	2.87	Yes	^*	^*
				B	^*	^*	4.14	Yes	4.14	Yes	1.73	No
3	C. botulinum type A	NCTC 7272	I	A	3.03	Yes	0.69	No	^*	^*	2.70	Yes
4	C. botulinum type F	NCTC 10281	I	F	0.88	No	1.36	No	4.73	Yes	^*	^*
5	C. butyricum type E	ATCC 43755	V	E	0.45	No	^*	^*	1.37	Yes	0.42	No
6	C. botulinum type C	NCTC 8264	III	C	2.41	Yes	1.91	Yes	1.04	Yes	^*	^*
7	C. botulinum type D	NCTC 8265	III	D	0.24	No	1.09	No	1.54	Yes	^*	^*
8	C. botulinum type Ab	NRCBCl 91	I	A	0.51	No	2.15	Yes	2.83	Yes	^*	^*
				B	^*	^*	3.80	Yes	1.86	Yes	0.31	No
9	C. botulinum type B	NCTC 3815	I	B	5.28	Yes	0.41	No	0.87	No	^*	^*
10	C. botulinum type E	NRCBCl 222	II	E	^*	^*	3.43	Yes	2.83	Yes	0.93	No
11	C. botulinum type B	NRCB Cl 36	II	B	^*	^*	8.33	Yes	3.17	Yes	1.27	No

Method that produces lowest Ct values.

Naturally Contaminated Samples

Naturally contaminated samples were collected from the NRCB during outbreaks or cases of botulism that occurred in Italy. These samples were tested using the method developed and published by CDC²⁷ and by the methods published by Fach et al.¹⁷ and Anniballi et al.¹⁵ Each leftover from positive samples tested at the NRCB was immediately frozen and stored at −20°C at the end of the analysis. At the time of this study, 25 naturally contaminated samples (Table 2) were defrosted, and 3 g of each sample was cultured in 30 ml TPGY broth under anaerobic conditions at 30°C for 24 hours. At the end of incubation time, each culture was divided into 3 different portions of 10 ml and centrifuged at 4,000×g for 5 min at 4°C. Each pellet was washed twice in 10 ml of phosphate buffered saline (pH 7.4). After washing, each pellet was resuspended in 10 ml of TE buffer, divided into 5 aliquots of 2 ml, and submitted to DNA extraction as reported below.

Table 2.

List of naturally contaminated samples submitted to DNA extraction and Bonferoni's multiple comparison tests applied to the Ct values obtained by real-time PCR amplification

					Methods (Ct Value)
					Chelex		Phenol-Chloroform-Isoamyl Alcohol		NucliSENS^®		DNeasy^® Blood and Tissue Kit
No.	Strain	Collection	Type of Botulism or Source	bont Type	Mean Diff.	P<0.05	Mean Diff.	P<0.05	Mean Diff.	P<0.05	Mean Diff.	P<0.05
1	Canned meat	C. botulinum type B	Foodborne	B	^*	^*	1.05	No	0.71	No	2.92	Yes
2	Canned tuna in oil	C. botulinum type B	Foodborne	B	^*	^*	1.08	No	3.61	No	6.10	Yes
3	Enema	C. botulinum type B	Infant	B	1.46	Yes	1.95	Yes	1.33	Yes	^*	^*
4	Canned chicory	C. botulinum type B	Foodborne	B	5.43	Yes	^*	^*	2.30	Yes	0.42	No
5	Canned asparagus cream	C. botulinum type F	Foodborne	F	0.29	No	1.57	Yes	2.49	Yes	^*	^*
6	Hay	C. botulinum type B	Survey	B	0.23	No	3.44	Yes	4.34	Yes	^*	^*
7	Intestinal content	C. botulinum type C	Animal-Avian	C	1.89	Yes	1.02	No	0.37	No	^*	^*
8	Canned mushrooms in oil	C. botulinum type B	Foodborne	B	1.56	No	^*	^*	4.31	No	1.99	No
9	Canned homogenized meat	C. botulinum type A	Foodborne	A	^*	^*	0.24	No	5.01	Yes	5.70	Yes
10	Faeces	C. botulinum type B	Infant	B	2.61	Yes	4.61	Yes	4.82	Yes	^*	^*
11	Canned asparagus	C. botulinum type F	Foodborne	F	1.60	No	2.29	Yes	3.56	Yes	^*	^*
12	Faeces	C. butyricum type E	Infant	E	^*	^*	1.46	Yes	2.07	Yes	0.02	No
13	Faeces	C. botulinum type F	Foodborne	F	0.62	No	0.97	No	0.33	No	^*	^*
14	Intestinal content	C. botulinum type D	Animal - Cattle	D	0.14	No	1.74	Yes	3.58	Yes	^*	^*
15	Canned bacon	C. botulinum type B np	Foodborne	B	1.90	Yes	7.40	Yes	6.74	Yes	^*	^*
16	Faeces	C. botulinum type B np	Foodborne	B	0.50	No	4.00	Yes	2.34	No	^*	^*
17	Fresh beans	C. botulinum type BF	Foodborne	B	0.52	No	4.92	Yes	7.86	Yes	^*	^*
				F	2.58	Yes	1.32	No	1.54	No	^*	^*
18	Faeces	C. botulinum type BF	Foodborne	B	^*	^*	2.83	Yes	5.95	Yes	1.34	No
				F	1.23	No	6.78	Yes	3.82	Yes	^*	^*
19	Faeces	C. botulinum type A	Foodborne	A	0.62	No	4.70	Yes	2.65	Yes	^*	^*
20	Peppers and tuna in oil	C. botulinum type A	Foodborne	A	0.90	No	0.54	No	1.33	No	^*	^*
21	Faeces	C. botulinum type C	Animal, Avian	C	3.71	Yes	2.69	Yes	3.98	Yes	^*	^*
22	Larvae	C. botulinum type C	Animal, Avian	C	2.12	Yes	0.23	No	2.59	Yes	^*	^*
23	Liver	C. botulinum type C	Animal, Avian	C	1.32	Yes	0.13	No	0.46	No	^*	^*
24	Liver	C. botulinum type C	Animal, Avian	C	1.94	No	2.47	Yes	2.13	Yes	^*	^*
25	Intestinal content	C. botulinum type C	Animal, Avian	C	^*	^*	4.31	Yes	3.50	Yes	0.55	No

Method that produces lowest Ct values.

DNA Extraction Methods

Four different DNA extraction methods were evaluated using 2-ml aliquot cell suspension described above. All DNA samples extracted using the methods reported below were stored at −70°C until use.

Method 1: Chelex 100 Matrix

Method 1 consisted of DNA extraction using Chelex^® 100 matrix (Sigma-Aldrich, Munich, Germany) as reported by Fenicia et al. with some modification.¹¹ Briefly, each 2-ml aliquot of strain or sample culture was centrifuged at 12,000×g for 5 min. Each pellet was treated with 200 μl of a 6% Chelex® 100 matrix solution at 56°C for 20 min, vortexed, treated at 99°C for 8 min, and immediately chilled on ice. The DNA was harvested after centrifugation at 12,000×g for 5 min, and stored at −70°C until use.

Method 2: Phenol-Chloroform-Isoamyl Alcohol

Method 2 was a phenol-chloroform-isoamyl alcohol–based extraction. One aliquot of each strain and sample culture was centrifuged at 12,000×g for 5 min and resuspended in 500 μl of TE buffer in a 2-ml tube. Each tube was first treated with 100 μl of a solution containing 10 mg ml⁻¹ of lysozyme and incubated at 37°C for 60 min, and then with 100 μl of a solution containing 10 mg ml⁻¹ of proteinase K and incubated at 60°C for 60 min. The DNA was extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) solution and precipitated overnight at −20°C with absolute ethanol, in presence of 0.2 mol L⁻¹ NaCl. The DNA was harvested by centrifugation at 14,000×g for 20 min, washed with ethanol 70% solution, and dissolved in 50 μl of TE buffer containing 10 mg ml⁻¹ of RNase DNase-free. After RNase treatment (37°C for 60 min), DNA was resubmitted to extraction with phenol:chloroform:isoamyl alcohol (25:24:1) solution, precipitated at −70°C for 30 min, and washed in 70% ethanol solution. Finally, the DNA was dissolved in 200 μl of TE buffer and stored at −70°C until use.

Method 3: NucliSENS® miniMAG

Method 3 was the direct application of a NucliSENS^® miniMAG kit provided by bioMérieux (bioMérieux, Lyon, France). The DNA extraction using this method was carried out according to the manufacturer's instructions. All samples were diluted up to a final volume of 200 μl.

Method 4: DNeasy® Blood & Tissue Kit

Method 4 was the direct application a DNeasy^® Blood & Tissue kit provided by Qiagen (Qiagen, Hilden, Germany). The DNA extraction using this method was carried out according to the manufacturer's instructions.

Spectrophotometer Measurements

Quality and concentration of extracted DNA was assessed using Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). DNA absorbance was measured in the elution buffers provided for each extraction method, and the spectrophotometer was blanked with the corresponding buffer before measurement. Absorbance ratio at 260/280 nm was used to assess the purity. Samples with a 260/280 nm ratio of 1.8 to 2.0 are considered pure or relatively free or clean of contaminants. Samples with lower values might be contaminated with protein, phenol, or some other UV-absorbing coextracted materials.^26,28

Agarose Gel Electrophoresis

The integrity of extracted DNA was assessed by gel electrophoresis. Specifically, 10 μl of each sample was analyzed in a 0.8% agarose gel containing Atlas Sight DNA Stain (Bioatals, Tartu, Estonia) and visualized by UV illumination. The presence of an intense band in the gel corresponding to the upper line of molecular weight marker GeneRuler 1 kb Plus (Fermentas, Glen Burnie, MD, USA) indicates intact genomic DNA with minimal degradation. Degraded or sheared DNA and RNA contamination can be visualized as a smear toward the lower molecular weight portion of the gel.²⁹

Real-time PCR

To assess the amount of amplifiable DNA obtained by means of the extraction methods compared in this study, the real-time PCR protocols published by Fach et al.¹⁷ and Anniballi et al.¹⁵ were carried out. Real-time PCR results were expressed as Ct (threshold cycle) values. A Ct value is the fractional cycle in which the increase in the fluorescence generated by the accumulation exceeds 10 standard deviations of the mean baseline fluorescence, with a selected range of cycles. Ct value is inversely proportional to the amount of target nucleic acid in the sample (ie, the lower the Ct level the greater the amount of target nucleic acid in the sample).

Statistical Analysis

To determine whether the variability of each evaluation criterion (260/280 nm absorbance ratio, Ct values) was significant, values were compared using one-way analysis of variance (ANOVA). When significant differences were identified post hoc analysis, Bonferroni's multiple comparison test was performed. Statistical significance level was set at 0.05. All analysis was performed using Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA).

Results

In Supplementary data (Tables 3 and 4, www.liebertonline.com/bsp) the mean concentration, absorbance 260/280 nm ratio, and Ct values obtained for the strains and the naturally contaminated samples tested were reported.

Quality and Concentration of Extracted DNA

The 4 extraction methods tested showed statistically significant differences in terms of DNA yields (P<0.05). The highest mean DNA yield was obtained with method 1, followed by method 4, while method 2 yielded the lower amount of DNA. Although the amount of DNA was higher for method 1, the nucleic acid extracted results were poor in terms of purity, showing low absorbance 260/280 nm ratio and suggesting the presence of contaminants such us carbohydrates and proteins. The purest DNA was obtained by means of method 4. Method 2 yielded pure DNA for 5 out of 11 strains and 5 out of 25 samples.

Integrity of Extracted DNA

Gel electrophoresis revealed that method 1 produced smeared DNA; methods 2 and 4 produced weak bands corresponding to the upper line of molecular weight marker GeneRuler 1 kb Plus, and very few or no smears. Method 3 produced DNA contaminated with RNA visible as a weak band at 2100 bp (data not shown).

Amplifiable DNA

Regarding the DNA quality for molecular applications, all 4 methods provided sufficient template for PCR, although methods 1 and 4 gave better results than methods 2 and 3. The 11 strains analyzed also included 3 bivalent strains that harbor 2 gene encodings for BoNTs and provided a total of 14 comparisons.

Method 1 produced the lowest Ct values in 5 comparisons (35%), and in another 5 comparisons the results were not statistically different (P<0.05) with respect to those produced by the extraction method that gave the lowest Ct values. Method 2 produced the lowest Ct values for 1 comparison, while in 4 other comparisons, the Ct values were not statistically different (P<0.05) with respect to those produced by the best method for these comparisons. Method 3 produced the lowest Ct values for 1 comparison and a comparison that was not statistically different in another case. Method 4 produced the lowest Ct values in 7 comparisons, and in 5 other comparisons the results were not statistically different (P<0.05) with respect to those produced by the best method for these strains.

The 25 naturally contaminated samples analyzed also included 2 samples contaminated with strains harboring 2 gene encodings for BoNTs, and a total of 27 comparisons were established. Method 1 produced the lowest Ct values in 6 comparisons, and in another 12 comparisons the results produced were not statistically different (P<0.05) from those provided by the best method. Method 2 produced the lowest Ct values in 2 comparison and not significantly different results in 9 comparisons against the best method. Method 3 produced the lowest Ct values with respect to the others, although in 9 comparisons the results were not statistically different with respect to those provided by the most suitable method. Method 4 produced the lowest Ct values in 19 comparisons and in a further 5 comparisons did not provide statistically significant differences against the most suitable method.

Discussion

In past decade molecular diagnostic methods have become routine in clinical laboratories as well as in laboratories that perform food analysis. The effectiveness of the molecular methods are strongly influenced by the DNA extraction—in fact, it is widely accepted that DNA extraction can influence the sensitivity of PCR-based methods for levels of DNA yield, purity, and integrity.^30-32

Lower purity of nucleic acids due to the PCR inhibitors may result in inefficient PCR amplification. PCR inhibitors may denature or precipitate DNA or bind magnesium ions, reducing the enzymatic activity of DNA polymerases.³⁰ Several substances such as DNA extraction reagents (eg, EDTA, ethanol, isopropanol, phenol, SDS, sodium acetate, sodium chloride), acid plant polysaccharides (eg, dextran sulphate, alginic acid), fats, and proteins may exert PCR inhibition effects. Some of these inhibition substances may be harbored by raw food materials.³³

In this study we investigated 4 of the most common DNA extraction procedures used in molecular biology diagnostic laboratories. The overall comparison of all parameters evaluated to establish the most suitable method for detection and genotyping of C. botulinum strains by means of PCR-based approaches shows that method 4 (DNeasy^® Blood & Tissue kit) is the best method for both detection and genotyping techniques. In fact, this method provides the most pure, most intact, and more amplifiable DNA compared to the others. However, method 1 (Chelex^® 100 matrix) seems to be suitable for PCR-based methods intended for laboratory diagnosis of suspected outbreaks of botulism, because it is faster and cheaper compared to method 4, and for samples in which the mean of Ct values obtained are statistically different (P>0.05) with respect to the best method, no lack of PCR amplification was shown. In addition, molecular methods for laboratory diagnosis currently are based on a microbial enrichment step prior to PCR, and so the differences in amplification seem to not influence the analytical results.

Footnotes

Acknowledgments

This research was supported by the framework of the EU project AniBioThreat (Grant Agreement: Home/2009/ISEC/AG/191) with financial support from the Prevention of and Fight against Crime Programme of the European Union, European Commission—Directorate General Home Affairs. This publication reflects the views only of the authors, and the European Commission cannot be held responsible for any use that may be made of the information contained therein.

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Supplementary Material

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Evaluation of DNA Extraction Methods Suitable for PCR-based Detection and Genotyping of Clostridium botulinum

Abstract

Material and Methods

Strains

Naturally Contaminated Samples

DNA Extraction Methods

Method 1: Chelex 100 Matrix

Method 2: Phenol-Chloroform-Isoamyl Alcohol

Method 3: NucliSENS® miniMAG

Method 4: DNeasy® Blood & Tissue Kit

Spectrophotometer Measurements

Agarose Gel Electrophoresis

Real-time PCR

Statistical Analysis

Results

Quality and Concentration of Extracted DNA

Integrity of Extracted DNA

Amplifiable DNA

Discussion

Footnotes

Acknowledgments

References

Supplementary Material