Comparison of Three Methods for Extraction of Mycobacterium Avium Subspecies Paratuberculosis DNA for Polymerase Chain Reaction from Broth-Based Culture Systems

Abstract

Conventional and real-time polymerase chain reaction (PCR) assays were used to measure the recovery of DNA from Mycobacterium avium subspecies paratuberculosis (MAP) extracted with 3 different methods (MagMAX^™, DNeasy^®, and phenol-chloroform) after growth in a broth-based culture system. Of the 304 samples tested, bacterial DNA was detected in 197 (65%) of samples after MagMAX, 156 (51%) after phenol-chloroform, and 123 (40%) after DNeasy extractions. By acid-fast stain, 177 (58%) of the samples yielded acid-fast–positive bacilli, of which 4 were PCR negative by the 3 extraction methods. The results demonstrated that the amplifiable MAP DNA, as evidenced by the number of PCR-positive cultures and amplicon intensity on ethidium bromide–stained agarose gel, was best for MagMAX, intermediate for phenol-chloroform, and least for DNeasy. When subjected to real-time polymerase chain reaction, the MagMAX extracts produced the best results, thereby making it an excellent kit for the efficient extraction of MAP DNA from the broth-based culture system.

Mycobacterium avium subspecies paratuberculosis (MAP) infection in cattle and several other domestic and wild animals causes chronic granulomatous enteritis. 1,6 Clinical signs include weight loss, fatigue, diarrhea, decreased milk production, and mortality, resulting in substantial economic loss. 1 Recently, MAP was associated with Crohn disease in humans, thus making it a pathogen of public health concern. 2

For decades, identification of MAP-infected animals was dependent on fecal culture on solid media, which takes up to 16 weeks to conclude because of the slow growth rate of the bacteria. The need for a faster turnaround time prompted the development of broth-based, nonradiometric methods that enhance the growth of the organism as well as an instrument that continuously monitors bacterial growth and produces signals when positives are detected. 1 Unlike solid media, the broth-based methods take a maximum of 5 weeks to conclude. Because the instrument fails in some instances to signal positive growth when low numbers of organisms are present, acid-fast stain is performed on all samples, and the identity of acid-fast–positive bacilli is confirmed by polymerase chain reaction (PCR) using MAP species-specific primers. However, since the introduction of broth-based culture methods, there have been concerns about inability to amplify by PCR the DNA extracted from MAP after growth in the egg yolk–rich dye containing broth (O. Okwumabua, personal communication, 2008). In addition, MAP has a cell wall that impedes lysis and good recovery of nucleic acids. For accurate reporting of results, these concerns are of major consequence to laboratories that perform broth-based techniques because the PCR method is currently the method of choice for culture confirmation. Among other factors, good yield and purity of DNA is essential for efficient performance of PCR assay. For these reasons, the current study sought to find a DNA extraction method that was easy to use, removed inhibitors, produced good bacterial lysis, and, as a consequence, increased PCR sensitivity. For this purpose, 3 methods of extraction were compared, namely, the MagMAX^™ Total Nucleic Acid Isolation Kit, a the DNeasy^® Blood and Tissue Kit, b and phenol-chloroform. 5

A total of 304 fecal samples were used in the present evaluation. Fifty samples were obtained from the National Veterinary Services Laboratory (NVSL; Ames, IA) as part of the Johne's national annual proficiency test administered to veterinary diagnostic laboratories for certification. Thirty-six of the 50 samples were confirmed positives and 14 were confirmed negatives for MAP. Two hundred fifty-four samples were collected from cattle with clinically proven or suspected MAP infection submitted to the Wisconsin Veterinary Diagnostic Laboratory (Madison, WI) for culture. Each fecal sample was processed by the double incubation method, inoculated into broth culture bottles as described elsewhere, 1 and incubated. c,d Acid-fast stain was then performed e following standard procedures when the culture system signaled positive growth and at the end of the incubation period (42 days) for samples producing no growth signal on the instrument.

Table 1.

Comparative results of positive polymerase chain reaction (PCR) assay on Mycobacterium avium subspecies paratuberculosis (MAP) DNA extracted using MagMAX^™, DNeasy^®, and phenol-chloroform cultured in broth-based systems.

No. positive by PCR		No. negative by PCR
Extraction method	No. of samples evaluated	No. positive by acid fast	No. negative by acid fast
MagMAX *	197/304	197/177	107/127
DNeasy †	123/304	123/177	181/127
Phenol-chloroform	156/304	156/177	148/127

*

Applied Biosystems, Foster City, CA.

†

Qiagen Inc., Valencia, CA.

For DNA extraction, 500 μl of culture was used as the uniform volume for all protocols and centrifuged at 16,000 × g for 20 min. The supernatant was discarded, and the resulting pellet was used. Extraction with MagMAX and DNeasy was performed according to the manufacturer's instructions. For the phenol-chloroform extraction method, the cell pellet was resuspended in 100 μl of sample buffer consisting of 1 M Tris (pH 8.0), 1 mM ethylenedi-amine tetra-acetic acid, and 1.0% Triton-X 100 and placed in a heat block at 100°C for 30 min to lyse the cells. 4 The lysate was allowed to cool to room temperature followed by centrifugation at 10,000 × g for 1 min. The cell-free supernatant containing DNA was transferred to clean sterile Eppendorf tubes f and processed using the standard phenol-chloroform–isoamyl alcohol method. 5 Finally, the DNA pellet was washed with 70% ethanol, vacuum dried, and dissolved in 20 μl of water. Conventional PCR amplification reactions were performed in a total volume of 50 μl containing 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl₂, 50 mM KCl, 0.001% gelatin, 200 μM of each deoxynucleo-side triphosphate, 1 μM of each MAP-specific P90 and P91 IS900-based primers, 3,7 2.5 U of Taq polymerase, g and 5 μl of the extracted DNA. The PCR assay was carried out in a commercial PCR system consisting of 5 min of preincubation at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C. Final extension was performed for 7 min at 72°C. DNA extracted from a known MAP-positive fecal sample was used as the positive control template. The negative control was a reaction mixture containing all reagents and extraction from a known MAP-negative fecal sample. The PCR products were visualized after electrophoresis on a 1% agarose gel following standard procedures. The real-time PCR was performed with a commercial Johne's real-time PCR kit h,i following the manufacturer's protocol, while DNA sequencing was performed using the dideoxychain termination method. 5

Figure 1.

Example of ethidium bromide–stained agarose gel showing the approximately 400-bp Mycobacterium avium subspecies paratuberculosis band after electrophoresis of polymerase chain reaction products using DNA extracted with the MagMAX^™ and phenol-chloroform methods. A, MagMAX, and B, phenol-chloroform. DNeasy is not shown. Lane M: ØX174/HaeIII molecular weight standards; lanes 1–9: clinical samples; lane 10: negative control; lane 11: positive control.

Results are summarized in Table 1. Of the 304 cultures, 177 (58%) yielded bacilli that were acid-fast positive and exhibited typical MAP cell type. Following DNA extraction by the MagMAX method, 197 (65%) were PCR positive, of which 24 were acid-fast negative. Four samples that were acid-fast positive yielded PCR-negative results. Sequencing and analysis of the PCR product of the 24 PCR-positive but acid-fast–negative samples revealed 100% nucleotide sequence identity with the DNA of MAP using a BLASTn search (http://www.ncbi.nlm.nih.gov/blast/BLAST.cgi). This may indicate the presence of a low number of bacteria in some of the culture broth below the limit of detection by the acid-fast method. The cultures that were acid-fast positive but PCR negative most likely were Mycobacteria spp. other than MAP. Similar observations have previously been reported. 1 Extraction by the phenol-chloroform method yielded 156 (51%) of amplifiable MAP DNA from the samples, whereas PCR of the DNeasy extract amplified DNA for MAP from 123 (40%) of the samples. In both cases, the PCR-positive samples were also acid-fast positive. With respect to the samples with known MAP status obtained from NVSL, the 36 true-positive samples were PCR positive for MAP, whereas the 14 negative samples remained negative after the MagMAX extraction method, suggesting efficient MAP DNA extraction. In comparison, 30 of the 36 known positive samples yielded amplifiable MAP DNA after the phenol-chloroform extraction method, whereas only 26 of the 36 known positive samples were PCR positive after DNeasy (data not shown). The known negative samples were PCR negative following extraction with both phenol-chloroform and DNeasy methods. Because the true-positive samples that were PCR negative with both methods contained low numbers of MAP as determined by the NVSL, it is likely that both methods produced levels of MAP DNA that were suboptimal for PCR detection.

Thus, the amplifiable MAP DNA, as evidenced by the signal intensity on the conventional PCR and sensitivity as measured by the number of positives, was best for the MagMAX method, intermediate for phenol-chloroform, and least for the DNeasy (Table 1; Fig. 1). The possibility that the DNA extracted using the MagMAX method could be used as template for real-time PCR was also tested. This method of amplification also produced good results, suggesting that the MagMAX is a good reagent for the extraction of MAP DNA grown in the broth-based culture system. It is worth mentioning that since the conclusion of the present evaluation, the authors′ laboratory has switched from using the phenol-chloroform method to using the MagMAX extraction kit and have processed more than 7,000 samples with results of exceptional quality and reliability.

In summary, of the 3 extraction methods evaluated, the MagMAX method provided the greatest amplifiable MAP DNA. In addition, the MagMAX procedure was technically simple, was not time-consuming, and results were highly reproducible. It is therefore recommended for extraction of MAP DNA.