The following mycobacteria are routinely isolated from tissues submitted for M. bovis diagnostic culture at the National Veterinary Services Laboratories: M. smegmatis, M. terrae, M. goodii, M. fortuitum, M. kansasii, M. wolinskyi, M. simiae, M. peregrinum, M. intracellulare, M. chelonae, M. avium subsp avium, and M.avium subsp paratuberculosis. DNA from these strains were used to determine the specificity of two previously published PCR primer pairs IS41/43 1 and IS6110 2 . Both sets of primers generated a PCR product for M. bovis, M. terrae, M. goodii, and M. wolinskyi (Table 1). The IS41/43 primer pair also detected M. fortuitum; whereas, IS6110 primer pair cross-reacted with M. peregrinum and M. chelonae.

The cross reactivity of previously published primers with commonly isolated environmental mycobacteria necessitated the development of primers with greater specificity. Primers and probe for real-time PCR were developed that targeted IS6110 (IS6110_T). The primers were tested for specificity using the mycobacteria described above (Table 1). The IS6110_T primer pair generated a PCR product from M. bovis, M. wolinskyi and M. goodii using conventional PCR; however, using the primers and probe in a real-time PCR assay resulted in a positive reaction to only M. bovis and M. wolinskyi (Table 1). A 317 bp region of M. wolinskyi and M. bovis genomes that contained the region amplified by the primers used in this paper were sequenced to determine if a test with higher specificity could be developed using this region. The amplified region from M. wolinskyi was 100% identical to that of M. bovis. Portions of the 16S gene was sequenced to confirmed the identity of M. wolinskyi.

The limit of detection for IS6110_T was 100 fg of M. bovis DNA. The limit of detection was determined using serial dilutions of M. bovis DNA. Clinical tissue samples will contain bovine (or other species being tested) DNA that may inhibit the real-time PCR reaction. To determine the effect of non-target DNA on the real-time PCR reaction, increasing amounts of bovine DNA was spiked with M. bovis DNA. M. bovis was detectable in up to 1 μg of bovine DNA. At 2 μg of bovine DNA inhibition of the PCR was detected.

To test the utility of this assay to detect M. bovis in clinical samples, DNA was isolated from tissue homogenates that were culture positive for M. bovis or M. smegmatis. Real-time PCR using primers for M. bovis and bovine β-actin was performed on each sample in duplicate. β-actin served as a control to detect inhibition of the PCR reaction. A sample was considered positive when a PCR product was detected in both duplicates from β-actin and IS6110. Initial studies using both conventional and real-time PCR assays only detected 2/10 and 1/10 samples respectively. It was hypothesized that the low level of detection was due to relatively little M. bovis DNA relative to bovine DNA present in samples. To test this hypothesis, DNA from 4 homogenized tissues that were culture positive for M. bovis and one that was culture positive for M. smegmatis were chosen. Real-time PCR reactions were prepared with increasing amounts of DNA from these samples (100 ng to 1000 ng). One of the four M. bovis culture positive samples was PCR positive when 100 ng and two of four when 250 ng of DNA was used in the PCR reaction (Table 2). Increasing DNA concentration did not result in detection of the remaining samples.

To increase the sensitivity of the assay, a nested PCR strategy was developed. Initially, conventional PCR was performed using primer IS41 and the IS6110_T reverse primer to generate a 334 base pair product that encompasses the region targeted by the real-time PCR assay. Five microliters of the initial PCR reaction was used as the template in the real-time PCR assay. The samples described above were tested using the nested assay. M. bovis DNA was detected in all four of the samples from infected tissues.

Additional tissue homogenates were obtained to test the diagnostic potential of this assay. DNA was isolated from tissues of M. bovis infected cattle (n = 30) and non-M. bovis infected animals (n = 18). Of the 30 M. bovis culture positive tissues 20 were PCR positive; whereas, no PCR product was detected in the non-M. bovis infected tissues (Table 3).

The following reference strains obtained from ATCC: M. smegmatis (ATCC 35797), M. terrae (ATCC 15755), M. goodii (ATCC 700504), M. fortuitum (ATCC 6841), M. kansasii (ATCC 12478), M. wolinskyi (ATCC 700010), M. simiae (ATCC 25275), M. peregrinum (ATCC 14467), M. intracellulare (ATCC 13950) and M. chelonae (ATCC 35752). M. bovis, M. avium, and M. avium subspecies paratuberculosis were well characterized clinical field isolates. Mycobacteria were grown in grown in Middlebrook 7H9 medium supplemented with 10% oleic acid-albumin-dextrose complex (Becton Dickinson Microbiology Systems, Franklin Lakes, NJ . Mycobactin J was added to culture media when growing M. paratuberculosis. DNA was isolated using the following method. Mycobacteria from 1.5 ml o f culture were washed once with 1 × Tris-EDTA (TE) buffer and resuspended in 500 μl TE. Twenty microliters of 100 mg/ml Lysozyme (Sigma, St. Louis, MO) was added and incubated for a minimum of 3 hours or overnight at 37°C. SDS was added to a final concentration of 1% followed by the addition of 150 μg of Proteinase K (Roche Applied Science, Indianapolis, IN) and incubated for 3 hours at 65°C. Following protein digestion, 100 μl of 5 M NaCl was added. One hundred microliters of CTAB/NaCl (274 mM hexadecyltrimethylammonium bromide/0.7 M NaCl, Sigma). The resulting solution was mixed thoroughly by inverting tube then incubated at 65°C for 10 minutes. An equal volume of Phenol/chloroform/isoamyl alcohol (25:24:1) was added and an emulsion formed by vortexing then centrifuged at 16,000 × g for 5 minutes at room temperature.

The aqueous phase was removed to a fresh tube and equal volume of isopropanol was added and the samples were placed at -80°C. Precipitated DNA was collected by centrifugation at 16,000 × g for 150 minutes at 4°C. The isopropanol was removed and 1 ml of ice-cold 70% ethanol was added. The sample was centrifuged at 16,000 × g for 5 minutes and the ethanol removed. The samples were then dried at room temperature until dry. The DNA pellet was resuspended in PCR grade water.

Forty-eight (42 bovine, 5 cervine, 1 porcine) homogenized lymph nodes submitted as part of USDA's National Bovine Tuberculosis Eradication Program were obtained from the Mycobacteria and Brucella Section at the National Veterinary Services Laboratories during 2005 and 2006. Homogenization and decontamination of tissues was performed according to established protocols 7 in preparation for recovery of M. bovis by culture. Homogenates not used to inoculate culture media were frozen at -80°C until used in PCR assays. Five hundred microliters of the homogenate was heat inactivated by incubating the sample in a screw-topped microcentrifuge tube at 80°C for 40 minutes. After 20 minutes the sample was mixed. Particulate matter (including mycobacteria) was collected by centrifuging the sample for 10 minutes at 13,000 × g. The supernatant was discarded and the pellet resuspended in 500 μl ASL buffer (Qiagen, Valencia, CA) followed by a 5 min incubation at 95°C. After cooling to 56°C, 125 μg of Proteinase K (Sigma, St. Louis, MO) was added to samples. Buffer AL (Qiagen) was then added and the sample was incubated overnight at 56°C. Liberated DNA was isolated using the Qiamp Blood Mini Kit according to the manufacturer's directions except that 500 μl of cold 100% ethanol was added before the sample was applied to the column.

The following primer pairs were tested IS41/43 1 , IS6110 2 , and primers designed in house IS6110_T forward (5'-AGTTTGGTCATCAGCCGTTC-3') and IS6110_T reverse (5'-CGAACTCAAGGAGCACATCA-3') using Primer3 8 . The following optimized protocols were used with 50 μl PCR reactions.

IS41/43: A 50 μl reaction was prepared using 1 × PCR reaction buffer (final MgCl₂ 2 mM), 1 U FastStart Taq, 2.5 mM each dNTP's (Roche), 5 μg BSA (Ambion), 2.5 pmol of each primer, and with 0.5 μl DNA. A touch down PCR method was used to reduce false priming and increase specificity. The following conditions were used: an initial activation step of 94°C for 2 min. The following cycle was repeated 7 times: 94°C for 45 seconds, 72°C for 1 min (-1°C/cycle), then 72°C extension for 2 min. Amplification was continued by cycling through the following conditions for an additional 28 times: 94°C for 1 minute, 65°C for 1 min then 72°C for 2 min. A final 10 min extension was performed.

IS6110_T: 50 μl reactions were prepared as for the IS41/43 primers except that 1 pmol of each primer was added. PCR with these primers was performed using the following thermocycler conditions: an initial activation step of 94°C for 2 min. The following cycle was repeated 14 times: 94°C for 45 seconds, 65°C for 1 min (-1°C/cycle), then 72°C extension for 2 min. Amplification was continued by cycling through the following conditions an additional 28 times: 94°C for 1 minute, 50°C for 1 min then 72°C for 2 min. A final 10 min extension was performed.

IS6110 Primers: A 50 μl reaction was prepared with the following components: 1 × Gene Amp PCR Buffer II (final MgCl₂ concentration 2.5 mM), 1.25 U AmpliTaq Gold (Applied Biosystems), 2.5 mM each dNTP (Roche), 2.5 pmol each primer, and 0.5 μl template.

All PCR products were analyzed on a 1.5% agarose gel (Invitrogen) stained with ethidium bromide and visualized with a Bio-Rad Gel Doc XR.

Detection of Mycobacterial DNA by real-time PCR was performed using the IS6110_T primers and a 5' Hex labeled probe (5'-AGCCACACTTTGCGGGCACC-3') with a 3' Iowa Black FQ quencher (Integrated DNA Technologies). Taqman Universal PCR Mastermix (Applied Biosystems) was used according to the manufacturer's directions with a final primer concentration of 0.4 μM each and 0.1 μM probe. The real-time PCR was run in an ABI7500. Detection of β-actin was used as a PCR control.

PCR reactions to detect mammalian β-actin were performed parallel with each sample. The primers 5'- TCCCTGGAGAAGAGCTACGA -3' and 5'- AGGAAGGAAGGCTGGAAGAG -3' and FAM labeled probe 5'- TCACCATCGGCAATGAGCGG -3' with a 3' Iowa Black FQ quencher (Integrated DNA Technologies) were designed using Primer3Plus 9 . β-actin PCR was performed as described for IS6110_T. β-actin was included as a positive control for the PCR reaction and to detect successful DNA extraction from tissue homogenates. Lack of amplification was assumed to indicate that the PCR reaction was inhibited. For a sample to be considered positive PCR products from both the IS6110 PCR and β-actin PCR needed to occur.

The initial outside PCR reaction was carried out as described for IS6110_T (conventional PCR) except 2.5 pmol of the primers IS41 and IS6110_T reverse with 5 μg bovine serum albumin (Ambion). The second step was carried out as described above.

IS6110 Products: PCR products were excised from a 1% agarose gel after electrophoresis. The PCR products were extracted from the gel and purified using a Gel extraction Kit (Qiagen) according to the manufacturer's instructions. Sequencing was performed by the Genomics facility at the National Animal Disease Center using standard techniques.

16S Gene: Primers T39 and T13 from Talaat et al. 10 were used to PCR amplify a portion of the 16S Gene. The PCR product was purified using a MinElute PCR Purification Kit (Qiagen) according to the manufacturer's instructions. The purified product was sequenced as above. The sequence was identified as M. wolinskyi using the Ridom Databse with 100% identity 11 .

Calculation of the diagnostic specificity and sensitivity along with 95% confidence intervals (CI) were done using epiR package (version 0.9-26) in R (version 2.11.1) 12 .