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Appl Environ Microbiol. May 2008; 74(9): 2751–2758.
Published online Mar 7, 2008. doi:  10.1128/AEM.02534-07
PMCID: PMC2394907

New Triplex Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Feces[down-pointing small open triangle]

Abstract

In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqManmgb and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 102 CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.

Mycobacterium avium subsp. paratuberculosis is the causative agent of ruminant paratuberculosis (Johne's disease), which has become a worldwide problem. There is controversy regarding its zoonotic capacity and potential role in the human Crohn's disease (14). Because of these reasons, a rapid, cost-effective, and automated diagnosis of this pathogen is a high priority task not only for animal breeders but also for the food production industry and for public health institutions. Culture-based detection of M. avium subsp. paratuberculosis is time-consuming, labor-intensive, and therefore not suitable. The PCR has been shown to be a powerful tool in microbiological diagnostics (12, 43). Guidelines for diagnostic quality assurance have been set by the International Organization for Standardization (7, 8). Standardized PCR and real-time PCR methods should fulfill numerous criteria, including a high detection probability with regard to the investigated matrix, the sample preparation, and DNA extraction as well as high specificity, robustness, and user-friendly protocols. In this context the real-time PCR technology offers the possibility for a one-step and closed-tube reaction (13).

As a molecular reference marker for the confirmation of M. avium subsp. paratuberculosis, the insertion sequence IS900 is commonly used (15, 24). Because of a considerably high sequence similarity with IS900-like elements or other genetic elements, cross-reactions might give false-positive results (16, 22; for a review of diagnostic tests, see reference 26). According to numerous authors, PCR analysis was unable to match the sensitivity of fecal culture for identifying minute quantities of M. avium subsp. paratuberculosis (49, 56). An increased sensitivity of the PCR analysis can be achieved by improved DNA extraction protocols guaranteeing the efficient removal of PCR inhibitors such as phytic acid, polyphenolics, polysaccharides, and hemin (4, 5, 18, 37, 52).

The aim of the present study was the development and careful validation of a new real-time PCR assay, considering the existing guidelines for PCR- and real-time PCR-based detection methods. The assay should offer the potential to be used as a stand-alone application for the detection of M. avium subsp. paratuberculosis from bovine fecal samples without additional PCR confirmation tests. For this purpose, the M. avium subsp. paratuberculosis marker sequences F57 and ISMav2 were combined with an internal amplification control (IAC) into a triplex real-time PCR assay. Broad-range applicability was assessed by considering PCR reagents from different suppliers. Robustness testing included two different fluorogenic probe formats and two different real-time thermocycler models, both representing widely used technologies. In addition, the fecal sample preparation and DNA extraction protocol was optimized. The applicability of our method was compared to those of the cultural gold standard and IS900 nested PCR (12) by testing 108 bovine fecal samples of unknown M. avium subsp. paratuberculosis status.

MATERIALS AND METHODS

Bacterial reference strains.

For estimation of the specificity of the developed real-time PCR assay, a total of 205 strains were used (Table (Table1).1). The 105 M. avium subsp. paratuberculosis strains collected for sensitivity testing contained two official type collection strains (DSM 44133 and DSM 44135) and 103 M. avium subsp. paratuberculosis field strains of bovine (n = 95), ovine (n = 5), and human (n = 3) origin. These strains had not been characterized for the marker genes F57 and ISMav2 before. The 34 non-M. avium subsp. paratuberculosis strains and the 65 nonmycobacterial strains used for specificity testing were selected because of their close genetic relationship to M. avium subsp. paratuberculosis or because they are found in the same environment and grow under similar conditions. The Mycobacterium strains were cultured on Herrolds egg yolk medium (HEYM) (Becton Dickinson, Heidelberg, Germany) with Mycobactin J (Synbiotics Corporation, France). The other control strains were grown on required solid media.

TABLE 1.
Mycobacterium avium subsp. paratuberculosis, non-M. avium subsp. paratuberculosis, and nonmycobacterial strains used for sensitivity and specificity testing

Preparation of DNA samples.

The preparation of the reference DNA from the mycobacterial strains was performed by using a protocol for gram-positive bacteria in the Qiagen DNeasy blood and tissue kit (Qiagen, Hilden, Germany). The procedure was slightly modified by including a mechanical cell homogenization and disruption with the Fastprep Ribolyzer (Q-biogene, Heidelberg, Germany) to achieve efficient cell lysis. In case of gram-negative control strains, the protocol for gram-negative microorganisms in the Qiagen DNeasy blood and tissue kit (Qiagen, Germany) was used. Approximately 107 CFU was used as a template in the PCR assay.

For the determination of the detection probability, assay precision, and robustness, M. avium subsp. paratuberculosis DNA standards from different M. avium subsp. paratuberculosis strains were prepared. Single colonies of the bovine strains 423 and 428, the ovine strain JD131 and the human strain SN5 were grown separately in mycobacterial growth indicator tubes (Becton Dickinson) containing oleic acid-albumin-dextrose-catalase enrichment and PANTA (both from Becton Dickinson) and Mycobactin J as recommended by the supplier. The DNA for each M. avium subsp. paratuberculosis standard was extracted by using 1-ml aliquots of the suspension according to the modified protocol described above. UV spectroscopic measurement of the total DNA quantity and quality was performed on a BioMate3 (Thermo Scientific, WI). The bacterial cells in 1 ml of each M. avium subsp. paratuberculosis suspension were mechanically sheared by repeated drawing and spilling through a syringe needle (gauge 26G3/8). Cell numbers were calculated after counting the cells in an aliquot of the M. avium subsp. paratuberculosis stock solution in a Tuerk counter chamber and comparative cultivation on Middlebrook 7H10 (Becton Dickinson) agar plates. Serial dilutions of M. avium subsp. paratuberculosis cells from 100 to 10−7 were prepared. DNA was extracted from 1-ml aliquots of each dilution as described above.

Primer, TaqManmgb probe, and LNA probe design.

The design of sequence-specific oligonucleotide primers was based on the M. avium subsp. paratuberculosis reference sequences for F57 (accession numbers X70277 and AE016958) and ISMav2 (accession numbers AF286339 and AE016958) published in the National Center for Biotechnology (NCBI) GenBank (Table (Table2).2). Similar sequences were identified according to the scientific literature, by comparative searches of GenBank and the Comprehensive Microbial Resource (CMR) database of The Institute for Genomic Research (TIGR) and were added to the alignments. The TaqManmgb probes for both primer sets were adopted by using Primer Express version 2.0 (Applied Biosystems, Darmstadt, Germany) with respect to the guidelines from Applied Biosystems and labeled at the 5′ end with VIC (for F57) (Applied Biosystems) or 6-carboxyfluorescein (FAM) (for ISMav2) (Applied Biosystems). The 3′ end contained a minor groove binder (mgb) and the nonfluorescent Eclipse DarkQuencher (Applied Biosystems). Both probe sequences were also synthesized as locked nucleic acid (LNA) probes (Eurogentec, Cologne, Germany) labeled with Yakima Yellow or FAM and the Black Hole Quencher (BHQ1) (all from Eurogentec). All oligonucleotide and probe sequences were submitted to the NCBI and TIGR databases for specificity testing, including broad-range and comparative genome basic local alignment search tool (BLAST) analysis.

TABLE 2.
Oligonucleotide primers, fluorogenic probes, and internal amplification control sequences used in this studya

IAC.

An IAC was selected as described previously (1). Briefly, the IAC was synthesized in one PCR, using the plasmid pUC19 vector as a template (M11662; Promega). The oligonucleotide sequences were identical to the F57 diagnostic primers possessing 5′ overhanging ends, whereas their 3′ ends were complementary to the pUC19 plasmid sequences. Separate TaqManmgb probes were designed according to the pUC19 vector sequence and labeled at the 5′ end with NED (Applied Biosystems). The PCR product was purified, and adjustment of the number of copies for use in the conventional PCR assays and the real-time PCR assays was done as described previously (34). The optimal copy number was assessed to be 175.

Triplex real-time PCR assay.

The optimized 50-μl PCR mixture for the triplex real-time PCR assay using TaqMan probes contained 300 nM of the primers F57-F/F57-R, 200 nM of the primers ISMav2-F/ISMav2-R, 250 nM of the target probes (F57 and ISMav2), 175 copies of the IAC, 25 μl of 2× qPCR MasterMix Plus without uracil-N-glycosylase (UNG) (Eurogentec), and a 5-μl aliquot of the DNA sample. The PCRs were performed in a 96-well plate format on the ABI Prism 7000 sequence detection system (Applied Biosystems). Thermal cycling conditions comprised a hot-start DNA polymerase activation at 95°C for 10 min, 50 cycles of denaturation at 95°C for 15 seconds, and annealing and extension at 60°C for 1 min. Each measurement was performed in duplicate, and the threshold cycle (CT), defined as the fractional cycle number at which the amount of amplified target reached a fixed threshold, was determined. Additional melting curve analysis was performed with the QuantiTect Sybr green PCR kit (Qiagen), the Absolute QPCR Sybr green mix (ABGene, Hamburg, Germany), and the Sybr green PCR master mix (Applied Biosystems) according to the manufacturers' instructions. DNAs from bovine, ovine, and human M. avium subsp. paratuberculosis strains were investigated.

Determination of detection probability.

The detection probability of the real-time PCR assay was first determined by analyzing serial dilutions of known M. avium subsp. paratuberculosis DNA standards prepared from the bovine strains 423 and 428, the ovine strain JD131, and the human strain SN5. Serial dilutions prepared from the M. avium subsp. paratuberculosis stock solution ranged from 107 to 101 CFU per ml. Second, 5-μl aliquots of DNA extracted from serial dilutions of homogenized M. avium subsp. paratuberculosis cells were used as a template for real-time PCR amplification. All experiments were done three times in triplicate format. The copy numbers for F57 and ISMav2 corresponding to the lowest limit of detection (LOD) were calculated. Briefly, the mass of the M. avium subsp. paratuberculosis genome in picograms was calculated by dividing the M. avium subsp. paratuberculosis genome size of 4,829,781 bp (33) through the number of copies per gene of interest.

Assessment of amplification efficiency, precision, and robustness.

Assessments of the amplification efficiency and the precision of the assay under optimized conditions were performed by triplicate analysis of serial dilutions of a known M. avium subsp. paratuberculosis DNA standard. The same operator repeated the experiment three times on different days with the same dilutions. The arithmetic mean of the CT values and the corresponding standard deviation (SD) were calculated for each sample. Standard curve construction was performed for both PCR marker genes. The slopes were used for the calculation of amplification efficiency (E) by using the equation E = 10(−1/slope) − 10 (9).

The robustness of the assay was investigated as follows. Duplicates of the serial dilutions of M. avium subsp. paratuberculosis DNA of a known standard were run with optimized and suboptimal concentrations of the PCR reagents. This included 10% more or less of the qPCR MasterMix Plus without UNG (Eurogentec) and variations of the annealing temperature of 62°C and 65°C. In addition, the influence of different TaqMan PCR reagents was investigated by considering six ready-to-use products from five different suppliers according to the manufacturer's instructions. The PCR reagents were TaqMan Universal PCR Mastermix (Applied Biosystems), the QuantiTect Multiplex PCR kit (Qiagen), Absolute QPCR mix (ABgene), qPCR MasterMix Plus plus UNG and qPCR MasterMix Plus without UNG (both from Eurogentec), and the LightCycler TaqMan Master (Roche, Mannheim, Germany). Analysis was conducted in triplicate format and done three times.

Assessment of the LOD on the ABI Prism 7000 sequence detection system and the LightCycler 1.0 real-time PCR thermocycler.

In general, the experiments were performed on the ABI Prism 7000 sequence detection system (Applied Biosystems) as described above. Comparative assessment of the LOD on the LightCycler 1.0 (Roche) was conducted with the serial dilution of an M. avium subsp. paratuberculosis DNA standard. The optimized 20-μl PCR mixture for the duplex real-time PCR assay using TaqMan probes contained 400 nM of the primers F57-F/F57-R or 300 nM of the primers ISMav2-F/ISMav2-R, 250 nM of the target probes (F57 and ISMav2), 175 copies of the IAC, 7.5 μl of the LightCycler TaqMan Master (Roche), and a 5-μl aliquot of the DNA sample. The PCRs were performed in a 32-capillary rotor. Thermal cycling conditions comprised a hot-start DNA polymerase activation at 95°C for 10 min, 50 cycles of denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 1 min, and extension at 72°C for 10 seconds. A cooling step was added at 40°C for 30 s. Fluorescence data were collected with the acquisition mode “single” during the extension with channel 1. Each measurement was performed in duplicate, and the CT was determined.

Preparation of DNA samples from bovine fecal samples.

For the real-time PCR assay for the detection of M. avium subsp. paratuberculosis from bovine fecal samples, two DNA sample preparation protocols were evaluated by analyzing artificially contaminated bovine fecal samples. Serial dilutions were prepared from the M. avium subsp. paratuberculosis stock solution of bovine strain 423, ranging from 106 to 101 CFU per ml. Beforehand the detection probability was assessed by determining the influence of an additional 5-μl aliquot of DNA isolated from three bovine fecal samples. The samples had been confirmed as M. avium subsp. paratuberculosis negative by culture. The DNA was prepared according to the modified protocol described below. Comparative analysis of the serial dilutions of the known M. avium subsp. paratuberculosis DNA standards with and without the background DNA was done in duplicate and repeated twice.

DNA extraction was done by using a modified protocol of the QIAamp DNA stool minikit (Qiagen), as follows. After preheating of 140 ml ASL buffer at 70°C, 350 μl DX buffer was added and the solution was mixed. One gram of bovine feces was mixed with 5 ml of the DX-ASL buffer to obtain a homogenous suspension. The mixture was subsequently incubated at 95°C for 10 min, and 1.3 ml of the supernatant was added to a 2-ml lysing matrix D tube (Q-biogene). Afterwards, mechanical cell disruption was done with a FastPrep-120 (Q-biogene) by four repetitions of 20 seconds at a speed setting of 6 followed by mixture of the tube contents. To separate the solid phase from the liquid phase, tubes were centrifuged at 5,000 × g for 5 min, and 1.2 ml of the resulting supernatant was transferred to a new 2-ml Eppendorf tube. After addition of one Inhibitex tablet (Qiagen) per sample to remove PCR inhibitors, the sample was mixed for 1 min and subsequently centrifuged at 15,800 × g for 6 min; 300 μl of each supernatant was then transferred to a new 1.5-ml Eppendorf tube which already contained 20 μl proteinase K (20 mg/ml; Qiagen) and mixed with 300 μl AL buffer. Proteinase K was incubated at 70°C for 5 min and subsequently at 95°C for 10 min. Further processing was done according to the kit manual. Eluted DNA was stored at 4°C for direct use or stored at −20°C.

Analysis of naturally contaminated samples of unknown M. avium subsp. paratuberculosis status.

A total of 108 bovine fecal samples of unknown M. avium subsp. paratuberculosis status were analyzed in duplicate by triplex real-time PCR as described above, by IS900 nested PCR (12), and by conventional microbiological culture (6). A 0.75% solution of hexadecylpyridiniumchloride was used for overnight decontamination before inoculation of tubes of HEYM with Mycobactin J. Fecal samples were collected from cattle herds suspected of having paratuberculosis, as identified by Pourquier paratuberculosis enzyme-linked immunosorbent assay (Institute Pourquier, Montpellier, France) on individual milk samples collected per animal, or from clinically infected animals admitted to the clinic for ruminants and pigs, Justus Liebig University, Giessen, Germany.

Statistical analyses.

Statistical analyses used the program packages BMDP for XP, release 8.1 (17), and BiAS for Windows, release 8.2 (3). The comparison of the PCR master mix results was done by a three-way analysis of variance for nested designs and mixed-effect models using the program BMDP8V. This test was followed by a pairwise comparison of the master mixes according to the Tukey test (BMDP7D). The statistical terms relative sensitivity, specificity, and diagnostic accuracy were calculated as described elsewhere (2) and were completed by calculation of the 95% confidence intervals with the program BiAS.

RESULTS

Selectivity.

Table Table11 summarizes the results of the sensitivity and specificity testing. All 105 M. avium subsp. paratuberculosis strains were identified correctly with the primer and oligonucleotide probe sets designed for F57 and ISMav2. The sequences of the primers, the TaqManmgb, the LNA fluorogenic probes, and the IAC are given in Table Table2.2. The sizes of the amplified PCR products were 62 bp (F57), 164 bp (ISMav2), and 105 bp (IAC). No amplification was observed using the 34 non-M. avium subsp. paratuberculosis strains and the 65 nonmycobacterial strains. CT values for various DNA extractions from the bovine, ovine, and human M. avium subsp. paratuberculosis strains tested did not differ significantly between the different sources and ranged between 14.87 and 21.43 for F57 and between 14.71 and 20.86 for ISMav2 generated with the TaqManmgb probes (Table (Table3).3). The end point fluorescence values (delta Rn) normalized against the passive reference dye ROX were 0.68 (SD, 0.05) for F57 and 1.19 (SD, 0.04) for ISMav2. The LNA probes gave CT values of 15.27 and 21.75 (F57) and 13.46 and 19.82 (Table (Table3)3) and end point fluorescence values (delta Rn) of 1.01 (SD, 0.1) for F57 and 2.33 (SD, 0.04) for ISMav2. For both probe chemistries, a baseline was set manually at 3 to 12 and a threshold line of 0.06 was used. Melting curve analysis conducted after Sybr green-based amplification gave melting temperatures of 80.4°C for the F57 amplicon, 85.4°C for the ISMav2 amplicon, and 84°C for the IAC.

TABLE 3.
CT values generated with the TaqManmgb and LNA probes for bovine, ovine, and human M. avium subsp. paratuberculosis DNA extractions

Determination of detection probability.

The broad linear range of detection was 107 CFU/ml to 10 CFU/ml of M. avium subsp. paratuberculosis. The 100% lower LOD was assessed with six different PCR master mixes from five different suppliers. It corresponded to 0.1 picogram bovine, ovine, or human M. avium subsp. paratuberculosis DNA per PCR, indicated as a serial dilution of 10−7 g of M. avium subsp. paratuberculosis DNA per PCR (Tables (Tables44 and and5).5). This was equivalent to detection of 19 copies of F57 and 57 copies of ISMav2. These results were obtained with the TaqMan Universal PCR Mastermix (Applied Biosystems), the Absolute QPCR mix (ABgene), the qPCR MasterMix Plus plus UNG and qPCR MasterMix Plus without UNG (both from Eurogentec), and the LightCycler TaqMan Master (Roche). Intra-assay SDs ranged between 0.21 and 0.35 for F57 and ISMav2, while the interassay SD was 0.37 to 2.5 CT values. No significant differences for each of the marker genes were observed by analysis of variance for 10−7 CFU per ml, the lowest dilution investigated. For 100 and 10−6 CFU per ml, overall significant differences were found (P < 0.0001). Pairwise comparison according to the Tukey test gave significant differences (P < 0.01) between all reagents investigated except between Absolute QPCR mix (ABgene) and the QuantiTect multiplex PCR kit (Qiagen).

TABLE 4.
CT values generated with the TaqMan Universal PCR Mastermix (Applied Biosystems) and the qPCR MasterMix Plus without UNG (Eurogentec) in the presence of 175 IAC copy numbers
TABLE 5.
CT values generated with four different ready-to-use PCR master mixes

The addition of background DNA obtained from M. avium subsp. paratuberculosis-negative bovine fecal samples did not affect the LOD or the amplification efficiency. In artificially contaminated fecal samples, an LOD of 100% for 103 CFU and 102 CFU of M. avium subsp. paratuberculosis per g bovine feces was achieved with the modified protocol of the QIAmp stool minikit (Qiagen, Hilden).

Assessment of amplification efficiency, precision, and robustness.

After optimization of the PCR master mix and the PCR setup, the amplification efficiency for both PCR marker genes was evaluated with regard to serial dilution of an M. avium subsp. paratuberculosis DNA standard. The amplification efficiency for the F57 primer and fluorogenic probe set was 98.2%, and that for the ISMav2 primer and fluorogenic probe set was 97.8%. The correlation coefficient (r) for both values was >0.99. Results were obtained with the 2× qPCR MasterMix Plus without UNG (Eurogentec). Precise amplification over the upper and lower LODs was shown by three consecutive runs, including triplicate analysis within each run, by using an M. avium subsp. paratuberculosis DNA serial dilution. The CT values for the F57 primer and fluorogenic probe set (fluorescent dye, VIC), the ISMav2 primer and fluorogenic probe set (fluorescent dye, FAM), and the IAC (fluorescent dye, NED) corresponded to the initial concentration of target DNA. CT values for F57 ranged between 15.02 and 20.49, and those for ISMav2 ranged between 13.84 and 20.43 (Tables (Tables44 and and55).

An increase of 10% of the optimized PCR reagents lowered the PCR efficiency (data not shown), while a decrease of 5% had no significant effect on the assay. Increasing the annealing temperature to 62°C showed an increase of the CT values of about 0.75 and a slight reduction of the lower LOD. CT values increased by about three cycles with an annealing temperature of 65°C and a 70% lower LOD.

Application of the assay with different thermocycler models.

The performance of the TaqMan assay with different real-time PCR thermocycler models was assessed by comparative analysis of a serial dilution of M. avium subsp. paratuberculosis DNA on the Applied Biosystems 7000 Prism sequence detection system and the LightCycler 1.0. The LightCycler 1.0 does not support the simultaneous detection of the fluorescent dyes VIC (F57) and FAM (ISMav2) due to technical limitations. Therefore, both primer and oligonucleotide sets were applied in separate runs. The LightCycler 1.0 allowed the detection of the lower LOD of 0.1 picogram M. avium subsp. paratuberculosis DNA per sample.

Analysis of naturally contaminated fecal samples.

Growth of M. avium subsp. paratuberculosis was observed and confirmed by real-time PCR and IS900 nested PCR with 53 of the 108 bovine fecal samples investigated (Table (Table6).6). According to the CFU/g feces detected, samples were divided into groups: 0 CFU/g (0), <10 CFU/g (+1), 10 to 50 CFU/g (+2), and >50 CFU/g (+3). Nineteen and 11 samples were positive by culture and direct triplex real-time PCR of the +2 and +3 groups, respectively. One of the +2 samples was not detected by IS900 nested PCR, resulting in a sensitivity of 90.9% (95% confidence interval, 58.7 to 99.8%). In the +1 group, the triplex real-time PCR gave 3 false-negative results out of the 13 false-negative results shown by IS900 nested PCR. Diagnostic specificity was 100% for both PCR methods. The sensitivity was 43.5% (95% confidence interval, 23.2 to 65.5%) with IS900 nested PCR and 87.00% (95% confidence interval, 66.4 to 97.2%) with the real-time PCR assay. This corresponded to a diagnostic accuracy of 87.03% with nested PCR and 97.22% with real-time PCR analysis.

TABLE 6.
Comparative statistical analysis of 108 possibly naturally contaminated bovine fecal samples

DISCUSSION

In this study, a new triplex real-time PCR assay was developed for the detection of M. avium subsp. paratuberculosis in bovine fecal samples. The overall analysis time was approximately 24 h, in contrast to up to 12 weeks of incubation time for the traditional culture method used. The high selectivity of the oligonucleotide primers and probes was confirmed by percent sequence identity comparisons using two different databases (NCBI GenBank and TIGR CMR database) and sequence alignments obtained from genetic elements with high similarity. Analysis of 105 M. avium subsp. paratuberculosis strains, 34 representative non-M. avium subsp. paratuberculosis mycobacteria, and 66 nonmycobacterial strains revealed high selectivity of the triplex assay. No false-positive or -negative results were reported.

F57 and ISMav2 have been described as M. avium subsp. paratuberculosis-specific markers (41, 48). Nested PCR was performed with oligonucleotide primers for the single-copy gene F57 (53). Conventional PCR primers for the three-copy element ISMav2 were used for analysis of bulk milk samples (44, 46, 47). Therefore, F57 and ISMav2 were selected as two well-characterized candidate genes offering the potential for the specific detection of M. avium subsp. paratuberculosis. In addition, other genetic elements have been recently identified and described as possibly being M. avium subsp. paratuberculosis specific (32, 40). The important characterization of diagnostic specificity based on developed oligonucleotide primers or probes was presented for the single-copy elements ISMap02 (45) and Hsp X (20). References to the following IS900 primers were made in more than 30 scientific publications: IS900 and 150C, IS900 and 921 (55), p36 and p1 (38), MK5 and MK6 (19), P90 and P91 (36), and P21 and P8 (36). Their applicability for routine diagnosis is limited, because of possible cross-reactions with other mycobacterial strains (16, 22, 50). Optimized nested PCR primer pairs for IS900 that do not show cross-reactions with IS900-like elements have been published and were therefore selected as a PCR control system (12). For diagnostic quality assurance, the use of an IAC is thought to be mandatory in diagnostic PCR (28). Several of the recently published real-time PCR methods are still hampered by not considering an IAC (23, 27, 31, 39). The IAC included in the PCR presented here could reliably exclude false-negative PCRs, especially in the presence of small amounts of M. avium subsp. paratuberculosis DNA (105 to 107 picogram DNA per PCR). For a high excess of M. avium subsp. paratuberculosis DNA, the amplification of the IAC template was suboptimal due to the competitive PCR.

Precision of the amplification was demonstrated in consecutive runs. The SDs of the CT values ranged between 0.5% and 3% (F57 PCR product), 0.7% and 0.9% (ISMav2 PCR product), and 0.53% and 0.55% (IAC PCR product). A decrease of the amplification efficiency can occur due to inadequate primer and probe design, nonoptimized PCR reagents, and amplification conditions. The addition of an IAC might cause PCR inhibition (2, 34). The standardized PCR setup was shown to be well optimized, because the addition of an IAC did not result in a disadvantageous effect on the LOD or amplification efficiency during multiplexing.

All ready-to-use TaqMan reagents enabled the reliable detection of the investigated M. avium subsp. paratuberculosis strains. The TaqMan Universal PCR Mastermix (Applied Biosystems) and the qPCR MasterMix Plus without UNG (Eurogentec) gave very similar results and enabled the lowest LOD of 0.1 picogram DNA per PCR, corresponding to 19 copies of F57 and 57 copies of ISMav2. None of the six PCR master mixes investigated offered a significant improvement in sensitivity (Tables (Tables44 and and5).5). DNA polymerase enzymes and buffers can vary substantially, with some being more prone to inhibition by harsh inhibitors of feces than others. No disadvantageous effect was reported for the selected ready-to-use master mix used in the standardized protocol. Besides the cost-effective handling of diagnostic reagents, the possible effect of different fluorescent compounds and PCR reagents on the performance of the PCR method has to be considered for each diagnostic test. TaqManmgb probes and LNA probe chemistries remained stable over 50 PCR cycles, and no instability resulting in a slowly increasing fluorescence signal in nontemplate controls was observed. No differences were observed in the sensitive detection of DNA derived from bovine, ovine, and human M. avium subsp. paratuberculosis strains. The fluorescent dye Sybr green offers the possibility for a simple and reasonably priced evaluation of newly developed primer pairs. Specificity of the PCR product is confirmed by melting curve analysis (58). The three Sybr green ready-to-use master mixes investigated in this study did not show differences in the detectability of M. avium subsp. paratuberculosis strains of bovine, ovine, or human origin, but the fluorescent signal of Sybr green was slightly inhibited in the analysis of DNA extracted from fecal samples (data not shown). Sybr green should therefore be used only for the confirmation of possible M. avium subsp. paratuberculosis colonies obtained by classical cultivation.

International standard providers encourage method developers to validate methods on a variety of instruments to prove that the analytical method is not limited due to different fluorescent compounds or instrument specificity. Several companies offer thermocycler models for the detection of TaqMan fluorogenic probes. The reactions can be performed in 96-well plates. The LightCycler 1.0 is typically used with hybridization probes. The fluorescent signal is measured through capillaries, and the temperature is adjusted with a heater fan instead of heat blocks. Therefore, the ABI 7000 Prism sequence detection system and the LightCycler 1.0 represent two technically different thermocycler platforms which are widely used. The presented real-time PCR assay gave identical LODs of 0.1 picogram M. avium subsp. paratuberculosis DNA on both instruments, with a probability of 100%. It should be mentioned that in contrast to the LightCycler 2.0, the LightCycler 1.0 cannot detect the two separate emission spectra of FAM and VIC in a duplex or even triplex PCR. For this purpose, the oligonucleotides and fluorescent probes developed for the marker genes F57 or ISMav2 have to be combined separately with the IAC primer and probe set. Promising further experiments are under way using the improved six-channel detection system of the LightCycler 2.0, enabling the application of the complete triplex assay in the configuration described above.

Investigation of bovine fecal samples by culture is the gold standard for the estimation of the prevalence of M. avium subsp. paratuberculosis on the herd level (21, 42). For PCR applications, the extraction of DNA from small amounts of different bacteria from fecal samples is hampered due to different PCR inhibitors (57). In addition, M. avium subsp. paratuberculosis is known to form clumps and to be highly resistant to chemical and enzymatic lysis (12). The robust real-time PCR assay was combined with a modified DNA extraction procedure to achieve maximum sensitivity for the detection of M. avium subsp. paratuberculosis from bovine fecal samples. Addition of nonspecific background DNA isolated with the optimized protocol from bovine feces did not decrease the LOD of the PCR. Numerous methods have been used for the extraction of M. avium subsp. paratuberculosis DNA from fecal samples, including immunomagnetic separation (31), buoyant density centrifugation (25), addition of resins (35), DNA sequence capture extraction (54), and silica membrane-based kits (11). The mechanical homogenization of the sample and disruption of the M. avium subsp. paratuberculosis cells included in the modified QIAamp DNA stool minikit protocol in this study showed an advantageous effect on the LOD (data not shown). The lower LOD in artificially spiked samples was 100% for 103 CFU/per gram feces and 100% for 102 CFU/g feces, compared to an LOD of 70% for 103 CFU/per gram feces with the standard protocol. Mechanical shearing of the fecal samples suspected to contain M. avium subsp. paratuberculosis by using a bead beater resulted in the formation of foam from the ASL buffer. This could not be reliably removed by centrifugation and hampered further processing. The addition of DX buffer to the ASL buffer reduced the foam formation significantly.

In addition, real-time PCR requires the investigation of the interaction of a probe dye with feces in order to remove any possible quenching effect of the matrix on the fluorescence activity of the probes. Silica membrane columns have been previously shown to provide a convenient method for DNA purification and especially removal of PCR inhibitors (29, 30). The analytical sensitivity obtained from spiking experiments is comparable to the results of other studies. An LOD of 100% for an IS900 real-time PCR was achieved with a silica membrane-based kit at 500 CFU/g feces (10). An LOD of 100% was described for 100 CFU/g feces (11) in combination with an F57 real-time PCR (51). The analysis of 108 naturally contaminated fecal samples of unknown status revealed a statistically better sensitivity and accuracy than IS900 nested PCR (Table (Table6).6). In accordance with the results of classical cultural investigation, 50 of 53 samples were confirmed to be M. avium subsp. paratuberculosis positive. However, for three of the samples it was not possible with this real-time PCR to determine definite concentrations of M. avium subsp. paratuberculosis in fecal samples. Similarly, those samples and 10 additional samples were not identified by using IS900 nested PCR. These three samples were poorly contaminated and produced growth of 2, 5, and 8 CFU, respectively, on HEYM agar. Although HEYM slants do not allow exact quantification, the detection limit obtained in this study was comparable to that in the spiking experiments and should be sufficient for detection of M. avium subsp. paratuberculosis in fecal samples contaminated at high or low levels. One sample contained M. avium subsp. avium as confirmed by sequence analysis. No false-positive reactions with this or any other sample were obtained with the real-time PCR.

Different strategies have been proposed to be suitable for the reliable detection of M. avium subsp. paratuberculosis by using real-time PCR, including use of conventional IS900 PCR primers (31), the combination of two separate real-time PCR runs for IS900 and F57 (27), and the use of F57-derived oligonucleotide primers and hybridization probes restricted to the LightCycler (51). The main advantages of our triplex real-time PCR assay can be summarized as the combination of the reliability of two different marker genes (F57 and ISMav2) and an IAC and its detailed validation according to the requirements made by international standard providers (7, 8). The latter includes applicability on different thermocycler models. Although not described yet and carefully excluded by the strains considered during this study, scientific evidence of possible cross-reacting strains might arise, as described for the former reference marker IS900. The application of this assay makes it very unlikely that cross-reactions with both marker genes will be obtained. Being not restricted to a distinct supplier or provider of PCR reagents makes the method easier to adopt for individual needs, and the IAC guarantees diagnostic quality assurance. Considering these factors in combination with the optimized DNA extraction protocol for fecal samples offering potential for automized sample preparation, we think that the assay presented here contributes to the improvement of routine diagnostic procedures for M. avium subsp. paratuberculosis.

Acknowledgments

This work was supported by the AZ III1.2-905 10-32 project funded by the Ministry of Science and Arts, County Hessen, Germany.

We thank K. Simon and C. Walter for excellent technical assistance. In addition, we gratefully acknowledge Tim Bull's Crohn's Disease Group, St. George's University London, London, United Kingdom; K. Doll, Clinic for Ruminants and Pigs, Justus Liebig University, Giessen; Dieter Klein, Landesuntersuchungsamt Rheinland-Pfalz, Faculty of Veterinary Medicine, Koblenz, Germany; Heike Köhler, National Reference Laboratory for Paratuberculosis, Friedrich Löffler Institute, Reference Center for Paratuberculosis, Jena; Irmgard Moser, National Veterinary Reference Laboratory for Tuberculosis, Friedrich Löffler Institute, Jena; and Elvira Richter, National Reference Center for Mycobacteria, Research Center Borstel, Borstel, Germany.

Footnotes

[down-pointing small open triangle]Published ahead of print on 7 March 2008.

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