MIMET-04608; No of Pages 5 Journal of Microbiological Methods xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth

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Verlaine J. Timms, Hazel M. Mitchell, Brett A. Neilan ⁎

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School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia

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a r t i c l e

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Article history: Received 3 December 2014 Received in revised form 10 March 2015 Accepted 17 March 2015 Available online xxxx

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Keywords: Crohn's disease Inflammatory bowel disease MAP M. paratuberculosis Mycobacterium paratuberculosis Paratuberculosis

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The aim of this study was to investigate DNA extraction methods and PCR assays suitable for the detection of Mycobacterium paratuberculosis in bovine tissue. The majority of methods currently used to detect M. paratuberculosis have been developed using bovine samples, such as faeces, blood or tissue and, in many cases, have been based on detection from pooled samples from a herd. However most studies have not compared PCR results to culture results. In order to address this problem, four DNA extraction protocols and three PCR assays were employed to detect M. paratuberculosis in bovine tissue. Given that culture is reliable from cows, the results were then compared with the known M. paratuberculosis culture status. The following DNA extractions were included, two commercial kits, a boiling method, an in house extraction based on a published method and enrichment by sonication. The three PCR assays used included single round IS900 and f57 assays and a nested IS900 assay. In addition, another PCR assay was validated for the detection of any Mycobacterial species and a universal bacterial 16S rRNA gene assay was used to detect sample inhibition. The in-house DNA extraction was the most consistent in extracting good quality DNA compared to all other methods. The use of two PCR markers, IS900 and f57, and a universal PCR enabled the correct samples to be identified as M. paratuberculosis positive. In addition, when compared to the culture result, false-positives did not occur and PCR inhibition was readily identified. Using an in house DNA extraction coupled with the IS900 and f57 PCR markers, this study provides a reliable and simple method to detect M. paratuberculosis in both veterinary and spill over infections. © 2015 Published by Elsevier B.V.

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1. Introduction

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Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the only M. avium Complex (MAC) subspecies to exclusively infect the gastrointestinal tract, causing Johne's disease in cattle and other livestock (Whittington et al., 2012). Studies have found M. paratuberculosis by PCR in 52%–92% of patients with Crohn's disease, compared to its incidence in 5%–26% of controls (Bull et al., 2003; Autschbach et al., 2005). This association of M. paratuberculosis with Crohn's has led to investigations to detect it in the human gut. Current approaches used to detect M. paratuberculosis have been based on investigations of Johne's disease in cattle. The classical detection method used in livestock is culture, which has been shown to have a wide range of sensitivity and specificity depending on the stage of disease, type of sample taken and the culture method and, in addition, diagnosis can take many months.

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Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis

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⁎ Corresponding author at: School of Biotechnology and Biomolecular Science, Room 344, Level 3 Bioscience Building, UNSW, Sydney 2052, Australia. E-mail addresses: [email protected] (V.J. Timms), [email protected] (H.M. Mitchell), [email protected] (B.A. Neilan).

Given that the isolation of M. paratuberculosis from humans is sporadic, molecular detection is the preferred method of choice. Although PCR has dominated this field in both livestock and human infections, the success of this approach is, however, greatly affected by sample quality (Radomski et al., 2013; Christopher-Hennings et al., 2003). The quantity of DNA extracted and sample quality are affected by the tough mycobacterial cell wall as well as the amount of nonmycobacterial DNA, proteins and inhibitors present in the sample. In all mycobacteria, the peptidoglycan layer of the cell wall is surrounded by a hydrophobic arabinogalactan–peptidoglycan–mycolic acid layer. In MAC, this layer is surrounded by a second layer, containing serovarspecific glycopeptidolipids (Belisle et al., 1991; Inderlied et al., 1993; Wayne, 1993), or in the case of M. paratuberculosis, lipopentapeptide (L5P) (Biet et al., 2008). The effects of the cell wall composition on the success of DNA extraction is demonstrated in studies using direct extraction, which usually involves bead-beating, that result in a 10-fold higher concentration of DNA compared to cell extraction procedures without a mechanical step (Radomski et al., 2013; Cook and Britt, 2007). A further study has reported that sonication to “enrich” mycobacterial DNA by removing background bacteria and other cells prior to bead-beating, was more successful than bead beating alone (Granger et al., 2004). Another study reported a 40% increase in the

http://dx.doi.org/10.1016/j.mimet.2015.03.016 0167-7012/© 2015 Published by Elsevier B.V.

Please cite this article as: Timms, V.J., et al., Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.03.016

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M. paratuberculosis strain ATCC19698 was grown for 4–6 weeks on a slope of Middlebrook 7H10 agar supplemented with 10% oleic acid–albumin–dextrose–catalase (OADC) (Difco) and 2 μg mL − 1 mycobactin J (Allied Monitor).

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2.2. Bovine samples

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Bovine tissue samples were obtained and catalogued from the OIE and National Reference Laboratory for Johne's Disease, Department of Primary Industries, Attwood, Victoria. The M. paratuberculosis status of the sample was determined by the OIE and National Reference Laboratory for Johne's Disease using culture and any isolates were identified as M. paratuberculosis based on mycobactin dependence and the presence of the IS900 gene (by PCR). Upon being sent, all biopsies were frozen at −80 °C until required. To prepare tissue for DNA extraction, approximately 150 mg (wet weight) of tissue was cut from each biopsy then

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All DNA extraction methods were first tested using six replicates of 141 1–2 colonies of a pure culture of M. paratuberculosis ATCC19698. 142 2.3.1. DNA extraction kits Two kits were used, the PowerPlant™ DNA Isolation Kit (MO BIO Laboratories Inc.) and the DNeasy Blood and Tissue Kit (QIAGEN). Samples were processed as per the instructions in each kit.

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2.1. Bacterial growth conditions

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2.3.2. Extraction by boiling Crushed tissue was placed in a tube containing 100 μL sterile H2O. The tube was then placed in a heat block set to 100 °C for 15 min, then ice for 15 min. Tubes were then centrifuged at 5000–6000 ×g for 5 min. The supernatant containing DNA was removed and placed in a clean tube. Two variations to this method were also attempted by adding 1% Triton X-100 or 0.1% NP40 to the H2O before the boiling step.

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2.3.3. Sonication and enrichment of M. paratuberculosis in bovine tissue A previous study by Granger et al. (2004) reported sonication to enrich mycobacterial cells in tissue samples. Given this, we adapted Granger's method in an attempt to increase the quantity and quality of DNA obtained from bovine tissue samples. Prior to the enzymatic and phenol/chloroform extraction, bovine samples were placed on ice, after which 200 μL of TE buffer was added and the biopsy crushed using sterilised crushers (Eppendorf). One percent (1%) Triton X-100 was then added to the tube after which the samples were sonicated using a bench-top sonicator (Soniclean Pty. Ltd.) for a range of times (2–10 min) to determine the ability of sonication to destroy non-mycobacterial cells. Following sonication, connective tissue and cellular debris were removed by centrifugation at 1000 ×g for 10 min at 4 °C, after which the supernatant was used for enzymatic and phenol/chloroform extraction.

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2.3.4. In-house DNA extraction This extraction procedure was modified from a published method (Bull et al., 2003) and formed the basis of M. paratuberculosis detection in human tissue in that study and another (Autschbach et al., 2005). In this study it is referred to as the in-house extraction method. A colony of pure M. paratuberculosis or the tissue debris from crushing the biopsies of bovine tissue (see above) were added to a mini-bead beater tube, with 600 μL of breaking buffer (50 mM Tris HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl), 0.6% SDS and 200 μg Proteinase K (Astral Scientific) and the tubes were incubated at an angle of 45° at either 37 °C or 50 °C for between 12 and 24 h with shaking (200 rpm). After chilling the tubes on ice for 5 min, the tissue was mechanically disrupted in a Bead Beater (FastPrep FP120, Savant) at 6.5 ms−2 for 45 s. The tubes were then chilled again for 15 min and the supernatant transferred to a clean tube. The beads were then washed with 100 μL of breaking buffer, and the wash added to the first supernatant. After adding 500 μL of phenol, the tubes were vortexed for 20 s then centrifuged at 10,000 ×g for 1 min. The aqueous layer was then transferred to a new tube containing 600 μL phenol–chloroform–isoamyl alcohol (25:24:1) and vortexed for 20 s and then centrifuged as before. Again, the aqueous layer was transferred to a new tube, containing 550 μL chloroform–isoamyl alcohol (24:1), which was vortexed for 30 s and centrifuged as before. The aqueous layer was then transferred to a new tube containing 90 μL of 10 M ammonium acetate and mixed. Eight hundred micolitres of ice-cold 100% ethanol was then added and left for 1 h on ice, after which the tubes were centrifuged at 10,000 ×g for 20 min. After washing the pellets twice in 70%

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ground between sterile slides. The bovine tissue was used as the sample 137 in the standard enzymatic and phenol/chloroform extraction method 138 (in-house method). 139

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amount of DNA extracted using a pre-treatment step involving enzymatic incubation to weaken the mycobacterial cell wall (Park et al., 2014). The most extensively used target for detecting M. paratuberculosis in livestock and humans is the IS900 marker. The insertion sequence 900 is 1451 bp in length, encodes a 399-amino acid putative transposase (p43) on one strand and an unknown open reading frame on the complementary strand and is present in 14–18 copies in the M. paratuberculosis genome (Bull et al., 2000; Li et al., 2005). The f57 segment is also used to detect M. paratuberculosis and is a 620 bp single copy segment, originally isolated from a mycobactin-independent strain of M. paratuberculosis (Poupart et al., 1993; Coetsier et al., 2000). In a comparison of 13 PCR assays, the single round IS900 PCR assay was found to be the best option for sensitivity and specificity and the single round f57 assay was also found to be specific for M. paratuberculosis (Mobius et al., 2008; Vansnick et al., 2004). The nested IS900 assay can detect 0.01 pg of DNA (corresponding to 10 genomes) when extracted from a pure culture while the f57 assay can detect 0.1 pg of DNA (corresponding to 100 genomes). However, the sensitivity and specificity of the IS900 assay are unknown when applied to DNA extracted from tissue while for f57, one study used Taqman real-time PCR to amplify this fragment in C57BL/6 mice infected with M. paratuberculosis K10 and could detect down to 10 genomes (Radomski et al., 2013). Until recently, there was no standard protocol proposed for extracting mycobacterial DNA from tissue and therefore comparison between studies using different extraction techniques was not possible as each method produces DNA of different quality (Radomski et al., 2013). In a search for other specific diagnostic markers, large sequence polymorphisms (LSPs) were compared between M. avium and M. paratuberculosis (Semret et al., 2004). The absence of the LSP designated as LSPA8 was shown to be 100% specific for M. paratuberculosis, while its presence indicated that the isolate was M. avium, rather than other mycobacteria. The absence or presence of LSPA8 can be determined by PCR, a different sized product is obtained depending on the presence or absence of the sequence (Semret et al., 2005). As these assays cannot be supported by microbial cultures from human tissue or faeces, samples with PCR inhibitors can be mistaken as “M. paratuberculosis negative”. When testing bovine tissue however, results can be compared to a cultured isolate result. To date, few studies have validated their assay with known positive or negative tissue samples (Imirzalioglu et al., 2011). This study tests a variety of DNA extraction methods and optimises four PCR assays using bovine tissue and compares the results with the M. paratuberculosis culture status.

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Please cite this article as: Timms, V.J., et al., Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.03.016

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2.5. PCR validation

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All PCR assays were first optimised on DNA extracted from a culture of M. paratuberculosis ATCC19698 using each of the DNA extraction methods outlined above. Further validation of the IS900, f57, LSPA8 and mycobacterial 16S (myco16S) rRNA gene assays were performed using 20 bovine mucosal biopsies catalogued as either M. paratuberculosis culture positive or negative. The primers used in the PCR assays are listed in Table 1. All of these primers had been previously validated and shown to be highly specific (Bull et al., 2003; Autschbach et al., 2005; Mobius et al., 2008; Vansnick et al., 2004; Semret et al., 2005; Tasara et al., 2005; Weisburg et al., 1991). Titrations of MgCl2 (0.75–5 mM) and primer concentrations (0.1–10 μM) were performed and a range of annealing temperatures (50–69 °C) tested to establish the most sensitive and specific assay for each primer set when applied to intestinal tissue. The detection limits of the PCR assays were assessed using serial dilutions of DNA (100 ng to 0.1 fg μL−1) extracted from M. paratuberculosis culture using the proteinase K and phenol/chloroform extraction procedure outlined above. To control for the presence of inhibitors, any samples negative for all PCR assays were subjected to a universal 16S rRNA gene PCR assay using the following conditions: 2.5 mM MgCl2, 200 μM dNTPs, 1 × reaction buffer, 1 U of Taq polymerase, 0.4 μM of 16S rRNA gene primers (Table 1) and 2 μL of DNA added together, and made up to 20 μL with sterilised distilled water. The PCR conditions were the following: 95 °C for 3 min, then 94 °C 30 s, 56 °C 1 min, 72 °C 1 min for 30 cycles then 7 min at 72 °C. Automated sequencing to identify PCR products from the M. paratuberculosis specific PCR assays was carried out using the PRISM BigDye™ cycle sequencing system v3.1 and ABI 3730 capillary Applied Biosystem.

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Table 1 Sequences of primers used in this study, including amplicon size and original reference.

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Nested IS900 (L/AV) 1st stage

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Nested IS900 2nd stage Single round IS900 f57 Mycobacterial 16S rRNA gene (myco16S) LSPA8 flanking sequence A

LSP 8 internal sequence Universal 16S rRNA gene

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The results of PCR optimisation are summarized in Table 2. Each PCR reaction was made up to 20 μL per tube and included 1 × reaction buffer and 20–100 ng DNA. All PCR assays were finished on a holding temperature of 20 °C until removed from the machine.

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Several DNA extraction methods were compared, two kits, DNA extraction by boiling, enrichment of mycobacterial cells by sonication and the in-house extraction method. Each DNA extraction method was compared on DNA amount, the 260/280 and 260/230 ratios and the result when subjected to all PCR assays. While the DNA extracted using the kits produced pure DNA the amount of DNA obtained was inconsistent, ranging from 60 to 600 ng from a pure culture of M. paratuberculosis. In our hands the boiling method produced no detectable DNA and was not used to extract M. paratuberculosis DNA from bovine tissue. In contrast, the in-house extraction method based on an incubation temperature of 37 °C at the enzymatic step, produced 33–64 μg of DNA. This extraction method with the addition of an enrichment step using a sonication time of 1 min also produced DNA with sufficient purity according to the 260/ 280 and 260/230 ratios. Given the consistent results of the latter methods, the in-house extraction and in-house extraction with sonication were applied to the bovine samples in which M. paratuberculosis culture status had been determined. Comparison of the total DNA obtained from the bovine tissue using the in-house extraction method and the same extraction method with an added enrichment step showed that for the in-house extraction method, total DNA ranged between 6000 and 150,000 ng, while with the added enrichment step, it ranged from 60–37,560 ng. Comparison of the DNA purity showed that using the in-house extraction method the 260/280 ratio ranged from 1.8 to 1.94 while the 260/230 ratio ranged from 0.76 to 2.16. The added enrichment step, resulted in a similar 260/280 ratio ranging from 1.71 to 2.81 and the 260/230 ratio from 0.05 to 1.23. The ranges calculated for both methods do not include bovine sample 13 as this sample consistently had a low DNA concentration (180–270 ng) and DNA purity (260/280 ratio of 2.1 and a 260/230 ratio of 0.3). Given the quality and quantity of DNA obtained using these methods, both were used to conduct the IS900, f57 and myco16S PCR assays.

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The concentration and quality of each DNA sample was measured using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies) and the ratios 260/280 nm and 260/230 nm inspected for the presence of organic matter and solvent residues respectively. DNA purity was determined as satisfactory if the 260/280 ratio fell between 1.6 and .2.2 and the 260/230 ratio fell between 1.8 and 2.2 as per the manufacturer's instructions. Approximately 20–100 ng of DNA was used in each PCR reaction as concentrations outside this range gave sporadic PCR amplification results.

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ethanol, the tubes were left to dry at room temperature for 1 h. Pellets were then re-suspended in 30 μL of TE buffer.

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Primer name

Sequence (5′–3′)

Amplicon size (bp)

Reference

L1 — forward L2 — reverse AV1 — forward AV2 — reverse Ptb1 forward Ptb1 reverse F57 — forward R57 — reverse S16p1 — forward S16p2 — reverse LSPA8flF — forward LSPA8flR — reverse LSPA8iF — forward LSPA8iR — reverse 27Fl — forward 1494R — reverse

ctt tct tga agg gtg ttc gg acg tga cct cgc ctc cat atg tgg ttg ctg tgt tgg atg g ccg ccg caa tga act cca g gtc ggc gtg gtc gtc tgc tgg gtt gat gcg cgg cac ggc tct tgt tgt agt c cct gtc taa ttc gat cac gga cta ga tca gct att ggt gta ccg aat gt cgg gtg agt aac acg tgg cta gtc tgc ccg tat tct gca gag cgg tga cat c ata ccg cca acg aca tct tc acc gcc aga tgt ttc tca tc gac tcg gtg ctg ctg gtc aga gtt tga tcc tgg ctc ag tac ggc tac ctt gtt acg ac

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Bull et al. (2003)

Mobius et al. (2008)

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Semret et al. (2005)

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Semret et al. (2005)

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Weisburg et al. (1991)

Bull et al. (2003)

Vansnick et al. (2004) Tasara et al. (2005)

Please cite this article as: Timms, V.J., et al., Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.03.016

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Table 2 Optimised conditions and detection limits for each PCR assay.

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MgCl2 (mM) dNTPs (μM) Taq polymerase (Unit) Primer conc. (μM) Cycling conditions

PCR assay

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nIS900 2nd stage

f57

Myco16S

5 100 1 2.5 1 min 96 °C 15 s 96 °C 15 s 50 °C 1 min 72 °C 35 cycles 5 min 72 °C 0.01

1.25 50 1 2 1 min 94 °C 10 s 94 °C 20 s 50 °C 30 s 72 °C 35 cycles 7 min 72 °C –

1.25 100 0.4 5 1 min 94 °C 10 s 94 °C 20 s 58 °C 30 s 72 °C 30 cycles 7 min 72 °C 0.01

0.75 50 0.5 10 4 min 95 °C 45 s 94 °C 45 s 62 °C 45 s 72 °C 40 cycles 10 min 72 °C 0.1

1.25 100 0.4 5 3 min 95 °C 30 s 94 °C 1 min 56 °C 1 min 72 °C 30 cycles 2 min 72 °C 1

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Investigations into the link between M. paratuberculosis and Crohn's disease remain controversial due to conflicting results obtained by previous studies (Bull et al., 2003; Autschbach et al., 2005; Bentley et al., 2008; Bernstein et al., 2007; Ellingson et al., 2003). The major reasons for this disparity relate to the methods employed to detect M. paratuberculosis by PCR and the continued difficulty in culturing this organism. This study aimed to optimise the detection of M. paratuberculosis in bovine tissue from individual cows with known M. paratuberculosis status. To

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Based on culture, bovine samples 1–10 were shown to be negative for M. paratuberculosis and bovine samples 11–20 were positive for M. paratuberculosis. Consistent with the culture results, M. paratuberculosis was not detected in the culture negative samples by any of the PCR assays. For the culture positive samples, a marked difference was observed in the PCR results for PCR DNA templates extracted with the added sonication step and PCR DNA templates extracted using the in-house method without enrichment. Of the M. paratuberculosis culture positive bovine samples, 3/10 were positive with both the single round and nested IS900 assay, while no samples were positive with the f57 or myco16S assays when DNA extracted with sonication was used. In contrast, when DNA extracted with the in-house method was used, 8/10 were positive by the IS900 PCR (both the single round and nested assay), the same 8 samples were positive by the myco16S PCR and of these, 6 were positive by the f57 PCR. All PCR products were confirmed by sequencing. One M. paratuberculosis culture positive bovine sample was negative by PCR using the IS900 and f57 assays however it was positive with the universal 16S rRNA gene PCR. One further bovine sample (bovine 13), was reported as positive by culture however it failed to produce any an amplification product of the expected size including the universal 16S rRNA gene PCR. As outlined above, DNA extracted from this sample was consistently of low concentration (180–270 ng) and purity (260/280 ratio of 2.1 and a 260/230 ratio of 0.3), despite attempting several protocols. The calculated sensitivity of the IS900, nested IS900 and myco16S PCR assays was 80%, while the sensitivity of the f57 assay was 60%. All PCR assays had a specificity of 100% when results were compared to culture. According to the results obtained for serially diluted DNA extracted from M. paratuberculosis ATCC19698, the detection limits of the PCR assays in our hands were found to be the following: single-round IS900 PCR was 0.01 pg of DNA; nested IS900 PCR was 0.01 pg DNA (both equivalent to 10 M. paratuberculosis genomes); f57 assay was 0.1 pg of DNA (equivalent to 100 genomes) and the myco16S was 1 pg (equivalent to 1000 genomes). The LSPA8 PCR assay was extensively tested with all 10 positive samples however no product was obtained for any of the 10 bovine samples tested, from either the flanking or internal primers.

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achieve this, the optimal methods of DNA extraction from tissue samples were initially determined, and based on these findings, DNA samples extracted using the optimal extraction methods were investigated in four validated and widely used PCR assays for M. paratuberculosis (Autschbach et al., 2005; Mobius et al., 2008; Naser et al., 2004; Bhide et al., 2006). It is known that the quality of a DNA sample affects downstream applications, such as PCR (Bhide et al., 2006), and therefore it is imperative that the DNA extraction method employed to extract mycobacterial DNA from various samples provides a high quantity of pure DNA. To date, many studies have used commercial kits to extract M. paratuberculosis DNA from tissue (Alinovi et al., 2009; Collins et al., 2006). However, as reported by others (Radomski et al., 2013), commercial kits result in lower yields of DNA when compared with in-house methods. Indeed in the current study we showed that the amount of DNA extracted from a kit never exceeded 600 ng while the in-house method provided DNA amounts ranging from 33 to 64 μg. Sonication was added to the in-house method as a way of enriching the sample of mycobacterial cells by lysing all other cells. When sonication was used, the number of positive samples detected by PCR was reduced from 8/10 to 3/10. While unexpected, these results may indicate that sonication is detrimental to M. paratuberculosis in vivo or that it frees M. paratuberculosis DNA that is then degraded by extraction processes downstream compared to intracellular DNA in a sample that is not sonicated. This study aimed to optimise PCR conditions for use in tissue that has other components such as eukaryotic DNA and inhibitors. Once optimised, the sensitivity of these assays was determined. An endogenous internal positive control (IPC) was used to identify samples containing PCR inhibitors (Hoorfar et al., 2004). The 16S rRNA PCR assay is a well established assay and does not impair detection sensitivity by competing with the target DNA for reaction components. However the inclusion of a defined exogenous IPC such as that used in previous studies (Bull et al., 2003) could provide information about the lowest limits of detection of a sample. Of the ten culture-positive bovine samples only two were negative by PCR and of the 10 culture-negative bovine samples, no false positives were observed. One of the two culture positive bovine samples failed to produce a PCR band in any of the PCR reactions, including the universal 16S rRNA gene PCR control assay, highlighting the importance of amplification controls. An adequate quantity of pure DNA could not be extracted from this sample despite attempting several different methods. It is likely that PCR inhibition occurs in other studies of this nature and has significant consequences at an individual level. This study has provided important information regarding the optimal method for DNA extraction from both M. paratuberculosis cultures and bovine tissue samples as well the optimal PCR conditions required to detect M. paratuberculosis. Such information has the potential to significantly improve the PCR detection rates of M. paratuberculosis infection in bovine tissue samples. Although human tissue samples may present new challenges, the results of the current study provide a useful starting point.

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Please cite this article as: Timms, V.J., et al., Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.03.016

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We would like to thank Jacek Gwozdz and Marios Carajias (Department of Primary Industries, Attwood, Victoria) for catalogue and supply of the bovine samples used in this study. This work was funded by Endoscopy Services Pty Ltd and the Australian Research Council.

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Please cite this article as: Timms, V.J., et al., Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis, J. Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.03.016

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Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis.

The aim of this study was to investigate DNA extraction methods and PCR assays suitable for the detection of Mycobacterium paratuberculosis in bovine ...
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