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SID 5 Research Project Final Report

SID 5 (Rev. 3/06) Page 1 of 20

NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code SE3235

2. Project titleTo Improve the sensitivity of the Mycobacterium tuberculosis (MTB) complex typing technique of spoligotyping for direct application.

3. Contractororganisation(s)

Dr.G.M. Taylor, Windeyer Institute UCL, 46 Cleveland Street, London W1T 4JF.gm.taylor@ucl.ac.ukTel: 0207-679-9487                    

54. Total Defra project costs £ 58,285(agreed fixed price)

5. Project: start date................ 01 September 2008

end date................. 30th April 2009

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.Executive Summary.This project details attempts made to improve the sensitivity of the MTB complex DNA fingerprinting method of spoligotyping for direct application. Modifications were made to the existing conventional (cPCR) method and an asymmetrical variant of PCR, known as linear-after-the exponential phase (LATE) PCR was evaluated. This latter approach has the advantage of preferentially amplifying the biotinylated strands of the DR amplicons which are used in hybridisation to the immobilised spacers. Real time PCR platforms were used to monitor formation of the DR region products. The DNA intercalating dye SYBR Green was used to observe formation of double stranded products and dual-labelled fluorescent hydrolysis probes were used to follow amplification of biotinylated and non-biotinylated products resulting from elongation of the excess primer DRa and the limiting primer DRb respectively. In initial experiments, real time PCR methods for two of the DR spacers were developed and probe chemistries compared as tools for monitoring and optimising changes to both the cPCR and LATE PCR methods. The regions chosen for this purpose were spacers 23 and 38, loci which are retained in the majority of M. bovis isolates found in the United Kingdom.

Evaluation. Improvements to PCRs were judged from outputs from the real-time PCR platform software, such as the reaction efficiency, the correlation co-efficient (RSq) for standards versus cycle threshold (Ct), Y, the slope of line of best fit and end-point fluorescence values. The modified and LATE PCR methods were assessed and compared to the original protocol using serial dilutions of reference strains, degraded M. tuberculosis DNA from archaeological contexts and environmental samples containing PCR inhibitors. Comparisons were made using criteria from the real-time platformsdescribed above, conventional membrane spoligotyping and the Luminex platform.

Findings. The data obtained showed that the existing protocol can be improved either by using LATE PCR or a modified form of cPCR. Both methods resulted in approximately a 10 fold increase in sensitivity. Accurate spoligotypes were obtained from templates in the range 1-20 genome equivalents (GE) cf. around 20-200 GE for the unmodified cPCR. If required, both methods allow quantitative real-time PCR to be performed and permit samples and blanks to be monitored by fluorescent hydrolysis probes. The changes to the existing method are minimal and are compatible with high throughput.Implications and future work. The ability to overcome inhibition offered by both the modified cPCR and LATE PCR means that it should be possible to extend the range of samples successfully spoligotyped to environmental and clinical

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specimens widely recognised as problematical e.g. faeces and soils without the need to culture.

This was a short project with limited opportunity to apply the developed methods to a large number of isolates. It would therefore be advisable to extend the modified assays to a larger number and sources of direct isolates. Samples that could be included in such an undertaking include fixed histological tissues, lymph nodes with and without visible lesions (VL & NVL) and urine and blood from infected cattle. A more sensitive form of spoligotyping may prove to be applicable for genotyping mycobacteria shed from vaccinated badgers in field trial areas and would assist in distinguishing vaccine strains from wild-type infections. Finally, if adopted, either the modified cPCR or LATE PCR could shorten the culture time required for successful spoligotyping.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

SE3235 – Final Report.Background. Spacer-OLIGOnucleotide typing – ‘spoligotyping’ is a PCR-based molecular typing procedure for MTB complex isolates which determines polymorphism at the direct repeat (DR) locus in the TB genome (Kamerbeek et al, 1997). The method is particularly useful for typing strains with low copy numbers of the insertion sequence IS6110, used in RFLP typing methods (Mazurek et al, 1991; Goyal et al, 1997).At the DR locus, repetitive DNA sequences 36 bp long are separated by spacers of between 35-41 bp. Over time, spacers are subject to deletion events through recombination of the DR regions, generating a unique "fingerprint". Genetic change can occur at other loci on the genome and therefore whilst the spoligotype does not define the clone it is useful for tracking isolates involved in short transmission chains and disease clusters.

The current method uses primers which target the DR and amplify double stranded DNA. The reverse primer is biotinylated with the result that all reverse strands are also biotinylated. The PCR product is heat-denatured and allowed to hybridize to a membrane containing the 43 spacers applied in parallel lanes using a blotting apparatus. The labelled strand may either hybridize to the membrane or recombine with the opposite strand.The membrane is washed stringently and sites of specific hybridisation are visualised by immunohistochemistry and enhanced chemiluminescence (ECL) with data recorded on x-ray film. Sensitivity of the method is not problematical if applied to DNA prepared from cultures but when applied directly to clinical samples, spoligotyping may fail or produce incomplete patterns. Data from paucibacillary samples may be complicated by weak hybridisation signals from some spacers making interpretation subjective.

Direct spoligotyping fails in up to 50% of cases when applied to culture-positive cattle tissue with visible lesions (VLN) even in cases which are PCR positive using the IS1081 element (Project SE3008; Taylor et al, 2005). As the IS1081 method will detect 91% of culture-positive cases of M. bovis (project SE3008) the current spoligotyping protocol will fall far short of its potential if directly applied to samples without prior culture. In this earlier project, use of an “optimised” protocol (Parwati et al, 2003) did not result in an improved success rate, suggesting a more radical approach is required. Potentially, spoligotyping should be as sensitive as any PCR amplifying multi-copy targets. In practice, the multiplex nature of the PCR favours "allele dropout" reducing sensitivity, as does reannealing of biotinylated and unlabelled strands during hybridization in the miniblotter.

Sequence capture of the DR region has been applied to problematical cases where mycobaterial numbers are scarce or when inhibitors of the PR reaction may be present (Mangiapan et al, 1999). However, sequence capture is a relatively complicated technique for routine diagnostic laboratories and adds significantly to unit cost.

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Moreover, specific extraction limits downstream applications to analysis of the captured DR region, preventing analysis of other loci by PCR-based methods e.g. vntr typing.

Aims.The main stages of the current project are summarised below:

1. Primer redesign to get Tmelt (L) –Tmelt (X) to approximate to 5oC..

2. Keeping Tmelt (L) and Tmelt (X) as high as possible compared to average Tmelt of the amplicons (TmeltA).

3. Optimising ratio of the excess primer, X to limiting primer L (X:L primer ratio).

4. Monitoring of exponential & linear phases.

5. Project outputs: LATE spoligotyping protocol.

6. Comparison with original SOP, as run at VLA Weybridge.

Methods.Materials. Oligonucleotide primers were obtained from Operon which became Eurofins DNA (www.eurofinsdna.com) after the merger of Operon with MWG. Dual labelled fluorogenic probes were supplied by Sigma-Genosys. Chemicals and other reagents were supplied by VWR International or Sigma-Aldrich.

PCR.a). Conventional hot-start PCR (cPCR) was performed in a final volume of 25µl using the Excite Core kit from BioGene with minor modification to the manufacturer’s instructions. Each reaction contained 25 pmol of each primer in 1µl, 2.5 µl of 10x reaction buffer, 1.5-3µl Mg2Cl, 2.5µl bovine serum albumin (non-acetylated, BSA, Sigma B4287), 1µl of template and 0.5U of Taq polymerase. The volumes were made up to 25 µl with water (VWR). After an initial denaturation step (between 8 -15 min at 95 oC), 35-41 cycles of amplification were performed as follows: denaturation at 95o C for 10s, annealing at 52oC for 30 s, extension at 72oCfor 20 s. SYBR Green (2.5µl) was included in some experiments at a final dilution of 1/55 000, and reactions were performed and monitored on either a Corbett RotorGene 3000 or a 6000 real-time PCR platform. Melt analyses were performed with the RotorGene software and all products were also run on 3% agarose checker gels. When hybridisation probes were included, these were used at a final concentration of 100nM, through the addition of 1µl 2.5µM working stock of probe/ 25µl reaction.

b). Linear After the Exponential PCR (LATE-PCR). Primer ratios of the excess:limiting primer were investigated over the range 100:1 to 5:1. Formation of double stranded product was monitored with SYBR Green and individual strands with hybridisation probes. A number of real-time kits from different manufacturers were compared with the original BioGene excel core kit. The products compared included: the Brilliant II QRT-PCR

master mix kit from Stratagene (product 600809, the ABI Universal Master Mix Kit (product 4304437) and the Hotstart taq master mix kit from Qiagen (product 203445).The effect of inclusion of BSA on product formation was also studied, as was the optimum magnesium concentration. Product formation was followed up to a maximum of 55 cycles.

c). Quantitative real-time PCR (QPCR). This was applied using both cPCR and LATE PCR protocols. It was performed in a total reaction volume of 25µl using one or more of the dual labelled fluorogenic probes or, on occasion, with one of the probes combined with SYBR Green as described above. Six DNA standards were prepared from a working stock DNA extract of BCG Pasteur and spanned the range 30,000-0.3 genome equivalents (GE) / µl. In some experiments wild-type M. bovis strain AF2122/97 was used to prepare DNA standards and these covered the range from 2.18 x106 GE/µl to 2.18 GE/µ. Standards were run in duplicate or in triplicate. Assays were performed on the Stratagene 3005 Mx platform, or on one of the Corbett machines mentioned above. Non-template controls, with water in place of unknown or standard were assayed in triplicate.

d). Spacer 23 PCR. This was developed as a sentinel PCR to optimise the hybridisation probes for this amplicon, prior their use in cPCR and LATE methods with the DR primers. The sequences of the spacer 23 primers are shown in Table 1. The method was found to be tolerant of a range of magnesium concentrations and a suitable means of monitoring PCR efficiency. Figure 1a shows spacer 23 product (169 bp) amplified using the specific primers 22F & 24R compared with the typical range of product sizes obtained using the spoligotyping primers DRa and DRb (Figure 1b).

Table 1. Primers and probes: sequences and other physical properties.

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Primer name Sequence Tmelt

MWGTmelt

OPCMW G+C

%

DRa 5’-GGTTTTGGGTCTGACGAC-3’ 59.9 48 5562 56Dra 5’-BITEG 5’- GGTTTTGGGTCTGACGAC-3’ 56.0 48 6131 56DRb 5’-CCGAGAGGGGACGGAAAC-3’ 64.5 53 5562 67 DRb2 5’-CCGAGAGGGGACGGAAA-3’ 62 51 5318 6522F 5’-ACCGCAGACGGCACGATTGAGACAA-3’ 67.8 63 7694 5624R 5’-GAGCACGTCTCACCCAGCAGGCG-3’ 71.7 64 7019 70Spacer 23 (Cy3)

5’-AGCATCGCTGATGCGGTCCAGCTCGTCC-3’(BHQ1) 83.3 69 9605 64

Spacer 23 (FAM)

5’-ATCGCTGATGCGGTCCAGCTCGTCCG-3’(BHQ1) 82.4 68 9031 65

Spacer 23 RC (FAM)

5’-CGGACGAGCTGGACCGCATCAGCGAT-3’(BHQ1) 82.4 68 9098 65

Spacer 38 (HEX)

5’-TGGATGGCGGATGCGTTGTGCGCGCAA-3’(BHQ1) 88.3 69 9711 63

RC = reverse complement. OPC = oligonucleotide properties calculator at:

http://www.basic.northwestern.edu/biotools/oligocalc.html

Figure 1A. Spacer 23 PCR with specific product of 169 bp amplified with primers 22F and 24R. This was used to optimise the FAM probe method which was then used to monitor amplification of the DR region. Figure 1B. Multiple products leading to a smear when DR primers are used in spoligotyping.

Spoligotyping.a). Conventional spoligotyping. This was performed using the method of Kamerbeek et al, 1997. A pre-prepared membrane for this purpose was obtained from Ocimum Biosolutions B.V., The Netherlands. After hybridisation and stringent washing, the membrane was incubated with streptavidin POD conjugated antibody (product 11 089 153 001, Roche). Signals were detected using the Western Blotting Substrate kit (product 32209, distributed by Thermo Fisher) with exposure to X-ray film (Hyperfilm ECL 18 x 24 cms., GE Biosciences).

b). Luminex Platform. Some spoligotyping products from reference strains were analysed on the Luminex platform (Luminex®100™ IS Total System, Luminex Corporation, Austin, Texas) at VLA Weybridge. This is a high throughput platform which reportedly yields concordant results with the conventional protocol and is a fast and cost effective alternative to traditional spoligotyping (Cowan et al, 2004). This is being investigated at VLA Weybridge by Steve Anderson and Mike Chaplin. Limited reagent availability at VLA allowed only one experiment to be performed.

Quantification of partially-purified DNA from reference strains. This was determined using a Nanodrop ND-1000 Spectrophotometer. The BCG standard used in some experiments was provided by Warwick University as part of project SE3231. Calculation of genome equivalents in the working stock and dilutions thereof was based upon their calculations originally determined by flow cytommetry and subsequently corrected as follows: Mean genomic standards = 42.41 x flow cytometric standards.

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Results.1. Primer redesign. The original primers DRa and DRb (Table 1) were designed by Kamerbeek et al for conventional PCR and have been used since by a number of researchers. The first aim of the study was to engineer a difference of 5oC between the primers with DRb having a Tmelt about 5oC higher than the excess primer DRa. Study of the primer Tmelts using the oligonucleotide properties website

(http://www.basic.northwestern.edu/biotools/oligocalc.html) showed an existing imbalance of 5oC between the two primers, with DRb (limiting primer) having a Tmelt of 53oC and DRa (excess primer) having a Tmelt of 48oC. In theory, the existing primers should be well suited for LATE PCR and so no modifications were made. This raised the question of whether it would be advantageous to reduce the G+C content of DRb to more closely match DRa for use in the existing protocol. This was studied in this project by following product generation with the existing primers and also using DRa with a modified DRb (DRb2) in which the terminal C residue was removed, lowering the Tmelt to 51oC. This reaction was followed using SYBR Green and also with dual labelled fluorogenic probes (details below). Reference strains amplified with DRa/DRb and DRa/DRb2 primer combinations monitored with SYBR Green showed little difference in end point fluorescence or Ct (not shown). To get an idea of reaction efficiency, BCG standards were also amplified with the same two primer combinations and monitored with the FAM Spacer 23 hybridisation probe. In this experiment, the modified primer pair showed an RSq value of 0.996 (cf 0.991) and an efficiency of 94.8% (cf. 82.4%) for the conventional primers. This indicates there may be a small advantage to be gained by using the modified primer combination in the cPCR method when problematical samples are assayed. However, it is unlikely this would be necessary when DNA from cultures are tested as at present.

2. Monitoring of DR PCR amplicons in conventional and LATE protocols. The DNA intercalating Dye SYBR Green was used to follow the formation of double-stranded DNA in conventional PCR and in the early cycles of LATE PCR (Supplementary Figure S1). Dual-labelled linear fluorogenic hybridisation probes were used to monitor amplification of both the excess and limiting primer strands. A variety of probe chemistries were compared and chosen to allow simultaneous detection of the strands. To achieve this, probes complementary to the excess strand were designed to hybridise to spacer 23 and to spacer 38, as these spacers are conserved in the majority of MTB complex isolates. Early experiments evaluated a Cy3/BHQ1 combination for spacer 23, to allow simultaneous use with SYBR Green. However, data acquisition for this probe was poor on the RotorGene platforms (3000 and 6000) but acceptable on the Stratagene MxPro 3005P platform. Later experiments used FAM/BHQ1 and HEX/BHQ1 chemistries for spacers 23 and 38 respectively. Generally, higher fluorescence values were obtained with FAM/BHQ1 for spacer 23 compared to Cy3/BHQ1 and HEX/BHQ1 for spacer 38 probe. In some experiments, a probe with reverse sequence complementarity (RC) to spacer 23 was used to follow limiting strand formation. This was also FAM/BHQ1 labelled, requiring separate tubes to be used when run in the same experiment with spacer 23 FAM probe. The result of one of the assays is shown in Figure S3. It is clear that limiting primer stand is still formed under LATE PCR conditions used here but reaction efficiency, Rsq value and end point fluorescence values are all reduced (Figure S3a) compared to excess primer strand (Figure S3b). Formation of the limiting strand could also be monitored in the same tube using the RC spacer 23 FAM probe in combination with spacer 38 HEX probe if required. Generally, this was found to be problematical and revealed sporadic amplification of the limiting strand, particularly at lower starting template concentrations (Figure S4a). The reasons for this are not readily apparent. Additionally, it should be noted that end-point fluorescence values are not comparable when different probe chemistries are used.

Optimisation of a LATE PCR protocol for the DR region. Limiting primer concentration. Based on results obtained in a series of experiments with varying concentrations of limiting primer DRb, a ratio of 20:1 was chosen and used in subsequent experiments. This ratio is typical of those used in LATE PCR methods (Pierce et al, 2005). At this ratio, it was apparent that double stranded products were formed and efficiency of the linear phase was maintained when this was followed with the FAM probe. For a given template concentration efficiency of the reaction was lower when the ratio DRa:DRb was 32:1 or less and a right-shifting of cycle threshold (Ct) was observed (Figure 2).

Figure 2. Investigation of primer ratios.

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Investigation of Optimum Magnesium ion concentration. This was assessed using the single target spacer 23 assay (Figure 3) as well as the whole DR region using DRa and DRb primers (Figure 4). The probe method was broadly tolerant of magnesium ion concentration but higher end-point fluorescence values were obtained at 3mM Mg2+ and above.

Figure 3. Effect of Magnesium ion concentration on spacer 23 amplified with specific primers and monitored with the FAM probe.

Figure 4. Effect of Magnesium ion concentration on spacer 23 amplified as part of the whole DR region and monitored with the FAM probe. Note shallower amplification profiles when a number of amplicons are generated.

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Evaluation of RT-PCR master mix kits from various manufacturers. Kits from Stratagene, Applied Biosystems and Qiagen were compared with the Excite core kit from BioGene, a product which has previously been shown to be one of the most efficient and flexible systems for problematical samples (Taylor et al, 2005). The spacer 23 PCR was used to compare amplification efficiencies, Ct values and profiles of the exponential phase of amplification (see Figure 5). Based on these criteria, the Qiagen kit, supplemented with additional magnesium to a final concentration of 3mM, was chosen for subsequent use in conventional PCR (cPCR) and LATE PCR protocol.

Figure 5. Spacer 23 primers with various PCR kits.

Amplification of the whole locus with DR primers DRa and DRb similarly confirmed that a more efficient reaction was obtained with supplementary magnesium concentration to 3mM.

Figure 6. Qiagen hot start master mix kit used with (3mM) and without (1.5mM) supplementary magnesium.

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Comparison of hybridisation probe chemistries for spacers 23 and 38 for monitoring biotinylated strand formation.

The FAM and HEX labelled probes for spacers 23 and 38 respectively were compared using serial dilutions of BCG Pasteur over the range 30,000 to 0.3 genome equivalents. The experiment showed little difference between the two chemistries, with good standard curves being obtained by both methods. Efficiency of the FAM labelled probe was slightly lower at 81.5% compared to the HEX probe 85.5%. This is shown in Figure 7.

Figure 7. BCG standards amplified by LATE PCR and monitored with two probe chemistries.

Inclusion of BSA in the PCR master mix. Work done for project SE3231 on faeces, soil and other problematical environmental samples showed that BSA was a necessary addition to the PCR reaction mix if inhibitors were to be successfully overcome. Therefore, BSA was added to the optimum kit from Qiagen and the amplification compared to controls without BSA. The results are seen in Figure 8. with Non-acetylated BSA, 2.5 µl of a 10mg/ml stock solution, added to the optimum kit from Qiagen did not impair the amplification reaction and so this was used in all subsequent experiments.

Figure 8. Spacer 23 cPCR amplification monitored with the FAM probe. Effect of inclusion of BSA (Non-acetylated BSA, 2.5 µl of a 10mg/ml stock solution added to each 25µl reaction.

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Project outputs.

1. The LATE PCR Protocol. Data from the primer ratio experiments, comparison of commercial master mixes, magnesium optima, inclusion of BSA, cycle nos. and probe chemistries was combined to produce a LATE PCR protocol for the DR region.

1. LATE PCR is performed in a final volume of 25 µl using the Qiagen HotStarTaq master mix kit. This is a premixed solution containing HotStarTaq DNA Polymerase, PCR Buffer, and dNTPs. The solution provides a concentration of 1.5 mM MgCl2 and 200 µM each dNTP.

2. Each 25 µl reaction contains 1µl (25 pmol) of excess primer DRa, 1µl (1.25 pmol) limiting primer DRb, 12.5µl of master mix, 2.5µl bovine serum albumin (10mg/ml), 1µl of hybridisation probe (100nM final), an additional 1.5µl of magnesium chloride (25mM) and 1µl of template. The volumes are made up to 25 µl with molecular biology grade water.

3. Cycling parameters. After an initial denaturation step of 15 minutes at 95oC, 50 cycles of amplification are performed as follows: denaturation at 95o C for 10s, annealing at 52oC for 30s, extension and data acquisition at 76oC for 60s. The reaction can be monitored with either the FAM or HEX hybridisation probe chemistries (Table 1).

4. Initial experiments performed in this short project suggest spoligotypes can be reliably obtained using either the standard membrane apparatus or the Luminex platform.

2. Suggested modifications to the conventional PCR (cPCR) protocol.

For problematical samples, such as those likely to contain inhibitors e.g. soils, faecal samples or environmental material the following options are suggested.

1. Addition of extra magnesium to the Qiagen kit to provide a final concentration of 3mM.

2. Addition of non-acetylated BSA to the reaction tubes to a final concentration of 1 mg/ml (2.5 µl of a stock solution of 10mg/ml). The BSA should be non-acetylated to overcome co-purified PCR inhibitors and to avoid reducing reaction efficiency. The importance of this step is illustrated in Figure 9 which shows 3 badger latrine samples from project SE3231 which were positive for M. bovis using the RD4 Taqman assay (Figure 9a) but negative when spoligotyped at both VLA and UCL with the conventional spoligotyping protocol (Figure 9b). Addition of BSA resulted in the same 3 cases generating DR region amplicon (Figure 9c).

3. If desired, the reaction may be monitored using either the FAM/BHQ1 probe for spacer 23 or the HEX/BHQ1 prober for spacer 38 and terminated as required, e.g. when plateau is reached after exponential phase of amplification. These spacers are present in the majority of M. bovis strains. The spacer 23 FAM probe provides the cheaper option for this purpose.

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4. Use of the shorter DRb2 (Table 1) primer provides a nearer match with DRa in terms of Tmelt with slight gain in reaction efficiency (Figure S2b).

5. Cycle nos. can be increased up to 41 for paucibacillary samples.

Figure 9a. RD4 real-time PCR of Ring Trial 1B latrine samples from project SE3231. RD4 positivity indicates M. bovis DNA. Three of the 100 samples assayed are positive.

Figures 9b and 9c. Conventional spoligotyping PCR applied to Ring Trial 1B latrine samples at UCL without (upper panel b) and with inclusion of BSA, 1mg/ml final concentration (lower panel c).

Comparison of the existing and modified cPCR protocols.

Conventional PCR, with equimolar primer DRa and DRb concentrations and using the Qiagen hot starTaq Master Mix kit was compared to the same kit with the inclusion of additional magnesium (3mM) and with 1mg/ml non-acetylated BSA. For the comparison, M. bovis wild-type reference strain AF 2122/97 was used to prepare a dilution series ranging from 2.18 x 106 genome equivalents down to 2.18 copies. Thirty five cycles of amplification was performed and the HEX hybridisation probe complementary to spacer 38 was used to monitor the formation of products. The results are seen in Figure 10.

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Figure 10. Comparison of modified and original cPCR protocols for DR amplification.

a). Modified cPCR with 3mM Mg and BSA.

b) Current SOP.

The reactions showed similar correlation coefficients, slope and efficiencies but differed in end-point fluorescence values over the range of standards run. For example the value reached by top standard was 1726 units for original SOP cf. 3650 units for the modified reaction. Additionally, according to probe data, the minimum detection limit was 218 GE for the unmodified cPCR and 21.8GE for the modified cPCR. When LATE PCR (50 cycles) was used the minimum detection limit reduced to 2.18 GE (not shown).

Comparison of optimum cPCR and L ATE PCR protocols.

a). Minimum detection limits of dilution series of reference strains. A dilution series of the reference strains BCG Pasteur and H37Rv were prepared. The DR region was amplified with primers DRa and DRb using both the cPCR and LATE PCR methods. The dilution series were assayed in triplicate and product formation was followed with spacer probe 38. LATE PCR was shown to be approximately 10 x more sensitive than the standard protocol whether judged by detection of either the H37Rv or BCG Pasteur dilution series. The results are shown in Table 2. Signal was detected down to 1 genome equivalent (GE) with LATE PCR with the H 37Rv strain and to < 1GE with the M. bovis BCG Pasteur reference strain.

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Table 2. Dilution series assayed by cPCR and LATE PCR protocols.

Strain PCR method

10-3 10-4 10-5 10-6 10-7 10-8 10-9 10-10 10-11

1319 GE/µl

132 GE/µl

13.2 GE/µl

1.32 GE/µl

0.132 GE/µl

H37Rv cPCR + + + - - nd nd nd nd

H37Rv LATE + + + + - nd nd nd nd

12,700* GE/µl

1270* GE/µl

127* GE/µl

12.7* GE/µl

1.27* GE/µl

0.13* GE/µl

BCG Pasteur

cPCR + + + + + - - - -

BCG Pasteur

LATE + + + + + + - - -

GE = genome equivalents. * theoretical value based on the correction factor supplied by Warwick for converting flow cytommetry cell counts to GE (see DNA quantitation in Methods).

b). Assay of heavily fragmented DNA extracts. A total of 20 DNA extracts prepared from 18 separate human burials with osteological evidence of tuberculosis were screened for the presence of MTB complex DNA using the multi-copy marker IS1081. Ten extracts from 8 burials were found to be positive by IS1081 RT-PCR using a hybridisation probe and taken forward for further evaluation. They were then amplified with the DR primers using cPCR and LATE PCR methods. In each case the HEX labelled probe for spacer 38 was used to report positive samples. Using cPCR 4/10 extracts showed a positive profile, whereas when LATE PCR was used an additional 2 cases were found to be positive. The results are shown in Table 3.

Table 3. TB cases with fragmented DNA assayed by cPCR and LATE PCR.Burial No. Case No.

Figure 13Date IS1081

PCRCt Spoligo

cPCR Ct Spoligo

LATE PCRCt

67RL nd Mid-late 18th C Hungary + 35.54 - No Ct - No Ct225 nd “ + 38.92 - No Ct - No Ct106 nd “ + 37.87 - No Ct - No Ct78 1 “ + 30.11 + 31.94 + 38.23121 2 “ + 34.59 - No Ct + 44.0428A (1) 3 “ + 38 - No Ct + 43.2428A (2) 6 “ + 30.90 + 31.32 + 38.4368C (1) 4 “ + 32.06 + 27.74 + 34.9368C (2) 7 “ + 23.53 + 25.28 + 29.3772C 5 “ + 36.48 - No Ct - No CtTotals 10/10 4/10 6/10

c). Environmental Samples from Project SE3231.Assay of 100 badger latrine samples, distributed by Warwick University was conducted at the UCL centre, as part of the Ring Trial 1b component of that grant. Three of these were consistently shown to be positive by RD4 RT- PCR using a dual-labelled hybridiastion probe (Figure 9a). Conventional spoligotyping was unsuccessful unless BSA was included in the PCR master mix, but no additional positive cases were detected. Therefore, to confirm this low prevalence of positives, the LATE protocol was also applied to the same 100 latrine extracts. Only one additional sample, UCL 116, was found to be positive. This is Warwick barcode sample 120047.

d). Conventional spoligotyping. This was established at UCL using the membrane system purchased from Ocimum Biosolutions. Reference strains e.g. BCG and wild-type M. bovis showed the expected spoligotypes using the cPCR procedure.

1. Spoligotyping was applied to the relevant dilutions used in the minimum detection series to compare with hydrolysis probe findings. There was generally good agreement between observing a fluorescent signal during amplification and the ability to obtain a complete spoligotype. Conversely, failure to obtain a probe signal was usually associated with a nil result, or, on some occasions a partial spoligotype was obtained. When the modified cPCR and LATE PCR variants were compared, a full spoligotype was obtained at one lower order of magnitude

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of template concentration using the latter method. For example, a BCG working stock at 10 -7 gave the full fingerprint and 10-8 only a partial pattern using cPCR but with LATE PCR 10 -8 produced the correct spoligotype and the 10-9 dilution a partial pattern. This is illustrated in Figure 11.

Figure 11. Spoligotyping of serial dilutions of M. bovis BCG Pasteur amplified by modified cPCR (rows 1-5) and by LATE PCR (rows 6-9).

2. Environmental samples from SE3231 were spoligotyped after existing and modified cPCR protocols as well as after LATE PCR. As expected from the negative HEX probe data (not shown), the existing cPCR SOP failed to generate more than a few random spacers (Figure 12a). The modified procedure gave a type 17 pattern with equivocal spacer 15 in one of the four cases, a possible type 17 in a second isolate but missing spacers 15 and 21 and failed in the remaining 2 cases. In contrast, LATE PCR showed a type 17 in 3/4 cases and a possible type 54 or 35 in the fourth case where spacers 10-15 were observed. This was Warwick barcode sample 011982L (Figure 12b).

Figure 12a. Spoligotyping of RD4 positive latrine samples from project SE3231 using unmodified SOP failed to generate any reliable patterns. Rows 1,2 = blanks. Rows 3,4 = Warwick 012126C. Rows 5,6 = Warwick 011528H. Rows 7,8 = Warwick 012119E. Rows 9,10 = Warwick 011982L.

Figure 12b. LATE PCR spoligotyping applied to problematical environmental samples from project SE3231. Case 1 = Warwick 011982L. Case 2 = Warwick 012126C. Case 3 = Warwick 012119E. Case 4 = Warwick 011528H.

3. Fragmented DNA from archaeological contexts.. Strict adherence to the conventional Kamerbeek method with 35 cycles of amplification and without addition of BSA yielded partial patterns or generation of only a few spacers. It was considered likely that this was due to the presence of inhibitors of PCR in the extract as the modified cPCR resulted in near complete spoligotype patterns indicative of M. tuberculosis isolates in 6/7 extracts studied. In the remaining case, burial 72, one of the duplicates failed (case 5, Figure 13). Near identical results were obtained from the same samples using LATE PCR, but with all 7 extracts showing strong patterns. All cases were seen to be missing spacers 33-36, which is typical of major phylogenetic groups 2 or 3 (ref). As both modified cPCR and LATE PCR gave essentially the same results, it is likely that the inclusion of BSA in both protocols was the crucial factor rather than poor template quality.

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Figure 13a. Spoligotyping of degraded templates from human remains. Comparison of modified cPCR with asymmetric LATE PCR.

It is interesting that 3 cases that were negative by cPCR generated spoligotypes when analysed. These were burials 121, 28 and 72 (cases 2,3 & 5 in upper figure). Close inspection of the amplification plots for these extracts showed minimal increases in fluorescent signal which did not reach the threshold level set by the automatic software.

Figure 13b. Detail of cPCR run of archaeological samples showing threshold line (green) and sub-threshold increases in fluorescent signal from 3 cases.

e). The Luminex Platform. DR PCR products obtained from the modified cPCR and LATE procedures were taken to VLA Weybridge for analysis on the Luminex platform. The reference strains examined by LATE all showed higher fluorescence signals at the positive spacer sites compared to the cPCR procedure. One of these experiments, with M. bovis BCG Pasteur is shown below in Figure 9. The spoligotype was readily discernible with both protocols and on average the signal intensity at the 35 positive spacers was 126% higher (range -51% to 323%) than with the routine PCR.

Figure 14. Luminex platform:comparison of conventional and LATE protocols.

a). Conventional spoligotyping SOP, 35 cycles of amplificationBCG 10-5 BCG 10-6 BCG 10-7 BCG 10-8 b). LATE spoligotyping protocol, 50 cycles of amplification.BCG 10-6 BCG 10-7 BCG10-8 BCG 10-9

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Conclusions.This project has developed and compared an asymmetric form of PCR, known as LATE PCR with the existing protocol. LATE PCR was found to have a lower minimum detection limit than the current SOP and results in complete spoligotypes down to around 1GE. Roughly equivalent gains were found for a modified form of the existing cPCR where BSA and additional magnesium were included in the master mix. However, LATE PCR proved more effective for spoligotyping 3/4 environmental samples whereas modified cPCR failed. The increased number of cycles in LATE PCR (50) may have been a factor in this instance as evidence from the RD4 assay suggested samples were paucibacillary as well as inhibited. In contrast, modified cPCR (35 cycles) and LATE PCR (50 cycles) were both found to be effective in spoligotyping aDNA cases of tuberculosis in human remains and this was attributed to successful adsorption of PCR inhibitors through the use of BSA. Both cPCR and LATE PCR may be monitored with fluorescently labelled hydrolysis probes and the reaction terminated as required. Increase in fluorescence over a few cycles was associated with successful spoligotyping. Both modified cPCR and LATE PCR can be used in quantitative real-time PCR for M. tuberculosis complex organisms.

Implications.The ability to overcome inhibition offered by both the modified cPCR and LATE PCR means that it should be possible to extend the range of samples successfully spoligotyped to environmental and clinical specimens widely recognised as problematical e.g. faeces, soils without the need to culture. DNA extracts prepared from cattle lymph nodes testing positive by direct PCR for M. bovis, such as RD4, offer a realistic chance of providing spoligotypes for epidemiological studies and surveillance.

Future work. This was a short project with limited opportunity to apply the developed methods to a large number of isolates. It would therefore be advisable to extend the procedures (modified cPCR and LATE PCR) to a larger number and sources of direct isolates from reference strains, such as DNA prepared from the panel of 10 common spoligotypes. It would also be interesting to spoligotype DNA recovered from a number of other archival or degraded sources to permit further validation. Other samples that could be included in such an undertaking include fixed histological tissues, lymph nodes with and without visible lesions (VL & NVL) and urine and blood from infected cattle. A more sensitive form of spoligotyping may prove to be applicable for genotyping mycobacteria shed from vaccinated badgers in field trial areas and would assist in distinguishing vaccine strains from wild-type infections. Finally, if adopted, either the modified cPCR or LATE PCR should shorten the culture time required for successful spoligotyping and should be equally applicable to membrane or Luminex platforms.

Supplementary Figures and Tables. Figure S1.

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Figure S2. Standard curves obtained using a) primer combinations DRa & DRb and b) DRa & DRb2. Note slight improvements in reaction efficiency, R value and slope in graph b. a).

b).

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Figure S3. Monitoring of limiting primer strand with RC spacer 23 FAM labelled hybridisation probe (a) compared with formation of excess primer strand monitored with standard FAM probe for spacer 23. Reactions were performed in separate tubes with the same master mix components, less probes.

a).

b).

Figure S4. Monitoring of limiting primer strand with RC spacer 23 FAM labelled hybridisation probe (a) compared with formation of excess primer strand monitored with standard HEX probe for spacer 38 (b). Reactions were performed in same tubes.a).

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b).

References.

Cowan LS,Diem L, Brake MC, Crawford JT. Transfer of a Mycobacterium tuberculosis genotyping method, spoligotyping, from a reverse line-blot hybridization, membrane-based assay to the Luminex multianalyte profiling system. J.Clin.Microbiol. 42: 474-477, 2004.

Goyal M, Saunders NA, van Embden JDA, Young DB, Shaw RJ. Differentiation of Mycobacterium tuberculosis Isolates by Spoligotyping and IS6110 Restriction Fragment Length Polymorphism. J. Clin. Microbiol. 35: 647-651, 1997.

Kamerbeek J, Schouls L, Kolk A, Van Agterveld M, Van Soolingen D, Kuijper S, Bunschoten A, Molhuizen H, Shaw R, Goyal M, Van Embden J. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J.Clin.Microbiol. 35: 907-914, 1997.

Mangiapan G, Vokura M, Schouls L, Cadranel J, Lecossier D, Van Embden J, Hance AJ. Sequence capture-PCR improves detection of Mycobacterial DNA in clinical specimens. J. Clin. Microbiol. 34: 1209-1215, 1996.

Mazurek G H, Cave MD, Eisenach KD, Wallace RJ Jr., Bates JH, Crawford JT. Chromosomal DNA fingerprint patterns produced with IS6110 as strain-specific markers for epidemiologic study of tuberculosis. J. Clin. Microbiol. 29: 2030–2033, 1991.

Parwati I, van Crevel R, van Soolingen D, van der Zanden A. Application of spoligotyping to Noncultured M. tuberculosis bacteria requires an optimized approach. J. Clin. Microbiol. 41: 5350-5351, 2003.

Pierce KE, Sanchez JA, Rice JE, Wangh LJ. Linear-After-The-Exponential (LATE)-PCR: Primer design criteria for high yields of specific single stranded DNA and improved real-time detection. PNAS 102: 8609-8614, 2005.

Taylor GM, Worth DR, Palmer S, Jahans K and Hewinson RG. Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR. BMC Veterinary Research 3:12, 2007. doi:10.1186/1746-6148-3-12.

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

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