Materials and Methods

i. Patient group

Forty two sputum samples from patients suspected of having tuberculosis were studied at the Medical Microbiology Department of the Royal Free University College Medical School in London. All the patients (24 males and 18 females) samples were processed as per routine laboratory procedure in the department. The sputum samples were subjected to culture, microscopy,

in-house PCR targetting the MPB 64 gene and a commercial kit which was compared to the PCR on samples spotted onto filter papers in this study. All the samples were screened with an auramine fluorescent stain and the status of those deemed positive was confirmed by the Ziehl-Neelsen stain. The samples were cultured on both the Lowenstein-Jensen media and the BACTEC as per routine in the department. Part of the sputum was spotted onto filter papers after decontamination as will be described later in this chapter, dried in a biosafety cabinet at room temperature and kept into separate envelopes till ready for analysis.

ii. List of equipment

The sample preparation, DNA extraction, amplification and detection were performed in different rooms designated clean area, grey area, dirty area and detection room. Each of the above areas had its own equipment and there was no interchange of equipment to avoid contamination.

-Refrigerator 4° Celsius -Freezer -20 and -70° Celsius -Centrifuges

-Dedicated Gilsons, pipettes, tips, pastettes, microcentrifuge tubes and racks -Waterbath

-Vortex and microcentrifuge -Sterile pestle

-Designated box for DNA extracts, protective gowns and gloves.

-Bijou bottles(7ml), small polythene bags, disposable gowns and gloves -PCR thermocycler

-Biomaster (Launch diagnostics) or multi-channel pipette -Weighing balance, weighing boats and Duran bottles (250ml) -Microwave oven

-Autoclave tape, parafiim

-Submarine gel apparatus, 2x18 well combs, gel tray -Power supply

-Ultra violet glasses and photographic system/Kodak digital science ID system

-Scalpel and perspex box

-Southern blot apparatus, hybridisation oven and tubes and Saran wrap -Film cassette (8x10), 3 plastic trays (>8x10) for developer/fixer/wash and a sink.

iii. List of media and reagents

All the reagents were kept in the designated areas as earlier mentioned to avoid any contamination. The reagents used included:

-Respiratory specimen preparation kit (Roche International Ltd, Cat. No 0756784)

-puregene kit which was adapted for DNA extraction from filter papers -MTB positive control kit (Roche , cat no. 0756954)

-Mycobacterium amplification kit (Roche, cat no. 0756784) -Mineral oil (Sigma ,cat, No. 400-5)

-Licensed Tag DNA polymerase and buffers (Gibco BRL, cat. No. 18038-026) -Primers for MPB64 PCR, MPB1 and MPB2 (R and D Systems)

-dNTP's (Promega, cat.No.u 1240)

-Sterile distilled water (Baxter, F7114, via pharmacy Dept) -Primers: (stored àt -20°c for up to 1 year)

G1 MPB-1(20mer): 5'TCC GCT GCC AGT CGT CTT CC-3' G2 MPB-2(20mer): 5'GTC CTC GCG AGT CTA GGC CA-3' Reference: Shankar et al 1990

-Mycobacterium detection kit (Roche, Cat.No.0757462) -Deionised distilled water (ultrapure, Elga)

-Agarose (Molecular biology grade 15510-019, Gibco BRL) -Ethidium bromide (lOmg/ml, E l510, Sigma)

-pGEM DNA marker (O.lug/ul, Promega) -3MM Whatman paper

-Quickdraw blotting paper(Sigma)

-Hybond N+ nylon membrane (Amersham Int. Pic, RPN 3038) -Hyperfilm MP (8x10”) (Amersham Int. Pic, RPN 1678H)

-Gene Images Random Prime Labelling and Detection System (Amersham Int. Pic. RPN 3500)

-Kodak GBX Fixer and Developer (P7167 and P7042, 5gal, Sigma) -0.5x Tris-borate electrophoresis buffer (TBE)

-Concentrated stock solution, 5xTBE: 54g Tris base (Sigma), 27.5g Boric acid (Sigma) and 20ml 0.5M EDTA (Sigma) pH 8

This solution was stored at room temperature and was discarded if any precipitate formed.

-Gel loading buffer ( stored at 4°C ): 0.25% Bromophenol blue, 40%(w/v) sucrose in water

-5M NaCI (292.2g/L) -1M NaOH (40g/L)

-1M Tris pH 7.4 (132.2gTris Hcl, 19.4gTris Base per L) -lOx SSC used for southern blot transfer.

-5x SSC used to prewet blot prior to prehybridisation step.

(Concentrated stock solution 20x SSC-175.3g NaCI, 88.2g Sodium Citrate, adjusted pH to 7.0 and made up to 1L).

-Gene Images buffer A (12.11g Tris Base, 17.53g NaCI, adjust to pH 9.5 and make up to 1 litre).

-Kodak GBX Fixer and Developer dilute 1:10 for working solutions and discarded when they changed colour or became cloudy.

-Probe: Oligonucleotide MPB3 (30 Mer 5TGG ACC CGG TGA ATT ATC AGA ACT TCG CAG-3',R and D systems) labelled with Gene Images random prime labelling and detection system (Amersham Int.PIc.RPN 3500). Stored at -20°C for up to 1 year.

-Extraction: Negative controls supplied with respiratory specimen preparation Kit, positive controls supplied separately and working solutions of controls was used only once,

iv. Methods

The specimen preparation and DNA extraction was done in the category 3 laboratory and the manufacturers instructions were followed for the sputum samples. Respiratory specimen preparation kit used for all specimens was stored at 4°C.

a. Decontamination procedure

The sputum samples were decontaminated by the N-acetyl-L-cysteine-NaOH method and concentrated by centrifugation. Briefly, an equal volume of N- acetyl-L-cysteine-NaOH solution was mixed with the specimen and left at room temperature for 20 minutes. A phosphate saline buffer (67mM, pH 6.8 ) was added up to 50 ml, and the mixture was centrifuged at 3, 500 x g for another 20 minutes. Excess fluid was poured off and the sediment was used for microscopy and DNA extraction as described below.

b. DNA extraction

When the samples were received:

i. The specimen and patients details were filled onto a clinical TB PCR record sheet. All the specimens recieved were stored at 4°C until processed. The samples were centrifuged at 3000 rpm for 5 minutes to collect specimen from the side of the container.

ii. The sample was divided prior to extraction, allowing for a repeat if necessary, and stored at -20°C in the category 3 laboratory.

iii. An appropriate number of 1.5ml eppendorf tubes were labelled and placed in a rack after which SOOul of sputum wash solution was dispensed into each tube.

iv. lOOul of the decontaminated specimen was then added to each tube containing the sputum wash and vortexed.

For filter papers, 50ul of the decontaminated sputum was spotted onto filter papers, as earlier described for viral load in previous chapters, dried in a biosafety cabinet at room temperature and then put into individual envelopes. The DNA was then later extracted using the Purgene kit method as earlier described in chapter 6. For the Roche kit, the following DNA extraction steps were followed.