Reverse line blotting

The identity of the infecting B. burgdorferi s.l. genospecies in all ticks yielding a real-time PCR product was determined using reverse line blotting (RLB), when sequencing did not give a clear result or for samples after September 2016. This method was as described previously (Alekseev et al, 2001). For full recipes of solutions see appendix 2.

2.5.1 Activation of the membrane and linking of genospecies-specific

oligonucleotides to the membrane.

The Biodyne C membrane was activated by incubation with 5ml of 16% (w/v) EDAC (Sigma, Dorset) solution for 15 min at room temperature. The membrane was rinsed with distilled

H₂O for 2 min on a mechanized rocker then placed on the inverted top half of a 45 lane mini-blotter base (Immunetics, Boston). Once the position of the membrane had been verified, a blotting cushion, then the bottom half of the mini-blotter were placed on top of

it, completing the “sandwich”, which was held together by tightened screws.

Each oligonucleotide (Eurofins, Luxemburg) (Table 3) was diluted in 150µl of 0.5M NaHCO₃

(pH 8.4) to a concentration of 10pmol 150μl-1_{. To the mini-blotter 150µl of ink solution (1µ} of ink in 100µl of 2X SSPE) was added to the first lane. Then into each subsequent lane 150μl

of each oligonucleotide was added and in the last lane the ink solution. Once all the samples have been applied to the min-blotter it was incubated at room temperature for 1 min and then the oligonucleotides and ink were removed by aspiration in the same order they were applied.

Forceps were used to transfer the membrane from the mini-blotter into a tray containing 100ml of 0.1M NaOH, in which the membrane was washed for 9 min at room temperature on a mechanized rocker. The membrane was rinsed in distilled H₂O then incubated in 250ml

of 2XSSPE/0.1% SDS at 50°C for 10 min in a rocking oven. Next, the membrane was incubated in 100ml of 20 mM EDTA at room temperature for 15 min on the mechanized rocker, after which it was wrapped in cling film and stored at 4°C until needed.

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2.5.2 Generation of 23S-5S rDNA intergenic spacer region amplicons

Each sample was processed by PCR, each well contained a 10pmol µl-1 _{solution of biotin} labelled primer B-5SBor primer 23SBor (Table 3), 7.5µl of PCR grade H2O and 12.5µl of 2 x MyTaq Red Mix (Bioline Ltd, London). Finally, 2µl of DNA extract was added. Typically, PCRs were carried out on 96 well plates, exposed to the following thermal cycle: 94°C for 3 mins, 94°C for 20 sec, 60°C for 30 sec, 72°C for 30 sec. The annealing step then decreases by 1°C until it reaches 52°C and then it is repeated for 40 cycles. Then a 72°C extension step that lasted for 10 min. The product was immediately placed on ice. 10µl of each PCR product was then added to 150µl of 2X SSPE/0.1% SDS in a 1.5ml Surelock microcentrifuge tube (Starlabs Ltd). This was heat denatured by placing the sample on a heating block at 100°C for 10 min. Then the products were again immediately placed on ice to cool and left there for at least 5mins before centrifuging at 11,000 x g for 15 seconds, then returned to the ice.

2.5.3 Hybridisation of amplicons to oligonucleotides linked to the

membrane and detection of bound amplicons

The membrane was washed for 5 min in 250ml of 2XSSPE/0.1% SDS at 50°C in the rocking hybridizing oven. The membrane was then once again applied to the mini-blotter so the holes are perpendicular to the position of the applied oligonucleotides. Any residual fluid was removed from the holes of the mini-blotter by aspiration. 150µl of the diluted PCR product was added to the blotter again taking care not to introduce bubbles. This was left to hybridize for 45 min at 50°C keeping perfectly horizontal, after which the mini-blotter was removed from the oven and the PCR products aspirated. The membrane was removed from the mini-blotter using forceps and was washed in 250ml of 2X SSPE/0.5% SDS for 10 min at 60°C and this wash was repeated.

The membrane was allowed to cool to prevent inactivation. 5 µl streptavidin POD conjugate (Roche, Basel Switzerland) was added to 25ml of 2X SSPE/0.5% SDS, which had been

preheated to 42°C, and the membrane was incubated in this solution for 30 mins at 42°C in the rocking oven. Then the membrane was washed in 250ml of 2X SSPE/0.5% SDS for 10 min at 42°C in the rocking oven and this wash was repeated. The membrane was then washed in 250ml of 2 XSSPE (Sigma, Dorset) for 5 min at room temperature on the mechanized rocker

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and this wash was repeated. 5ml of ECL 1 and 5ml of ELC 2 (GE Healthcare,

Buckinghamshire) was added to the membrane in quick succession so the membrane was covered and incubated for 1 min with agitation. The membrane was then carefully wrapped in cling film and taped into a film cassette and taken to the dark room.

Working by red light only Amersham Hyperfilm, ECL (GE Healthcare, Buckinghamshire) was overlaid onto the blot in the cassette, which was sealed and left for 7 mins. The film was taken out of the cassette and placed in developer (Kodak, New York) for 1 min with constant movement, it was then placed in tap water for 1 min to rinse off the developer and then into fixative (Kodak, New York) for 1 min. The film was examined.