It was the moment David Cameron came face to face with the West Midlands MEP threatening to cause him one of the biggest headaches of his career.

Nikki Sinclaire collared the Prime Minister during the Conservative conference in Manchester – and asked him when his government would back a referendum on leaving the European union.

Mr Cameron faces the prospect of an awkward Commons vote when MPs debate calls for a referendum.

And it’s largely down to Ms Sinclaire – after she led a successful campaign to collect 100,000 signatures for a petition “to allow the British people a referendum on the United Kingdom’s continued membership of the European Union.”.

The coalition agreement, drawn up by the Conservatives and Liberal Democrats last year, promises that any petition securing 100,000 signatures will be considered for a formal debate in Parliament.

And Ms Sinclaire and allies have collected more than 100,000 names, including 49,000 in the West Midlands alone, after she took her EU battle bus decorated with the vivid colours of the union flag across the country.

The formal request for a debate was then made by Conservative MP David Nuttall, who told the Commons Backbench Business Committee: “I suggest that the matter is of enormous national interest”.

Some commentators have suggested Mr Cameron is increasingly out of step with his own party over the issue of Europe, and a serious vote on whether to hold a referendum – the first the Commons has had since 1975 – would reveal strong support for a referendum among MPs.

But the Prime Minister was on good form when Ms Sinclaire introduced herself in Manchester to ask when a referendum would take place.

She said: “I bumped into him by chance at the Hilton in Manchester while he was there for the Conservative conference.

“He didn’t want to talk about a referendum but he said he believed membership of the European Union was good for the UK.

“It was an amiable meeting.”

But she predicted that Mr Cameron would face growing pressure on Europe unless he gave in to demands for a referendum.

David Cameron talks tough on immigration, but the immigration that concerns people is coming from Eastern Europe and he knows he can’t do anything about that while we remain in the European Union.

“He talks about repatriating powers from the EU back to member states, but that cannot happen without the consent of every member of the EU, and it would probably require a referendum in Ireland and maybe other countries too.

“This campaign is going to keep on growing unless he listens. It is a real Achille’s heal for David Cameron.”

Ms Sinclaire is an independent MEP who was previously a member of UKIP. Her campaign for a referendum is backed by MPs including Labour’s Roger Godsiff (Lab Hall Green) and Conservative Richard Shepherd (Aldridge Brownhills), as well as UKIP West Midlands MEP Mike Nattrass.