Cabinet minister Michael Gove has ruled out the prospect of Nicola Sturgeon being granted a second independence referendum - even if her party wins a majority at Holyrood in 2021.
Mr Gove said such a vote would be a "waste of time" and a "distraction", insisting the SNP should instead be focusing on dealing with problems in Scotland's hospitals and improving the education system.
The Chancellor of the Duchy of Lancaster argued another ballot on the issue should not be allowed to take place as it would contribute to eroding trust in politicians, citing previous pledges from SNP leaders that the 2014 referendum would be a "once in a lifetime event".
Mr Gove's comments came after Alister Jack appeared to suggest an SNP majority in the next Holyrood election could give rise to a mandate.
The Scottish Secretary said on Tuesday the "democratic mandate for a Section 30 order", which would permit a legally binding referendum to take place, was a "matter for 2021".
He added: "We'll see whether or not the Scottish National Party get a majority then - and I mean the Scottish National Party, not in collaboration with other parties, not in any alliances, but a Scottish National Party majority."
But Mr Gove, campaigning with Mr Jack in Dumfries and Galloway on Wednesday, ruled out any prospect of a second independence vote.
He also suggested that by continuing to push for indyref2, the Scottish First Minister had become the Tories' strongest electoral asset in Scotland.
While polls suggest the Scottish Tories could lose some of the 13 seats they won in 2017, Mr Gove said the party's campaign north of the border was "going very well".
He added: "Our biggest electoral asset of course is Nicola Sturgeon, because Nicola Sturgeon's insistence on a second independence referendum, come hell or high water, is not going down well on the doorstep."
Mr Gove claimed the Scottish First Minister and the SNP would "use any reason, any excuse, any justification to say there should be a second independence referendum", adding if Ms
Sturgeon's party was to win a majority at Holyrood in less than two years' time "they will say that is another reason to have a referendum".
But he added: "We're not going to grant that. We will say no, because she said that this was a once in a lifetime event.
"It erodes trust in politics if you say it is a once in a lifetime event and then you go back on that."
Mr Gove, speaking during a visit to a farm outside Castle Douglas, added: "The SNP must justify their record on health, on education, on the economy and in that 2021 election we will be saying not only that we don't want a second independence referendum, that is a total distraction and waste of time, we will also be saying let's have a competent government in Scotland that can make sure that we manage our NHS effectively and improve school standards.
"People are continually asking the question that why is it when you've got a sick kids' hospital in Edinburgh that is ready to open but receiving no patients, when you've got the Queen
Elizabeth Hospital in Glasgow going through such difficult times, when Scottish education is moving backwards, why is she not concentrating on the day job?
"And a vote for the Scottish Conservative and Unionist Party is a vote for the only party which is resolutely opposed to another independence referendum, and we're finding strong support for that."