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The victims, who worked at a wine farm just outside the South African suburb of Somerset West, were on the back of the truck when it was struck by the commuter train at 7.16am local time.
In a statement, the Railway Safety Regulator said preliminary investigations suggested the truck driver had ignored warning signals at the level crossing.
Paramedics, who were reported by the South African Press Association earlier as saying that 27 people had been killed, said that three of those injured were in a critical condition.
Metrorail, the company operating the train, said that no rail passengers were injured in the crash.
The rail company said in a statement that the accident happened "when a truck and trailer carrying some 30 farm workers collided with Cape Town-bound train 3208. According to eyewitnesses, the truck stalled at the crossing."
"It was a horrible sight," train passenger Ray Weyer told South African radio. "I saw a truck in front of the train and there were a few bodies in front of it."
Mr Weyer said that passengers on the train heard a bang and saw smoke. He said the train driver was "in a terrible state" and had told them he didn't know what had happened.
Metrorail said the level crossing did not have barriers but had the statutory number of warning signs.
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