CBI sources said that the agency took over the probe from Rajasthan Police after a formal notification was issued by the state and central governments.
Rajasthan Chief Minister Vasundhara Raje had asked the Centre last year to order a CBI probe into the scam.
Rajasthan Police's FIR, filed in June last year immediately after BJP returned to power in the state, has named Gehlot, Pilot, Ravi Krishna and Karthi Chidmabaram, sons of senior Congress leaders and former union ministers Vayalar Ravi and P Chidambaram, respectively.
The irregularities in the implementation of the project included excess bills claimed by the firm and paid to them and an alleged financial irregularities to the tune of Rs 2.56 crore in operating the ambulance service during the previous Congress rule was claimed by the police.
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The Rajasthan Police, which also named officials of Ziqitza Healthcare, had filed the FIR on the basis of a probe into a complaint filed by former Jaipur Mayor Pankaj Joshi.
Gehlot, who was not immediately available for comments, had earlier said that the state government had failed to perform and the case was only to divert the attention of the people.
"When the FIR was lodged in June last year, it came to my knowledge that the officers were being pressurised to act in this manner," he had said earlier.