Control nucleic acids for multiple parameters

摘要

Methods are provided for the amplification of at least a first and a second target nucleic acid that may be present in at least one fluid sample using an internal control nucleic acid for qualitative and/or quantitative purposes.

主权项

1. A method for controlled amplification of at least a first and a second target nucleic acid from a plurality of biological samples, comprising:
a) adding an internal control nucleic acid to each of the plurality of biological samples in a number of reaction vessels corresponding to the number of biological samples, wherein the internal control nucleic acid is identical for each of the plurality of biological samples, and wherein the sequence of the internal control nucleic acid is different from the sequences of the target nucleic acids; b) purifying target nucleic acids from the plurality of biological samples and the internal control nucleic acid simultaneously in the reaction vessels; c) adding amplification reagents to the reaction vessels, wherein the amplification reagents for the first target nucleic acid comprise at least one set of primers specific for the first target nucleic acid and at least one set of primers specific for the internal control nucleic acid, and the amplification reagents for the second target nucleic acid comprise at least one set of primers specific for the second target nucleic acid and at least one set of primers specific for the internal control nucleic acid, wherein the primers specific for the internal control nucleic acid are different from the primers specific for the first and second target nucleic acids, and wherein the amplification reagents further comprise a polymerase with reverse transcriptase activity; d) incubating in the reaction vessels the target nucleic acids and the amplification reagents for a period of time and under conditions suitable for transcription of RNA by said polymerase with reverse transcriptase activity to occur, wherein the incubation of the polymerase with reverse transcriptase activity comprises the following steps in sequence: a first temperature of 55° C., followed by a second temperature of 60° C., followed by a third temperature of 65° C.; e) incubating the reaction vessels simultaneously under identical conditions sufficient for amplification to occur; and f) detecting and measuring signals generated by the amplification; wherein the signals generated by the amplification of the internal control nucleic acid are indicative of an amplification occurring in the reaction vessels even in the absence of signals generated by the amplification of the at least first and second target nucleic acids.