Generic PCR

Abstract

The present invention provides a method for the amplification of at least a first and a second target nucleic acid that may be present in a fluid sample. The invention further provides a kit and an analytical system for carrying out said amplification.

What is claimed is: 
     
         1 . A process for isolating and simultaneously amplifying at least a first and a second target nucleic acid that may be present in at least one sample, the process comprising the steps of:
 a) combining a solid support material and a sample for a period of time, under conditions sufficient to permit nucleic acids comprising the target nucleic acids to be immobilized on the solid support material;   b) isolating the solid support material from the other material present in the sample in a separation station;   c) purifying the nucleic acids in the separation station by separating the sample from the solid support material and washing the solid support material one or more times with a wash buffer;   d) contacting the purified nucleic acids with one or more amplification reagents comprising a polymerase with reverse transcriptase activity, in at least two reaction vessels, wherein at least a first reaction vessel comprises at least the first target nucleic acid and at least a second reaction vessel comprises at least the second target nucleic acid, and wherein the second target nucleic acid is absent from the first reaction vessel;   e) incubating in the reaction vessels the purified nucleic acids with the one or more amplification reagents for a period of time and under conditions suitable for transcription of RNA by the 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.; and   f) incubating in the reaction vessels the purified nucleic acids with the one or more amplification reagents for a period of time, under conditions sufficient for an amplification reaction indicative of the presence or absence of the first and second target nucleic acid to occur,   
       wherein the conditions for transcription and amplification in steps d) to f) are identical for the at least first and second target nucleic acids. 
     
     
         2 . The process according to  claim 1 , wherein at least two target nucleic acids are amplified in the same reaction vessel. 
     
     
         3 . The process according to  claim 1 , wherein the first target nucleic acid is absent from the second reaction vessel. 
     
     
         4 . The process according to  claim 1 , wherein the first target nucleic acid and the second target nucleic acid are from different organisms. 
     
     
         5 . The process according to  claim 1 , wherein the first and/or the second target nucleic acid is a non-viral nucleic acid. 
     
     
         6 . The process according to  claim 5 , wherein the first and/or the second target nucleic acid is a bacterial nucleic acid. 
     
     
         7 . The process according to  claim 1 , wherein in step e) the period of time is up to 30 minutes, 20 minutes, 15 minutes, 12.5 minutes, or 10 minutes. 
     
     
         8 . The process according to  claim 1 , wherein the polymerase with reverse transcriptase activity is a polymerase comprising a mutation conferring an improved nucleic acid extension rate and/or an improved reverse transcriptase activity relative to the respective wildtype polymerase. 
     
     
         9 . The process according to  claim 8 , wherein the polymerase with reverse transcriptase activity comprising a mutation is selected from the group consisting of:
 a) a CS5 DNA polymerase   b) a CS6 DNA polymerase   c) a  Thermotoga maritima  DNA polymerase   d) a  Thermus aquaticus  DNA polymerase   e) a  Thermus thermophilus  DNA polymerase   f) a  Thermus flavus  DNA polymerase   g) a  Thermus filiformis  DNA polymerase   h) a  Thermus  sp. sps17 DNA polymerase   i) a  Thermus  sp. Z05 DNA polymerase   j) a  Thermotoga  neapolitana DNA polymerase   k) a Termosipho  africanus  DNA polymerase   l) a  Thermus caldophilus  DNA polymerase.   
     
     
         10 . The process according to  claim 1 , wherein the target nucleic acids comprise RNA and DNA. 
     
     
         11 . The process according to  claim 1 , wherein amplified fragments comprise up to 450 bases. 
     
     
         12 . The process according to  claim 1 , wherein a control nucleic acid is added to the sample and/or the purified nucleic acid at any of the steps. 
     
     
         13 . The process according to  claim 1 , further comprising the step of determining the quantity of the target nucleic acids after and/or during step f). 
     
     
         14 . The process of  claim 1 ,
 wherein said first reaction vessel further comprises primers and probes for at least said first target nucleic acid,   wherein said second reaction vessel further comprises primers and probes for at least said second target nucleic acid,   wherein said primers and probes for at least said first target nucleic acid are absent from said second reaction vessel, and   wherein said primers and probes for at least said second target nucleic acid are absent from said first reaction vessel.   
     
     
         15 . A process for simultaneously amplifying at least a first and a second target nucleic acid that may be present in a sample, the process comprising the steps of:
 a) contacting the target nucleic acids from the sample with one or more amplification reagents comprising a polymerase with reverse transcriptase activity, in at least two reaction vessels, wherein at least a first reaction vessel comprises at least the first target nucleic acid and at least a second reaction vessel comprises at least the second target nucleic acid, and wherein the second target nucleic acid is absent from the first reaction vessel;   b) incubating in the reaction vessels the target nucleic acids with the one or more amplification reagents for a period of time, under conditions suitable for transcription of RNA by the polymerase with reverse transcriptase activity to occur; and   c) incubating in the reaction vessels the target nucleic acids with the one or more amplification reagents for a period of time, under conditions sufficient for an amplification reaction indicative of the presence or absence of the first and second target nucleic acid to occur,   wherein the conditions for transcription and amplification in steps a) to c) are identical for the at least first and second target nucleic acids.   
     
     
         16 . An analytical system for isolating and simultaneously amplifying at least two target nucleic acids that may be present in a sample, the analytical system comprising the following modules:
 i) a separation station comprising a solid support material, the separation station being constructed and arranged to separate and purify a target nucleic acid comprised in a sample; and   ii) an amplification station comprising at least two reaction vessels, the reaction vessels comprising amplification reagents, at least a first purified target nucleic acid in at least a first reaction vessel and at least a second purified nucleic acid in at least a second reaction vessel, wherein the second target nucleic acid is absent from the first reaction vessel, and a polymerase with reverse transcriptase activity, the polymerase further comprising a mutation conferring an improved nucleic acid extension rate and/or an improved reverse transcriptase activity relative to the respective wildtype polymerase.