Simplified device for nucleic acid amplification adn method for using same

Abstract

The present invention relates to a disposable device ( 100 ) for amplifying at least one target nucleic acid present in a liquid and biological sample of interest, which consists of a solid body ( 2 ), at least one fluid channel ( 3 ) connecting an inlet ( 4 ), via which all or part of the sample of interest can be drawn up and/or discharged, and an outlet ( 5 ), which is itself connected to a means for the drawing up/discharging of the said sample of interest, the fluid channel ( 3 ) further comprising from the inlet ( 4 ) to the outlet ( 5 ): a first compartment ( 8 ) containing all or part of the thermostable constituents, a means ( 15 ) for mixing the constituents with the sample of interest, a second compartment ( 9 ) containing all or part of the non-thermostable constituents, and in addition, at least one zone intended for heating the said sample of interest ( 6 ) mixed with the said amplification constituents in order to allow the amplification of the target nucleic acid. The invention also proposes an amplification method using such a device. The said invention has a preferred application in the field of medical diagnosis.

1 . Disposable device ( 1 ) for amplifying at least one target nucleic acid present in a liquid and biological sample of interest ( 6 ), which consists of a solid body ( 2 ), at least one fluid channel ( 3 ) connecting an inlet ( 4 ), via which all or part of the sample of interest ( 6 ) can be drawn up and/or discharged, and an outlet ( 5 ), which is itself connected to a means ( 7 ) for the drawing up/discharging of the said sample of interest, the fluid channel ( 3 ) further comprising from the inlet ( 4 ) to the outlet ( 5 ):
 a first compartment ( 8 ) containing all or part of the thermostable constituents ( 12 ) required for producing the amplification,   a means ( 15 ) for mixing the constituents ( 12  optionally combined with  13 ) with the sample of interest ( 6 ),   a second compartment ( 9 ) containing all or part of the non-thermostable constituents ( 13 ) required for producing the amplification,   and in addition, at least one zone intended for heating ( 11 ) the said sample of interest ( 6 ) mixed with the said amplification constituents ( 12  combined with  13 ), in order to allow the amplification of the target nucleic acid.   
     
     
         2 . Device according to  claim 1 , for detecting amplicons, characterised in that it further comprises, in the second compartment ( 9 ), all or part of the detection constituents required for detecting the amplicons. 
     
     
         3 . Device according to  claim 1 , for detecting amplicons, characterised in that it further comprises, in the fluid channel ( 3 ), a third compartment ( 10 ) containing all or part of the constituents required for detecting the amplicons. 
     
     
         4 . Device according to  claim 3 , characterised in that the third compartment ( 10 ) is located between the second compartment ( 9 ) and the outlet ( 5 ) of the device ( 1 ). 
     
     
         5 . Device according to any one of  claims 1  to  4 , characterised in that the inlet ( 3 ) accommodates a cone ( 28 ) of a pipette ( 16 ) or the tip ( 3 ) of the pipette has a pipette-cone-shaped configuration. 
     
     
         6 . Device according to any one of  claims 1  to  5 , characterised in that the drawing up/discharging device ( 7 ) is of the piston type such as, for example, a pipette ( 7 ). 
     
     
         7 . Device according to any one of  claims 1  to  6 , characterised in that the cross-section of the channel ( 3 ) is constant and that the compartments ( 8 ,  9  and optionally  10 ) have a larger cross-section. 
     
     
         8 . Device according to any one of  claims 1  to  7 , characterised in that the inlet ( 4 ) communicates with at least two fluid channels ( 3 ). 
     
     
         9 . Device according to any one of  claims 1  to  8 , characterised in that the outlet ( 5 ) comprises at least two fluid channels ( 3 ). 
     
     
         10 . Device according to any one of  claims 1  to  9 , characterised in that the constituents ( 12  &  13 ) are formed of freeze-dried or dried biological compounds, soluble in the sample of interest ( 6 ). 
     
     
         11 . Device according to any one of  claims 1  to  10 , characterised in that the drawing up/discharging means ( 7 ) is an integral part of the disposable device ( 1 ). 
     
     
         12 . Device according to  claim 11 , characterised in that the drawing up/discharging means ( 7 ) comprises a cylinder ( 17 ) connected to the fluid channel ( 3 ) and a piston ( 18 ) moving within the cylinder ( 17 ) manually or by means of an actuator. 
     
     
         13 . Device according to any one of  claims 1  to  12 , characterised in that the mixing means ( 15 ) consists of the fluid channel ( 3 ), the routing of which comprises at least one baffle ( 19 ). 
     
     
         14 . A method for amplifying at least one target nucleic acid, present in a liquid and biological sample of interest, within a disposable device which comprises a solid body, at least one fluid channel connecting an inlet, via which all or part of the sample of interest can be drawn up and/or discharged, and an outlet, which is itself connected to a device for the drawing up and/or discharging of the sample of interest, 
       the fluid channel further comprising from the inlet to the outlet:
 a first compartment containing all or part of thermostable constituents required for producing the amplification, 
 a means for mixing the thermostable constituents, optionally combined with non-thermostable constituents, with the sample of interest, 
 a second compartment containing all or part of the non-thermostable constituents required for producing the amplification, 
 and at least one zone intended for heating the sample of interest mixed with the thermostable and non-thermostable amplification constituents, in order to allow the amplification of the target nucleic acid, 
 
       which method consists in:
 (a) drawing up via the inlet all or part of the sample of interest within the device, 
 (b) moving the said sample to the first compartment for dissolving the thermostable amplification constituents therein, 
 (c) mixing the sample and the thermostable constituents with the mixing means, 
 (d) applying a first temperature gradient in order to denature the nucleic acid of interest, 
 (e) moving the mixture to the second compartment for dissolving the non-thermostable amplification constituents therein, 
 (f) mixing the mixture and non-thermostable constituents with the mixing means, and 
 (g) applying at least one second temperature gradient in order to amplify the denatured nucleic acid. 
 
     
     
         15 . The method according to  claim 14 , characterised in that the thermostable amplification constituents of step (b) also contain restriction enzymes, which are not necessarily thermostable, but which allow the digestion of the nucleic acids of interest, which are deoxyribonucleic acids (DNA), prior to the application of the first temperature gradient of step (d). 
     
     
         16 . Method according to either of  claims 14  and  15 , characterised in that, after step (g), it consists in:
 (h) mixing mixture ( 6 + 12 + 13 ), 
 (i) moving the new mixture ( 6 + 12 + 13 ) to dissolve the detection constituents ( 14 ) therein, and 
 (j) detecting the presence of amplicons. 
 
     
     
         17 . The method according to  claim 14 , characterised in that the amplification is a PCR amplification, for which the first temperature gradient is between 90 and 100° C., and the second temperature gradients are an alternation of the temperature in three different steps:
 between 90 and 100° C. for the first denaturation temperature, preferably about 94° C., 
 between 50 and 60° C. for the second hybridisation temperature, preferably about 55° C., 
 between 70 and 75° C. for the third polymerisation temperature, preferably about 72° C. 
 
     
     
         18 . The method according to  claim 14 , characterised in that the amplification is a post-transcriptional amplification (NASBA or TMA), for which the first temperature gradient is between 60 and 70° C., preferably about 65° C., and the second temperature gradient is between 40 and 50° C. for the second polymerisation temperature gradient. 
     
     
         19 . The method according to  claim 21 , characterised in that the first temperature gradient is applied to the first compartment and/or to the mixing means and that the second temperature gradient(s) is/are applied to the mixing means and/or to the second compartment and/or to the third compartment. 
     
     
         20 . The method according to  claim 14 , characterised in that the first temperature gradient is applied for 5 to 20 minutes, and that the second temperature gradient(s) is/are applied:
 in the case of a PCR amplification:
 for the denaturation, for less than one minute, 
 for the hybridisation, for less than one minute, and 
 for the polymerisation, for less than two minutes, 
   in the case of a post-transcriptional amplification, for less than two hours.   
     
     
         21 . The method according to  claim 14 , characterized in that the device further comprises, in the fluid channel, a third compartment containing all or part of constituents required for detecting amplicons. 
     
     
         22 . The method according to  claim 21 , characterized in that the cross section of the channel ( 3 ) is constant and that the third compartment ( 10 ) has a larger cross-section. 
     
     
         23 . The method according to  claim 21 , for detecting amplicons, characterised in that, after step (g), it consists in:
 (h) moving the mixture to the third compartment to dissolve the detection constituents therein,   (i) mixing the mixture, and   (j) detecting the presence of amplicons.   
     
     
         24 . A method according to  claim 14 , characterised in that the drawing up/discharging means comprises a cylinder connected to the fluid channel and a piston moving within the cylinder manually or by means of an actuator. 
     
     
         25 . A method according to  claim 14 , characterised in that the mixing means consists of the fluid channel, the routing of which comprises at least one baffle. 
     
     
         26 . The method of  claim 20 , wherein the first temperature gradient is applied for 15 minutes. 
     
     
         27 . The method of  claim 20 , wherein the second temperature gradient applied in the case of a PCR amplification for the denaturation is from 2 to 20 seconds. 
     
     
         28 . The method of  claim 20 , wherein the second temperature gradient applied in the case of a PCR amplification for the denaturation is 5 seconds. 
     
     
         29 . The method of  claim 20 , wherein the second temperature gradient applied in the case of a PCR amplification for the hybridisation is from 2 to 20 seconds. 
     
     
         30 . The method of  claim 20 , wherein the second temperature gradient applied in the case of a PCR amplification for the hybridisation is 5 seconds. 
     
     
         31 . The method of  claim 20 , wherein the second temperature gradient applied in the case of a PCR amplification for the polymerisation is from 5 to 80 seconds. 
     
     
         32 . The method of  claim 20 , wherein the second temperature gradient applied in the case of a PCR amplification for the polymerisation is 10 seconds. 
     
     
         33 . The method of  claim 20 , wherein the second temperature gradient applied in the case of a post-transcriptional amplification is from 5 to 80 minutes. 
     
     
         34 . The method of  claim 20 , wherein the second temperature gradient applied in the case of a post-transcriptional amplification is
 about 60 minutes in the case of RNA target nucleic acids or   about 90 minutes in the case of DNA target nucleic acids.