Method of minimizing contamination in amplification reactions using a reaction tube with a penetrable membrane

• US 5,604,101 A
• Filed: 11/17/1995
• Issued: 02/18/1997
• Est. Priority Date: 10/22/1993
• Status: Expired due to Term

• Current Assignee: Abbott Laboratories
• Original Assignee: Abbott Laboratories
• Inventors: Zurek, Thomas F., Hanley, Kathleen A., Lee, Helen H., Pepe, Curtis J., Hofferbert, A. David
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/559,932
• Time in Patent Office: 459 Days
• Field of Search: 435/6, 435/91.2, 422/100, 422/101, 422/102, 215/247, 215/249, 215/250, 215/258, 215/354
• US Class Current: 435/6.16
• CPC Class Codes: B01L 3/50825 Closing or opening means, corks, bungs closures for containers B65D; means for removing stoppers B67B7/02
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046

Abstract

A disposable reaction vessel for performing nucleic acid amplification assay. The disposable reaction vessel has a penetrable cap that can be penetrated by an automated pipettor to aspirate a portion of an amplified reaction product. The disposable reaction vessel contains the reagents necessary to perform a nucleic acid amplification assay. A patient specimen is added to the unit dose reagents in the disposable reaction vessel and the penetrable cap is closed. The disposable reaction vessel containing the reaction mixture and the specimen undergoes amplification, typically by placing it in a thermal cycler. After amplification the intact disposable reaction vessel is transferred to an automated analyzer where an automated pipettor penetrates the closure membrane and aspirates a portion of the amplified sample for further processing, without removal of the reaction vessel cap. This avoids the generation of potentially contaminating aerosols or droplets.

Claims

1. In a method for amplifying and detecting nucleic acid materials comprising the steps of:

a) adding a sample suspected to contain a target nucleic acid material to an amplification vessel along with oligonucleotide probes or primers, at least one of which bears a detectable reporter group, for amplification of said suspected target nucleic acid to form a reaction mixture;

b) sealing the reaction mixture inside said vessel by closing a tightly sealing cap;

c) amplifying the target nucleic acid material within said vessel; and

d) detecting the presence of amplified target nucleic acid by detection of said detectable reporter group;

wherein the improvement comprises

i) providing said sealing cap with a membrane that is penetrable by a pipettor probe;

ii) removing a portion of the reaction mixture from said vessel for detection wherein said removing is effected by piercing said cap membrane with a pipettor probe aspirating said portion of the reaction mixture into said pipettor; and

iii) dispensing said portion in a distinct detection compartment without uncapping said vessel, thereby avoiding drops or aerosols of the amplified material which might contaminate the environment, unreacted samples or reagents.

2. The method of claim 1 wherein the improved method further comprises inactivating all nucleic acid material left in the vessel and in the detection compartment by dispensing thereto a nucleic acid inactivation reagent from a pipettor.

3. The method of claim 2 wherein said inactivating comprises the consecutive addition of a copper phenanthroline chelate and hydrogen peroxide solution.

4. The method of claim 1 wherein the membrane has a thickness ranging from 0.002 to 0.015 inches.

5. The method of claim 4 wherein the membrane has a thickness ranging from 0.005 to 0.009 inches.

6. The method of claim 1 wherein the pipetting probe is a metallic tube with a beveled tip.

7. The method of claim 6 wherein the outer diameter of said probe does not exceed 0.050 inches.

8. The method of claim 1 wherein the amplifying step comprises a polymerase chain reaction.

9. The method of claim 1 wherein the amplifying step comprises a ligase chain reaction.

10. The method of claim 1 wherein the improved method further comprises a step of placing the sealed amplification vessel in an automated pipettor probe instrument for automated detection, said placing step being prior to the removing of step ii.

11. The method of claim 10 wherein said removing and detecting steps are both performed by the automated instrument.

12. The method of claim 10 further comprising a step of inactivating all nucleic acid material left in the vessel and in the detection compartment by dispensing thereto a nucleic acid inactivation reagent, wherein said removing, detecting steps and inactivating steps are all performed by the automated pipettor instrument.