Method and device for detecting the presence of a single target nucleic acid in a sample

Abstract

A method comprises loading a sample portion into a first porous structure, subjecting the sample portion to an amplification step, and determining whether the sample portion contains at least one molecule of a target nucleic acid. If the sample portion contains a single molecule of the target nucleic acid, the sample portion would attain a detectable concentration of the target nucleic acid after a single round of amplification. Also, a microfluidic device comprising a porous sample structure and a sample portion positioned in the porous sample structure. Also, a microfluidic device comprising a porous sample structure and an amplification targeting reagent positioned in the porous sample structure.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation application of U.S. patent application Ser. No. 10 / 798,857 (the entirety of which is incorporated herein by reference), filed on Mar. 11, 2004, which is a divisional application of U.S. patent application Ser. No. 10 / 131,854, filed on Apr. 25, 2002, which is a divisional application of U.S. patent application Ser. No. 09 / 563,714, filed on May 2, 2000 (now U.S. Pat. No. 6,391,559), which is a divisional application of U.S. patent application Ser. No. 08 / 838,262, filed Apr. 17, 1997 (now U.S. Pat. No. 6,143,496).[0002]This application is also a continuation application of U.S. patent application Ser. No. 10 / 131,854 (the entirety of which is incorporated herein by reference), filed on Apr. 25, 2002, which is a divisional application of U.S. patent application Ser. No. 09 / 563,714, filed on May 2, 2000 (now U.S. Pat. No. 6,391,559), which is a divisional application of U.S. patent application Ser. No. 08 / 838,...

AI Technical Summary

Benefits of technology

[0018]According to embodiments of the invention, methods are provided for determining the existence and / or initial concentration of a target nucleic acid molecule in samples of about 1 microliter or less. According to some embodiments of the invention, methods are provided for a clinical diagnosis PCR analysis which can quickly and inexpensively detect a single target nucleic acid molecule.

Problems solved by technology

Unfortunately, problems exist in the application of PCR to clinical diagnostics.

Development has been slow due in part to: labor intensive methods for detecting PCR product; susceptibility of PCR to carryover contamination—false positives due to contamination of a sample with molecules amplified in a previous PCR; and difficulty using PCR to quantitate the number of target nucleic acid molecules in a sample.

Furthermore, the assays are not labor intensive and are easily automated.

These methods have the disadvantage that slight differences in amplification efficiency between the control and experimental nucleic acids can lead to large differences in the amounts of their products after the million-fold amplification characteristic of PCR and related technologies, and it is difficult to determine relative amplification rates accurately.

However, these assays also require assumptions about relative amplification efficiency in different samples during the exponential phase of PCR.

However, to accumulate detectable amounts of product from a single starting template molecule usually requires that two or more sequential PCRs have to be performed, often using nested sets of primers, and this accentuates problems with carryover contamination.

However, this strategy usually fails before getting to the limit of detecting single starting molecules due to the appearance of artifactual amplicons derived from the primers (so called “primer-dimers”) which interfere with amplification of the desired product.

Attempts have been made to miniaturize PCR assemblies but no one has developed a cost-effective PCR assembly which can carry out PCR in a nanoliter-sized sample.

Part of the problem with miniaturization is that evaporation occurs very rapidly with small sample volumes, and this problem is made worse by the need to heat samples to ˜90° C. during thermocycling.

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