Loc for detection of hybridization of nucleic acid sequences with PCR amplification using primers covalently attached to stem-and-loop probes

Abstract

A LOC device having a supporting substrate, primer-linked, stem-and-loop probes, each with a nucleic acid sequence that matches one of a plurality of target nucleic acid sequences, and a primer for elongating against the target nucleic acid sequence to form a complementary sequence such that during use the nucleic acid sequence matching the target nucleic acid sequence anneals to the complementary sequence to change a fluorescence emission from the probe in response to an excitation light, a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes, and, CMOS circuitry on the supporting substrate, the CMOS circuitry having operative control of the excitation light.

Description

FIELD OF THE INVENTION[0001]The present invention relates to diagnostic devices that use microsystems technologies (MST). In particular, the invention relates to microfluidic and biochemical processing and analysis for molecular diagnostics.CO-PENDING APPLICATIONS[0002]The following applications have been filed by the Applicant which relate to the present application:GBS001USGBS002USGBS003USGBS005USGBS006USGSR001USGSR002USGAS001USGAS002USGAS003USGAS004USGAS006USGAS007USGAS008US GAS009USGAS010USGAS012USGAS013USGAS014USGAS015USGAS016US GAS017USGAS018US GAS019USGAS020USGAS021USGAS022US GAS023USGAS024USGAS025USGAS026USGAS027USGAS028US GAS031USGAS032US GAS033USGAS034USGAS035USGAS036US GAS037USGAS038US GAS039USGAS040USGAS041USGAS042USGAS043USGAS044USGAS045USGAS046US GAS047USGAS048USGAS049USGAS050USGAS054USGAS055US GAS056USGAS057US GAS058USGAS059US GAS060USGAS061US GAS062USGAS063US GAS065USGAS066US GAS067USGAS068US GAS069USGAS070USGAS080USGAS081USGAS082USGAS083US GAS084USGAS085US GAS086USG...

AI Technical Summary

Benefits of technology

[0096]The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, amplifies the nucleic acid targets in the sample, identifies the sample's nucleic acid sequences via hybridization with primer-linked stem-and-loop probes using its integral image sensor, and provides the results electronically at its output pads, with the primer-linked stem-and-loop probes providing for a large number of optimal parallel amplification reactions to be run, also providing for high specificity, sensitivity, and reliability of detection of the target sequences.

Problems solved by technology

Insufficient stringency can result in a high degree of nonspecific binding.

Excessive stringency can lead to a failure of appropriate binding, which results in diminished sensitivity.

Despite the advantages that molecular diagnostic tests offer, the growth of this type of testing in the clinical laboratory has been slower than expected and remains a minor part of the practice of laboratory medicine.

This is primarily due to the complexity and costs associated with nucleic acid testing compared with tests based on methods not involving nucleic acids.

However, controlling fluid flow through the LOC device, adding reagents, controlling reaction conditions and so on necessitate bulky external plumbing and electronics.

Connecting a LOC device to these external devices effectively restricts the use of LOC devices for molecular diagnostics to the laboratory setting.

The cost of the external equipment and complexity of its operation precludes LOC-based molecular diagnostics as a practical option for point-of-care settings.

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