Simultaneous quantification of nucleic acids in diseased cells

Abstract

A process for assessing mitochondrial toxicity of a compound that includes contacting nucleic acids from a host with an amplification reaction mixture that contains at least two primers that provide detectable signals, wherein: a first primer provides a first detectable signal upon amplification of a host mitochondrial nucleic acid; a second primer provides a second detectable signal upon amplification of a host nuclear nucleic acid; and comparing the first and second detectable signals.

Description

[0001] This application claims priority to U.S. Provisional Application No. 60 / 241,488, filed on Oct. 18, 2000, U.S. Provisional Application No. 60 / 256,067 filed on Dec. 15, 2000 and U.S. Provisional Application No. 60 / 282,156, filed on Apr. 6, 2001.FIELD OF THE INVENTION [0002] This application is in the area of processes for the detection and analysis of viral infections and mitochondrial toxicity, and for processes for the identification of active compounds for the treatment of viral infections and processes to measure mitochondrial toxicity resulting from drug therapies. BACKGROUND OF THE INVENTION [0003] The detection and quantification of nucleic acid sequences is of importance for a wide range of applications. The most widely used method to detect nucleic acids are based on the polymerase chain reaction (PCR). PCR is used to amplify a segment of DNA flanked by stretches of known sequences. Two oligonucleotides binding to these known flanking sequences are used as primers for ...

AI Technical Summary

Benefits of technology

"The patent describes a method for simultaneously measuring the levels of nucleic acid in a host cell and a virus using real-time PCR. This technique allows for the calculation of a sensitivity assay that indicates the relative condition of the host cell and the virus. The method is economic, non-radioactive, rapid, accurate, reproducible, and amenable to large through-put. It can provide a dynamic range of quantification with linearity of over 5-7 logs. The method can also be used to evaluate the effectiveness of anti-viral agents and to assess the inhibitory effect of a compound on cellular DNA polymerases. Overall, the method provides a reliable and useful tool for the evaluation of anti-viral agents and the development of new treatments for viral infections."

Problems solved by technology

PCR itself is quite simple, but sample preparation can be laborious.

When the specimen contains a large amount of target nucleic acid, sample preparation is trivial.

But sample preparation is more difficult in most clinical specimens, particularly when a large volume specimen must be processed and only a few pathogens are present.

Complex protocols are often required.

PCR can be used to screen for drug resistance mutations, but it does not provide direct antibiotic susceptibility data.

Most published studies have not included a sufficient number of negative controls.

Detection is the most difficult PCR procedure, especially when the number of pathogens in the specimen is low.

This makes PCR for detection especially prone to carryover contamination.

Sample preparation may be laborious, as there is an attempt to process as large a specimen volume as possible.

Inhibitors of PCR occur naturally in many clinical samples, and are a major limitation.

PCR is quite sensitive, but it is not inherently quantitative.

Due to the ongoing replication of the virus, anti-retroviral drug resistance eventually develops, leading to therapy failure.

The design of an HIV-1 viral load test is a real challenge.

Most toxic events are reversible at an early stage, however lactic acidosis is often irreversible and can result in death.

Tissues with high ATP demand are most susceptible to mitochondrial toxicity.

Available tests for HCV infection are limited.

Initial serologic tests for HCV had poor sensitivity.

Second and third-generation serologic tests have improved sensitivity, but are still not completely dependable.

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