Method of probe design and/or of nucleic acids detection

a technology of applied in the field of oligoucleotide probe design and nucleic acid detection, can solve the problems of viruses not being able to culturing, elude detection in myriad ways, and etiologic agents often remain unidentified, and achieve the effect of efficient amplification

Inactive Publication Date: 2007-02-22
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for designing oligonucleotide probes for nucleic acid detection that can improve the efficiency and accuracy of amplification. The method involves identifying and selecting regions of a target nucleic acid with higher efficiency of amplification, and designing probes that can hybridize to these regions. The probes are designed using a method that takes into account factors such as geometrical amplification bias and the score of the amplification efficiency of each position in the target nucleic acid. The method can be used to detect target nucleic acids in biological samples with high accuracy.

Problems solved by technology

In clinical practice, however, the etiologic agent often remains unidentified, eluding detection in myriad ways.
For example, some viruses are not amenable to culturing.
At other times, a patient's sample may be of too poor quality or of insufficient titre for pathogen detection by conventional techniques.
Moreover, both PCR- and antibody-based approaches may fail to recognize suspected pathogens simply due to natural genetic diversification resulting in alterations of PCR primer binding sites and antigenic drift.
While the concept of using oligonucleotide hybridization microarrays as a tool for determining the presence of pathogens has been proposed, significant hurdles remain, preventing the use of these microarrays routinely (Striebel, H. M., 2003).

Method used

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  • Method of probe design and/or of nucleic acids detection
  • Method of probe design and/or of nucleic acids detection
  • Method of probe design and/or of nucleic acids detection

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0151] In the first set of experiments, a PDC containing 53555 40-mer probes from 35 viruses affecting human was used for 4 independent microarray experiments. These 53555 probes were chosen based on a 5-bps tiling of each virus and were not subjected to any of our probe design criteria. Thus, we would expect errors arising due to CG-content, cross-hybridization and inefficient amplification to be significantly more than that of a PDC with well-designed probes. We tested our analysis algorithm in such an adverse setting for 4 experiments.

[0152] In each experiment, a human sample with an unknown pathogen was amplified by the RT-PCR process using random probes and then hybridized onto the PDC. We subjected the probes for each of the 35 viruses on our PDC to the one-tailed t-test with significance level 0.05 and computed the Weighted Kullback-Leibler (WKL) divergence of their signal intensities to the signal intensities of all the probes on the chip to determine which virus was in the...

example 2

[0156] In the second set of experiments, the analysis algorithm correctly detected the actual virus in the 3 samples and also the negative sample. After designing good probes for our chip, the Weighted Kullback-Leibler divergence of the acutal viruses in Experiment 1, 2 and 3 was greater than that of the corresponding experiments without probe design. This means that the signal intensities from the actual virus were relatively higher than the background noise in the PDC. This showed that our probe design criteria had removed some bad probes from the PDC, which resulted in a more accurate analysis.

[0157] Again, we present results of the 4 experiments if we had just used the t-test with a level of significance 0.05. This time, the number of viruses detected to be present for each sample is shown in Table 4:

TABLE 4False positive detection of viruses usingt-test alone in a PDC with probe design.Sample Name35259_32435179_12235253_84135915_111Viruses69910DetectedUsing T-testFalse58810P...

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Abstract

It is provided a method of designing oligonucleotide probe(s) for nucleic acid detection comprising the following steps in any order: (i) identifying and selecting region(s) of a target nucleic acid to be amplified, the region(s) having an efficiency of amplification (AE) higher than the average AE; and (ii) designing oligonucleotide probe(s) capable of hybridizing to the selected region(s). It is also provided a method of detecting at least one target nucleic acid comprising the steps of: (i) providing a biological sample; (ii) amplifying the nucleic acid(s) of the biological sample; (iii) providing at least an oligonucleotide probe capable of hybridizing to at least a target nucleic acid, if present in the biological sample; and (iv) contacting the probe(s) with the amplified nucleic acids and detecting the probe(s) hybridized to the target nucleic acid(s). In particular, the method indicates the presence of at least a pathogen, for example a virus, in a human biological sample. The probes may be placed on a support, for example a microarray or a biochip.

Description

FIELD OF THE INVENTION [0001] The present invention relates to the field of oligoucleotide probe design and nucleic acids detection. The method according to the invention may be used for the detection of pathogens, for example viruses. BACKGROUND OF THE INVENTION [0002] The accurate and rapid detection of viral and bacterial pathogens in human patients and populations is of critical medical and epidemiologic importance. Historically, diagnostic techniques have relied on cell culture passaging and various immunological assays or staining procedures. More recently PCR-based assays have been implemented, allowing for more rapid diagnosis of suspected pathogens with higher degree of sensitivity of detection. In clinical practice, however, the etiologic agent often remains unidentified, eluding detection in myriad ways. For example, some viruses are not amenable to culturing. At other times, a patient's sample may be of too poor quality or of insufficient titre for pathogen detection by ...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C12Q1/68G06F19/00G16B25/20
CPCG16B25/00C12Q1/6813G16B25/20G16B99/00C12Q1/6806C12Q1/6811
InventorWONG, CHRISTOPHER W.SUNG, WING-KINLEE, CHARLIEMILLER, LANCE D.
OwnerAGENCY FOR SCI TECH & RES