Method and rapid test for detection of specific nucleic acid sequences

a technology of specific nucleic acid sequences and test kits, which is applied in the direction of microorganism testing/measurement, biochemistry apparatus and processes, etc., can solve the problems of insufficient use of visualization of pcr products by means of gel electrophoresis, inability of probes to function as primers during elongation, and inability to distinguish between correct products and amplification artifacts by intercalating dyes, etc., to achieve the effect of rapid

Inactive Publication Date: 2010-09-02
AJ INNUSCREEN GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0030]An objective of the present invention was to provide a universally usable method for specific detection of target nucleic acid sequences, which method can be performed very rapidly and also simply and furthermore which does not need any expensive instrumental systems. The method is intended to be suitable as a molecular genetic rapid test and to respect the requirements of diagnostic specificity assurance. In this regard it is important that only one specific amplification product be detected and that amplification artifacts can be unambiguously discriminated.

Problems solved by technology

The widespread use of visualization of a PCR product by means of gel electrophoresis is not sufficient for this purpose.
In addition, a phosphate group is also located at the 3′-end of the probe if necessary, so that the probe cannot function as a primer during elongation.
From the advantages, however, there also results an extreme disadvantage for application: In principle it is not possible by means of intercalating dyes to distinguish between correct product and amplification artifacts (such as primer dimers or defective products).
While primer dimers and other artifacts are being formed, they naturally also bind intercalating dyes and thus lead to an unspecific increase in fluorescence even in negative samples.
A great disadvantage, however, is that they are implemented on very expensive instrumental platforms, which have to unite the process of amplification and that of subsequent optical detection, in a manner corresponding to the problem, in one hardware solution.
Ultimately this explains the high financial expenditure that must be invested for the use of real-time PCR systems, Also ultimately, the operation of such instrumental systems requires a high degree of expertise.
Besides the time needed to perform the PCR, therefore, several hours of working time are also needed to perform the subsequent detection method.
Such a method usually needs 8 hours and therefore is also not suitable as a rapid test.
Once again, however, these methods are also laborious to perform, need a large number of procedural steps to be performed and therefore are not suitable as rapid tests.
These methods also are laborious and associated with very expensive instrumental platforms.
However, the method does not combine hybridization of the probe with the PCR process but instead performs the latter process as a separate procedural step.
However, the method suffers from a fundamental and dramatic error source.
This method may lead to false-positive results due to primer-dimer formation and mispriming.
Furthermore, amplification artifacts frequently lead to a false-positive signal in these cases.

Method used

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  • Method and rapid test for detection of specific nucleic acid sequences

Examples

Experimental program
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Effect test

example 1

Detection of Listeria monocytogenes DNA by Means of the Hybridization Method Integrated into the PCR and of Lateral Flow Detection. Comparison of an Unphosphorylated and a Phosphorylated Probe

[0057]Two types of labeled probes were tested against one another in the mixture. The first probe is FITC-labeled at the 5′-end, and the second probe is also singly phosphorylated at its 3′-end. The 3′-phosphorylation of the probe prevents it from being elongated by the Taq polymerase.

Mixture 1 (unphosphorylated hybridization probe)

PCR primer / probe

L. monocytogenes sense primer(SEQ ID NO: 1)(5′-CGC AAC AAA CTG AAG CAA AGG-3′)L. monocytogenes antisense primer(SEQ ID NO: 2)(5′-BIOTIN-TCC GCG TGT TTC TTT TCG AT-3′)L. monocytogenes probe(SEQ ID NO: 3)(5′-FITC-CCA TGG CAC CAC CAG CAT CT-3′)

Reaction mixture (amplification / hybridization)

Per sample:

sense primer (50 pmol / μL)0.1 μLantisense primer (50 pmol / μL)0.1 μLprobe (25 pmol / μL)0.1 μLdNTP Mix (12.5 mM)0.3 μL10X PCR buffer (MgCl2 included)1.5 μLTaq-DN...

example 2

Performance of the Method by Means of Asymmetric PCR and Check of Specificity of the Test on the Basis of Testing of Positive and Negative Starting Samples

[0066]The inventive method was used as an example for detection of Rickettsia DNA isolated from tick tissue. The specificity of the method was determined by means of parallel tests on Rickettsia-negative DNA samples, also isolated from tick tissue.

PCR primer probe:

Rickettsia sense primer:(SEQ ID NO: 5)5′-GGG ACC TGC TCA CGG CGG-3′Rickettsia antisense primer:(SEQ ID NO: 6)5′-Biotin-TCT ATT GCT ATT TGT AAG AGC GGA TTG-3′Rickettsia probe:(SEQ ID NO: 7)5′-FITC- CAA AGA AGT ATT AAA GGA ACT C-Pho-3′

Reaction mixture (amplification / hybridization)

Per sample:

sense primer (50 pmol / μL)0.05 μL antisense primer (50 pmol / μL)0.1 μLprobe (25 pmol / μL)0.1 μLdNTP Mix (12.5 mM)0.3 μL10X PCR buffer (MgCl2 included)1.5 μLTaq-DNA polymerase0.75 U DNA (positive or negative)1.5 μL (approx. 50 ng)PCR-grade H2Oadd 15 μl

[0067]The PCR was performed in the Spee...

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Abstract

A universally usable method for specific detection of target nucleic acid sequences, which method can be performed very rapidly and also simply and furthermore which does not need any expensive instrumental systems. The method is intended to be suitable as a molecular genetic rapid test and to respect the requirements of diagnostic specificity assurance. In this regard it is important that only one specific amplification product be detected and that amplification artifacts can be unambiguously discriminated. A nucleic acid amplification kit suitable for performing this method.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of PCT / EP2008 / 057857, filed Jun. 20, 2008, and claims priority to Germany 10 2007 029 772.8, filed Jun. 22, 2007, both of which are hereby incorporated by reference.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to a method and a test kit for detection of specific nucleic acid sequences with the steps of amplification, hybridization by means of probes, and detection of the hybridization event; wherein the detection of the hybridization event takes place on a solid phase outside the reaction vessel for amplification / hybridization.[0004]2. Description of the Related Art[0005]Genetic diagnostics has become an indispensable tool of modern medical laboratory diagnostics, forensic diagnostics, veterinary medical laboratory diagnostics or food and environmental diagnostics.[0006]Genetic diagnostics was revolutionized with the invention of PCR technology, with which...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q2600/158C12Q1/689
Inventor HILLEBRAND, TIMOGRASER, ELMARA
Owner AJ INNUSCREEN GMBH
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