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Method and kit for primer based amplification of nucleic acids

A nucleic acid and nucleotide technology, used in biochemical equipment and methods, determination/inspection of microorganisms, etc.

Active Publication Date: 2014-02-12
戴尔斯瑞克斯实验室
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The second problem is to identify optimized primer-to-locus ratios
This is a time-consuming process that needs to be performed for each batch of produced assays
Successful multiplex PCR is not guaranteed even after exhaustive optimization experiments

Method used

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  • Method and kit for primer based amplification of nucleic acids
  • Method and kit for primer based amplification of nucleic acids
  • Method and kit for primer based amplification of nucleic acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] In this example, nucleic acid sequences from each disease agent and the second disease agent were obtained and isolated as described herein. Nucleic acid from each pathogen was amplified by the Tem-PCR method described herein. The nucleic acid of each pathogen was placed in a separate reaction tube and the complete mix of primers specified in Table 2 was added along with the reagents for RT-PCR. For each pathogen, a separate reaction was performed at each of the three primer ratios specified in Table 5. Although any procedure known in the art for RT-PCR can be used, the following procedure was used in this example. Prepare a master rRT-PCR mix consisting of 5X RT-PCR buffer, a deoxynucleoside triphosphate (dNTP) mix (containing 400 μM of each dNTP), complete fractions of the primers shown in Table 2 at the ratios indicated in Table 5. mixture, and RT-PCR enzyme preparation. Ribonuclease inhibitors were also added at a concentration of 5-10 units / reaction if necessary...

Embodiment 2

[0086] In this example, nucleic acid sequences from each disease agent and the second disease agent were obtained and isolated as described herein. Nucleic acid from each pathogen was amplified by the Tem-PCR method described herein. The nucleic acid of each pathogen was placed in a separate reaction tube and the complete mix of primers specified in Table 3 was added along with the reagents for RT-PCR. The conditions for RT-PCR were as described in Example 1 above. The primer ratio used in this example was 1:1:1:1:10:40 (F out :F in :R in :R out :FSP:RSP).

[0087] Detection of amplified products containing target sequences was performed as described for the direct detection method using the Luminex beads described in Example 1 . Add the detection oligonucleotides for each pathogen listed in Table 3 to each detection reaction.

[0088]Table 6 shows the results of this experiment using the target enrichment and target amplification primers disclosed in Table 3. The row ...

Embodiment 3

[0092] In Examples 1 and 2 above, only 1 nucleic acid sample was added to each reaction (albeit in the presence of target enrichment primers specific for multiple pathogens). Table 7 shows the specificity of the TemPCR amplification method when nucleic acid samples from multiple pathogens are included in a single sample for multiplex amplification and subsequent detection. In this example, target enrichment primers for each organism listed in Table 3, as well as target amplification primers and nucleic acids from the indicated pathogens were included in each sample. Rows in Table 7 indicate the detection oligonucleotides detected in the multiplex detection step (note that detection oligonucleotides specific to the target sequences of all organisms listed in Table 3 are included in each detection reaction ), while the row in Table 6 indicates the identity of the pathogenic nucleic acid that was added to each sample. TemPCR amplification conditions and multiplexing were perform...

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Abstract

The present invention discloses a novel method for diagnosing or differentially diagnosing disease mediators and second disease mediators. The disclosed method uses a novel amplification strategy called TemPCR to allow sensitive and specific amplification of target sequences from any disease agent and / or secondary disease agent whose nucleic acid sequence is known. The TemPCR method uses at least one set of target enrichment primers (present in low concentration) specific to the disease agent or secondary disease agent to be detected and at least one pair of common target amplification primers (present in high concentration). At least one pair of said target enrichment primers includes binding sequences for target amplification primers. Thus, the use of the TemPCR method allows multiplex amplification reactions to be performed without the need for empirical optimization of multiplex amplification parameters. Methods for nucleic acid isolation and target sequence detection for use with the TemPCR method are also disclosed.

Description

Background of the invention [0001] An important issue for the effective prevention and control of any pathogen is the timely and accurate diagnosis of patients infected with the pathogen. One aspect of this diagnosis includes differential diagnosis. By differential diagnosis, it is meant to identify and identify those patients with a disease state or disease caused by the disease pathogen of interest, and those patients with a disease state condition caused by one or more second pathogens that lead to similar clinical manifestations. This differential diagnosis is critical because the symptoms / clinical manifestations observed in the disease caused by the target disease pathogen can also be observed in the disease caused by the second pathogen. Therefore, the potential for misdiagnosis is a significant concern. This misdiagnosis can lead to false positives, and false negatives. Each of these types of misdiagnosis has a detrimental effect on the prevention and control of dise...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686Y02A50/30C12Q2527/143C12Q2525/155C12Q2565/102C12Q2549/119C12Q2537/143C12Q2531/113
Inventor 韩健
Owner 戴尔斯瑞克斯实验室
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