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Fluorescent real-time detection reagent and method for simultaneously detecting multiple target genes

A target gene and target detection technology, applied in the field of fluorescent real-time detection reagents, can solve the problems of uncapping pollution, large product fragments, and reduced PCR efficiency

Active Publication Date: 2022-06-21
中国科学院上海免疫与感染研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the multiplex PCR reactions are based on the color of the fluorescent probe, the Tm value of the amplified product, or the size of the amplified product to distinguish different targets. Therefore, the number of targets detected is limited by the PCR instrument channel limit (4-6 ), and different targets are distinguished by the size of the amplified product, there is a risk of uncapped contamination, and at the same time, the PCR efficiency may be reduced due to the large product fragment

Method used

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  • Fluorescent real-time detection reagent and method for simultaneously detecting multiple target genes
  • Fluorescent real-time detection reagent and method for simultaneously detecting multiple target genes
  • Fluorescent real-time detection reagent and method for simultaneously detecting multiple target genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1. Templates for amplifying 9 genes by single-tube multiplex qPCR

[0106] In this example, bacterial KPC (SEQ ID NO: 1), NDM (SEQ ID NO: 2), ACC (SEQ ID NO: 3), VIM (SEQ ID NO: 4), EBC (SEQ ID NO: 4) were obtained by gene synthesis NO: 5), CIT (SEQ ID NO: 6), OXA48 (SEQ ID NO: 7), GES (SEQ ID NO: 8) and DHA (SEQ ID NO: 9), a total of 9 drug resistance gene fragments were used as templates. The primer and probe sequences of each gene are shown below, wherein the probes of KPC, NDM and ACC are labeled with a Texas red fluorescent group at the 5' end and a BHQ2 quenching group at the 3' end; probe 5 of VIM, EBC and CIT The CY5 fluorophore was labeled at the ' end and the BHQ3 quenching group was labeled at the 3' end; the probes for GES, OXA48 and DHA were labeled with the HEX fluorophore at the 5' end and the BHQ2 quenching group at the 3' end.

[0107] KPC-F: CGGCAGCAGTTTGTTGATTG (SEQ ID NO: 10)

[0108] KPC-R: CAGACGACGGCATAGTCATTT (SEQ ID NO: 11)

[0109] K...

Embodiment 2

[0142] Example 2. Detection of three mixed templates of Texas red, CY5 and HEX channels by single-tube multiplex fluorescence qPCR method

[0143] In this example, the plasmids of the synthesized genes KPC (SEQ ID NO: 1), NDM (SEQ ID NO: 2) and ACC (SEQ ID NO: 3) are used to mix at 1:1:1 to configure a mixed template Mix1, VIM (SEQ ID NO: 4), EBC (SEQ ID NO: 5) and CIT (SEQ ID NO: 6) plasmids, mixed at 1:1:1 to configure the mixed template Mix 2, OXA48 (SEQ ID NO:7) , GES (SEQ ID NO: 8) and DHA (SEQ ID NO: 9) plasmids, mixed at 1:1:1 and configured into a mixed template Mix 3, using multiplex qPCR to detect the mixed template, primer probes and examples used 1 is the same.

[0144] reaction system:

[0145] 2×PCR buffer (with enzyme) 12.5μl SYTO 9 (10μM) 1 Forward primer (10μM) 0.25*9 Probe (10μM) 0.1*9μl Reverse primer (10μM) 0.25*9μl template DNA10 6

3μl Sterilized distilled water Add to 25μl

[0146] The PCR rea...

Embodiment 3

[0152] Example 3. Sensitivity determination of single-tube multiplex fluorescence qPCR detection

[0153] This example uses the synthesized genes KPC (SEQ ID NO: 1), NDM (SEQ ID NO: 2), ACC (SEQ ID NO: 3), VIM (SEQ ID NO: 4), EBC (SEQ ID NO: 5) ), CIT (SEQ ID NO: 6), OXA48 (SEQ ID NO: 7), GES (SEQ ID NO: 8) and DHA (SEQ ID NO: 9), each of which was serially diluted to 10 1 -10 7 copies / μl template, 9 genes at different concentrations were detected by multiplex qPCR, and the primers and probes used were the same as those in Example 1.

[0154] reaction system:

[0155] 2×PCR buffer (with enzyme) 12.5μl SYTO 9 (10μM) 1 Forward primer (10μM) 0.25*9 Probe (10μM) 0.1*9μl Reverse primer (10μM) 0.25*9μl template DNA10 6

3μl Sterilized distilled water Add to 25μl

[0156] The PCR reaction program is as follows:

[0157] 95°C for 15 minutes; 40 cycles: 95°C for 3 seconds, 60°C for 20 seconds (to collect fluorescence signal...

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Abstract

The invention provides a fluorescent real-time detection reagent and method for simultaneously detecting multiple target genes. The present invention also provides a primer-probe pair for multiple target genes, a method for labeling the probes of multiple target genes with different Tm values ​​of amplified products with the same fluorescent group, combined application of fluorescence detection and Tm value analysis , to realize the detection of multiple target genes in a single reaction system. The method of the invention is simple, fast and sensitive.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and more particularly relates to a fluorescent real-time detection reagent and method for simultaneously detecting multiple target genes. Background technique [0002] Pathogen detection is to detect pathogens in the early stage of disease infection, provide timely treatment, and help prevent disease. Pathogen diagnosis is generally divided into bacterial, viral and fungal diagnosis. [0003] In recent years, with the widespread use of carbapenems, the number of multidrug-resistant gram-negative bacilli, especially carbapenem-resistant Enterobacteriaceae (CRE), has been increasing. , resulting in difficulties in clinical anti-infective treatment and hospital infection-related work. However, CRE is prone to multidrug resistance and high mortality during bloodstream infection, which is a serious threat to public health security. Therefore, the identification of CREs is crucial for...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686
Inventor 张驰宇李莹雪
Owner 中国科学院上海免疫与感染研究所
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