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mecA GENE AMPLIFICATION PRIMER PAIR, mecA GENE DETECTION KIT AND mecA GENE DETECTION METHOD

A detection method and primer pair technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as infection of susceptible hosts, and achieve high sensitivity and high precision results

Pending Publication Date: 2020-05-19
MITSUI CHEM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Staphylococcus aureus is more pathogenic than other coagulase-negative staphylococci, for methicillin-resistant staphylococcus aureus and coagulase-negative staphylococci, the Infection of susceptible hosts is still a big problem

Method used

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  • mecA GENE AMPLIFICATION PRIMER PAIR, mecA GENE DETECTION KIT AND mecA GENE DETECTION METHOD
  • mecA GENE AMPLIFICATION PRIMER PAIR, mecA GENE DETECTION KIT AND mecA GENE DETECTION METHOD
  • mecA GENE AMPLIFICATION PRIMER PAIR, mecA GENE DETECTION KIT AND mecA GENE DETECTION METHOD

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0164] (Example 1) primary screening

[0165] Genomic DNA extracted from the culture medium of MRSA (RIKEN CORPORATION transfer strain, JCM31453) was used as a template using the High pure PCR template preparation kit (Roche), primers of sequence number 15 and sequence number 16 were used, and the reaction solution shown in Table 1 was used The composition implements PCR to amplify the full length of mecA ORF. Then, a serial dilution of the amplification product of mecA was prepared, and a primer pair for comparison consisting of a forward primer consisting of SEQ ID NO: 13 and a reverse primer consisting of SEQ ID NO: 14 was used, and the reaction solution shown in Table 1 was used. PCR was performed to determine the concentration of the amplified product of mecA whose number of cycles was increased to about 20 cycles, and used as a template. It should be noted that the methicillin-resistant Staphylococcus aureus JCM31453 can be obtained through the Microbial Materials Devel...

Embodiment 2

[0167] (Example 2) secondary screening

[0168] Using 50 μg / ml human genomic DNA solution (invirogen), the concentration of genomic DNA extracted from the culture solution of MRSA (RIKEN CORPORATION transfer strain, JCM31453) using High pure PCR template preparation kit (Roche) was adjusted to increase the number of cycles to about 20 cycles of the solution, used as a template.

[0169] For 88 primer pairs, PCR was performed using the reaction solution composition shown in Table 1, and evaluation was performed. As the real-time PCR device, Rotor-Gene Q MDx 5plex HRM (QIAGEN) was used. The reaction conditions of PCR were: heating at 95° C. for 5 minutes, followed by 40 repetitions of 94° C. for 10 seconds, 65° C. for 10 seconds, and 72° C. for 30 seconds. In terms of evaluation, 57 pairs were screened that were confirmed to be improved compared to the results of the comparative primer pairs with respect to the number of rising cycles, the shape of peaks in HRM, the intensity ...

Embodiment 3

[0170] (Example 3) three screening

[0171]Using 50 μg / ml human genomic DNA solution (invirogen), the concentration of genomic DNA extracted from the culture solution of MRSA (RIKEN CORPORATION transfer strain, JCM31453) using High pure PCR template preparation kit (Roche) was adjusted to 10.4 molecules / reaction solution solution as a template. The 57 primer pairs were evaluated by performing PCR by triple assay using the reaction solution composition shown in Table 1. As the real-time PCR device, Rotor-Gene QMDx 5plex HRM (QIAGEN) was used. The reaction conditions of PCR were: heating at 95° C. for 5 minutes, followed by 40 repetitions of 94° C. for 10 seconds, 65° C. for 10 seconds, and 72° C. for 30 seconds. For evaluation, 9 pairs were screened where amplification was observed in both triple assays, peaks for the height of dF / dT in HRM (lane A), the presence of primer dimers, and the presence or absence of non-specific products were confirmed An improved primer pair com...

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Abstract

Provided is a primer pair of primers for methicillin-resistant gene detection for the purpose of achieving highly sensitive methicillin-resistant gene detection. Said primer pair comprises a combination of SEQ ID NO:3 and SEQ ID NO:7, a combination of SEQ ID NO:2 and SEQ ID NO:9, a combination of SEQ ID NO:1 and SEQ ID NO:8, a combination of SEQ ID NO:1 and SEQ ID NO:9, a combination of SEQ ID NO:4 and SEQ ID NO:11, a combination of SEQ ID NO:5 and SEQ ID NO:12, a combination of SEQ ID NO:6 and SEQ ID NO:10, or a combination of SEQ ID NO:6 and SEQ ID NO:12.

Description

technical field [0001] The invention relates to a primer pair, a kit and a detection method for simple, rapid and high-sensitivity detection of the mecA gene as a specific genetic element of methicillin resistance. Background technique [0002] Methicillin-resistant Staphylococcus aureus (hereinafter referred to as "MRSA") is the main pathogenic bacteria in nosocomial infections all over the world, and has been valued as the most important clinical drug-resistant bacteria in many countries. mecA, which is a gene associated with methicillin resistance of MRSA, encodes PBP (Penicillin Binding Protein)-2' (also referred to as PBP-2a). Generally, S. aureus mainly produces 4 kinds of PBPs. PBP is a protein involved in the synthesis of peptidoglycan of the cell wall and has transpeptidase activity. β-lactam antibiotics play a role by binding to the active site of PBP to inhibit the activity of transpeptidase and inhibit the synthesis of peptidoglycan. However, PBP-2' avoids the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12M1/00C12Q1/686C12Q1/689
CPCC12M1/00C12N15/11C12Q1/689C12Q1/686
Inventor 天野仰远藤绚子矢内久阳辻健太郎森重敬
Owner MITSUI CHEM INC
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