Ultra-sensitive electrochemical LCR sensor for detecting methicillin-resistant staphylococcus aureus in synovial fluid

A methicillin and staphylococcus-resistant technology, applied in the field of electrochemical LCR sensors, can solve the problems of difficulty in distinguishing single bases, false positives and false negatives, etc., and achieves low detection cost, simple detection operation process, and high sequence sensitivity. Effect

Pending Publication Date: 2021-04-27
THE FIRST AFFILIATED HOSPITAL OF FUJIAN MEDICAL UNIV
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  • Claims
  • Application Information

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Problems solved by technology

However, due to the polymorphism of the MecA gene, especially the change in the single-copy series, it is difficult to distinguish these single-base changes by PCR and RT-PCR based on primer amplification, resulting in false positive or false negative results

Method used

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  • Ultra-sensitive electrochemical LCR sensor for detecting methicillin-resistant staphylococcus aureus in synovial fluid
  • Ultra-sensitive electrochemical LCR sensor for detecting methicillin-resistant staphylococcus aureus in synovial fluid
  • Ultra-sensitive electrochemical LCR sensor for detecting methicillin-resistant staphylococcus aureus in synovial fluid

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Embodiment 1

[0035] An LCR-based electrochemical sensing technique for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in the synovial fluid of patients with periprosthetic joint infections (eg figure 1 shown), including the following steps:

[0036] (1) Find out various homolog series of MecA gene through NCBI, and select the common conservative series to design probes. The designed probe series are: MecA gene (completely matching DNA target strand, T-full DNA target) : 5'-CCTCTGCTCAACAAGTTCCAGATTACAACTTCACCAGGTT CAACTC-3'; capture probe (end modified SH-C6, CP): 5'-SH-TTTTTTTTTT GAGTTGAACCTGGTGA AGTTGTAA-3'; signal probe (5' end modified PO4 - , 3' end modified biotin biotin, SP): 5'-PO4-TCTGGA ACTTGTTGAGCAGAGG-Biotin-3'; Partial target probe 1 (Half-target 1, hT-1): 5'-CCTCTGCTCA ACAAGTTCCAGA-3'; Partial target probe 2 (Half-target 2, hT-2): 5'-CCTCTGCTCAACAAGTTC CAGA-3'.

[0037] (2) Add the specimens, reagents and ligase required for the amplification reaction to...

Embodiment 2

[0044] A method for detecting methicillin-resistant Staphylococcus aureus (MRSA) in the joint fluid of patients with periprosthetic infection based on sensing technology, the steps are as follows:

[0045](1) The three-electrode system was used for the measurement, the dsDNA-BSA-AuE modified electrode prepared in Example 1 was used as the working electrode, the Ag / AgCl was used as the reference electrode, and the platinum wire electrode was used as the counter electrode, and the electrochemical workstation was used for detection. The three-electrode system was immersed in 500 μL of TMB substrate solution, and the current-time curve (i-t, initial potential 100 mV, t=100 s) and cyclic voltammetry curve (CV, scan speed 0.1 V / s) were recorded.

[0046] (2) In the LCR reaction system, add 1.0 × 10 -13 - 1.0×10 -8 M a series of DNA target strands with different concentrations, and the obtained products are electrochemically detected by the method of step (1), and different curren...

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Abstract

The invention discloses an ultra-sensitive electrochemical LCR sensor for detecting methicillin-resistant staphylococcus aureus in synovial fluid. The ultra-sensitive electrochemical LCR sensor comprises the following steps of (1) designing two specific double-stranded short probes (S-dsDNA) according to conservative series of a MecA gene; (2) in an LCR reaction system, taking the MecA gene as a template, connecting two short series of probes through DNA ligase to form long double-stranded DNA (L-dsDNA), and then carrying out cyclic amplification by using an L-dsDNA template to form a large amount of L-dsDNA, and (3) fixing the formed L-dsDNA with sulfydryl and biotin modification on a BSA modified gold electrode through a gold-sulfur bond, dropwise adding avidin-PolyHRP to be combined with biotin on the L-dsDNA, finally putting the electrode into a base solution containing TMB and H2O2, and enabling H2O2 to oxidize TMB to generate an electrochemical signal under the catalysis of HRP. The sensor has the advantages of economy, quickness, high sensitivity, specificity and the like, can be used for detecting a single base mutation series, and realizes the detection of the MRSA in the synovial fluid of a patient infected around the joint prosthesis.

Description

technical field [0001] The invention relates to an ultra-sensitive electrochemical LCR sensor for detecting methicillin-resistant Staphylococcus aureus (MRSA) in the joint fluid of patients with joint prosthetic infection, especially relates to an electrochemical sensing technology based on LCR to detect joint Methods for methicillin-resistant Staphylococcus aureus (MRSA) in the joint fluid of patients with periprosthetic infections. Background technique [0002] Hip and knee periprosthetic infection (PJI) is a catastrophic and serious complication after artificial joint replacement, which occurs in about 1%-2% of patients after artificial joint replacement. Most PJIs are caused by Gram-positive bacteria including Staphylococcus species. Several studies have shown that about 74% of PJI patients are caused by methicillin-resistant Staphylococcus aureus (MRSA) infection. The traditional method of detecting MRSA is through bacterial culture. However, because many PJI patient...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6862C12Q1/14C12N15/11
CPCC12Q1/6862C12Q1/689C12Q2531/137C12Q2563/131C12Q2565/607C12Q2521/501
Inventor 张文明钟光贤陈锦元魏洪翔方心俞林建华
Owner THE FIRST AFFILIATED HOSPITAL OF FUJIAN MEDICAL UNIV
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