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System for simultaneously detecting methicillin-susceptible and methicillin-resistant staphylococcus aureus and using method thereof

A methicillin-resistant type, methicillin technology, applied in the field of biomedicine, can solve the problems of high probe cost, poor detection sensitivity, poor repeatability of results, etc., and achieve the effects of improving detection efficiency and saving operation time

Pending Publication Date: 2020-06-26
贵州中医药大学第二附属医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method takes about 2-4 days, including bacterial enrichment, selective culture, and subsequent systematic biochemical tests and drug sensitivity tests. Prone to misjudgment
With the rapid development of nucleic acid diagnostic technology, some PCR-based diagnostic technologies (such as ordinary PCR technology, fluorescent PCR technology) have been used for the detection of Staphylococcus aureus, but these methods rely on expensive equipment and require follow-up electrophoresis operation, the cost of synthesis of the required probes is high, and skilled operators are also required
For some backward laboratories, PCR-based diagnosis cannot be performed, and PCR-based diagnosis still has problems such as poor detection sensitivity and long detection process, which is not conducive to rapid detection and emergency detection

Method used

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  • System for simultaneously detecting methicillin-susceptible and methicillin-resistant staphylococcus aureus and using method thereof
  • System for simultaneously detecting methicillin-susceptible and methicillin-resistant staphylococcus aureus and using method thereof
  • System for simultaneously detecting methicillin-susceptible and methicillin-resistant staphylococcus aureus and using method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: m-LAMP-LFB simultaneously detects MSSA and MRSA (for samples with different DNA concentrations)

[0066] 1. Materials and equipment

[0067] Bacterial genomic DNA extraction kits (Bacterial genomic DNA extraction kits; QIAampDNA minikits) were purchased from Qiagen, Germany; Universal isothermal amplification kits and colorimetric indicators were purchased from Malachite Green; Biotin-labeled-deoxycytidine triphosphate (biotin-14-dCTP) was purchased from Bei-JingHaiTaiZhengYuan Company.

[0068] LFB materials include backsheets, sample pads, absorbent pads, conjugation pads, and NC membranes, all of which were purchased from Jie-Yi Biotechnology.Co.Ltd. anti-Dig (digoxigenin antibody), anti-FAM (carboxyfluorescein antibody) and biotin-BSA (biotinylated bovine serum albumin) were purchased from Abcam.Co.Ltd. Gold nanoparticles coupled streptavidin (dark red, (Dye streptavidin-coated polymer nanoparticles, 129nm, 10mg ml -1 , 100 mM boric acid, pH 8.5, conta...

Embodiment 2

[0081] Example 2: m-LAMP-LFB detects clinical samples

[0082] In order to further test the practicability of this method, 63 whole blood samples were tested in this embodiment, all of which were from patients suspected of Staphylococcus aureus infection (collected by a hospital affiliated to a University of Traditional Chinese Medicine). In this embodiment, traditional enrichment culture and bacterial drug susceptibility test (including blood culture, colony morphology observation, Gram staining, biochemical analysis and methicillin sensitivity test), PCR detection (using both famA and mecA) were adopted in the present embodiment. Gene-specific primers were used for ordinary PCR amplification respectively, that is, the primers used were femA-F3 in Table 1, femA-B3, mecA-F3, mecA-B3) and the m-LAMP-LFB method of the present invention. The m-LAMP amplification process and LFB detection process using m-LAMP-LFB are the same as in Example 1. The traditional bacterial enrichment ...

experiment example 1

[0088] Experimental Example 1: Determination of Optimum Reaction Temperature

[0089] The reaction temperature of LAMP is more important to the reaction efficiency. In this experimental example, the reaction temperature of LAMP was optimized. In this experimental example, the LAMP temperature of the famA gene and the mecA gene were tested (LAMP alone). The experimental method was the same as that described in the first paragraph of Comparative Example 1. The difference was that the temperature of the LAMP reaction was designed in a gradient, starting from 60°C Up to 67°C, set an experimental point every 1°C. LAMP amplification of femA gene, DNA template (concentration 10ng / μl) comes from S.aureus (MSSA, ATCC 25923), Pseudomonas aeruginosa (Pseudomonas aeruginosa, negative control), Enterococcusfaecalis (Enterococcus faecalis, negative control ) and double distilled water (blank control). LAMP amplification of the mecA gene, DNA templates (concentration 10ng / μl) from MRSA (AT...

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Abstract

The invention relates to the technical field of biomedicine, and particularly relates to a system for simultaneously detecting methicillin-susceptible and methicillin-resistant staphylococcus aureus and a using method thereof. The system comprises an m-LAMP unit and a detection unit; the m-LAMP unit is used for performing loop-mediated isothermal amplification on femA gene and mecA gene simultaneously in the same reaction system; the m-LAMP unit comprises a first primer combination for performing loop-mediated isothermal amplification on the femA gene and a second primer combination for performing loop-mediated isothermal amplification on the mecA gene; and the detection unit is used for detecting femA gene and mecA gene products after loop-mediated isothermal amplification. The system canrealize simultaneous detection of MSSA and MRSA, and can be applied to practice operations of mechanisms such as hospitals and the like for detection of MSSA and MRSA, and effective reference is provided for formulating a timely and accurate treatment scheme.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for simultaneously detecting methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (methicillin-resistant Staphylococcus aureus). , MRSA) systems and methods of use. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is one of the most common pathogenic bacteria in hospital and community environments, it can infect almost every tissue and organ in the body, causing a wide range of infectious diseases, such as soft tissue infection, skin infection pneumonia, mastitis, Osteomyelitis and endocarditis etc. Due to the non-standard application of antibiotics in recent years and the weak supervision of relevant institutions, the outbreak of drug-resistant strains and "super bacteria" has become a serious trend. Since Methicillin-resistant Staphylococcus aureus (MRSA) was first detected in 1961, its de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/14
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2565/607C12Q2537/1376C12Q2563/107
Inventor 陈旭李世军
Owner 贵州中医药大学第二附属医院
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