Triple real-time fluorescent PCR (polymerase chain reaction) detection primer, detection probe, detection kit and detection method for methicillin-resistant staphylococcus aureus

A methicillin- and staphylococcus-resistant technology, applied in the field of triple real-time fluorescent PCR detection primers, can solve the problems of low affinity and achieve reliable results, strong specificity, and high sensitivity

Active Publication Date: 2013-12-04
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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Problems solved by technology

mecA (Methicillin determinant A) is the drug resistance determinant gene of MRSA, which encodes PBP2a, which has a very low affinity with β-lactam antibiotics, and promotes the synthesis of bacterial cell walls and drug resistance. It has been clarified that the mecA gene only exists in staphylococci. Located on a

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  • Triple real-time fluorescent PCR (polymerase chain reaction) detection primer, detection probe, detection kit and detection method for methicillin-resistant staphylococcus aureus
  • Triple real-time fluorescent PCR (polymerase chain reaction) detection primer, detection probe, detection kit and detection method for methicillin-resistant staphylococcus aureus
  • Triple real-time fluorescent PCR (polymerase chain reaction) detection primer, detection probe, detection kit and detection method for methicillin-resistant staphylococcus aureus

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Embodiment 1

[0052] The MRSA standard strain ATCC33591 was inoculated into 7.5% tryptone soybean broth, and incubated overnight at 37°C with shaking. Take 2mL of the culture, centrifuge at 12000r / min for 3min to remove the supernatant, float the sediment with 1mL of deionized water, centrifuge at 12000r / min for 2min to remove the supernatant, repeat twice, and finally add 200μL of deionized water to extract DNA on the nucleic acid extractor for Fluorescent PCR amplification.

[0053] Real-time fluorescent PCR amplification system: reaction system 30μl, template DNA 4μl, 10×TaqMan buffer 5μl, 5mmol / LMgCl 2 2μl, 2.5mmol / L dNTPs3μl, TaqMan probe 20μmol / l each 1μl (including femA, mecA, nuc gene probes 1μl each, 3μl in total), primer 20μmol / l each 1μl (including femA, mecA, nuc gene detection 6 primers in total, 6 μl in total), UNG enzyme 0.55U / μl 0.2 μl, Taq polymerase 2.5U / μl 3 μl, deionized water 3.8 μl. The real-time fluorescent PCR reaction parameters were 95°C for 30s, 95°C for 5s, 60°...

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Abstract

The invention discloses a triple real-time fluorescent PCR (polymerase chain reaction) detection primer, a detection probe, a detection kit and a detection method for methicillin-resistant staphylococcus aureus. The detection primer and the detection probe are designed according to nuc, mecA and femA gene sequences respectively; through the detection primer and the detection probe, triple real-time fluorescent PCR detection is performed on a sample according to the detection method; in a primary amplification reaction, the detection on the specificity of the methicillin-resistant staphylococcus aureus in the sample to be detected is completed so as to simply, conveniently, rapidly and accurately judge whether the sample to be detected contains the methicillin-resistant staphylococcus aureus. When the triple real-time fluorescent PCR detection is performed on the sample according to the detection method, the detection is rapid and a process from sample preparation to detection result obtainment can be completed within 8-10 hours; interferences from false positiveness, cross contamination and the like can be avoided; the detection result is reliable, the detection sensitivity is high, and the detection specificity is high; and a beneficial tool is provided for epidemiological survey through the methicillin-resistant staphylococcus aureus.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, and in particular relates to triple real-time fluorescent PCR detection primers, probes, detection kits and detection methods for methicillin-resistant Staphylococcus aureus (MRSA). Background technique: [0002] Methicillin-resistant Staphylococcus aureus (MRSA) was first reported by Jevons in the UK in 1961, and now it has become a global pathogenic microorganism. AIDS) is also listed as the three most difficult infectious diseases in the world. At present, commonly used methods for MRSA drug resistance detection include disk diffusion method, broth dilution method, agar screening method, molecular biology diagnosis method, etc. [0003] Methicillin-resistant Staphylococcus aureus (Meth icillin-resistant Staphylococcus aureus, MRSA) and Methicillin-resistant coagulase-negative staphylococci (MRCNS) have become hospital and community infections common pathogenic bacteria, and the incid...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12N15/11C12R1/445
Inventor 袁慕云许龙岩张旺王志强阳静陈碧玲
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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