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Detection method of Staphylococcus aureus based on exo-rpa technology and its complete set of reagents

A staphylococcus, golden yellow technology, applied in the biological field, can solve the problems of low sensitivity and short amplification reaction time, achieve high sensitivity, get rid of the dependence of precision instruments, and good specificity

Active Publication Date: 2022-06-28
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recombinase polymerase-mediated amplification technology (RPA) is a constant temperature amplification technology that has emerged in recent years. Since the RPA amplification method does not require complicated temperature changes and the amplification reaction time is short, it can be combined with a paired detection fluorescent probe The exo-RPA method can get obvious positive results within 10 minutes at the shortest, which is suitable for on-site rapid detection. The disadvantage is that the sensitivity of RPA technology is slightly lower than that of real-time quantitative PCR (qPCR) detection method

Method used

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  • Detection method of Staphylococcus aureus based on exo-rpa technology and its complete set of reagents
  • Detection method of Staphylococcus aureus based on exo-rpa technology and its complete set of reagents
  • Detection method of Staphylococcus aureus based on exo-rpa technology and its complete set of reagents

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, the construction of Staphylococcus aureus exo-RPA detection method

[0042] The design of the sequence.

[0043] 1. Target sequence selection.

[0044] On the basis of previous research, the present invention selects the chromosomal specific sequence femB of Staphylococcus aureus (sequence shown in SEQ ID No. 1) as the target sequence after repeated screening and comparison, which can specifically detect Staphylococcus aureus. .

[0045] 2. Design of RPA amplification primer pair and RPA probe.

[0046] According to the above-mentioned chromosomal-specific gene femB sequence of Staphylococcus aureus, multiple RPA amplification primer pairs and RPA probes were designed: RPA-femB-F1, RPA-femB-F2, RPA-femB-F3, RPA-femB-F4, RPA -femB-R1, RPA-femB-R2, RPA-femB-R3, RPA-femB-R4 and RPA-femB-P. The specific sequence is shown in the following table:

[0047] Table 1 Candidate RPA amplification primer pairs and paired fluorescent probes of the present invention...

Embodiment 2

[0063] Example 2. Sensitivity and specificity evaluation of Staphylococcus aureus exo-RPA detection method

[0064] 1. Genome sensitivity and stability analysis

[0065] The detection was carried out according to the "three, Staphylococcus aureus exo-RPA detection method" in Example 1, and the template DNA was the concentration gradient of Staphylococcus aureus genomic DNA 100aM, 20aM, 10aM, 5aM, 1aM, 0.1aM, a total of 6 gradients, 2μL ddH 2 O was used as a negative control, and the sensitivity and stability were evaluated by 7 repeated experiments.

[0066] The result is as figure 2 As shown in A, take the average fluorescence value of the reaction time of 1 minute, 5 minutes, 10 minutes, 15 minutes and 20 minutes respectively, and the positive detection time of the reaction is 5-10 minutes; figure 2 B shows that the detection sensitivity of S. aureus genomic DNA can reach 1 aM, and the experimental repeatability is good, indicating that the detection method has high sens...

Embodiment 3

[0070] Example 3. Application of the method of the present invention in the detection of Staphylococcus aureus nucleic acid in simulated clinical samples

[0071] In order to verify the practicability of the exo-RPA detection method of Staphylococcus aureus, Staphylococcus aureus (USA300) was added to normal human whole blood with different numbers, respectively, and the samples containing 10 3 -10 1The whole blood of Staphylococcus aureus at the concentration of CFU / mL, and the same volume of PBS was added to the group was the blank control sample (BC group). Nucleic acids from whole blood mock samples were extracted with QIAamp DNA Mini Kit. The testing personnel performed blind testing on the above 12 samples: 3 μL of the extracted nucleic acid from the above samples was taken as a template, and the specific gene nuc of the samples was detected by real-time fluorescent PCR (COPPENS J, VAN HEIRSTRAETEN L, RUZIN A, et al. .Comparison of GeneXpert MRSA / SA ETA assay with semi...

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Abstract

The invention discloses a method for detecting Staphylococcus aureus based on exo-RPA technology and a set of reagents thereof. The set of reagents includes primer RPA-femB-F3, primer RPA-femB-R2 and probe RPA-femB-P; the nucleotide sequence of the primer RPA-femB-F3 is as shown in SEQ ID No.4, and the The nucleotide sequence of the primer RPA-femB-R2 is shown in SEQ ID No.7, and the nucleotide sequence of the probe RPA-femB-P is shown in SEQ ID No.10. The set of reagents has good sensitivity and specificity for clinical blood simulation samples, can effectively detect staphylococcus aureus, and provides an available rapid detection method for in vitro diagnosis of staphylococcus aureus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting Staphylococcus aureus and a complete set of reagents based on exo-RPA (fluorescence RPA) technology. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is a common zoonotic pathogen that can cause human and animal infections. It is an important foodborne pathogen that causes food poisoning and can also be transmitted through animals and animal foods. give. The bacteria are widely distributed in nature and can cause a variety of infections in humans and animals, such as human respiratory system, skin and soft tissue, urinary system infections, and endocarditis, and animals such as cow mastitis. In recent years, the isolation rate of Staphylococcus aureus (hereinafter referred to as Staphylococcus aureus) has increased significantly, and it has rapidly spread to the world. It has become one of the important pathogens of nosoc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/14C12N15/11
CPCC12Q1/689C12Q1/6844C12Q2600/166C12Q2531/119C12Q2521/507C12Q2522/101C12Q2537/1376C12Q2563/107C12Q2545/113
Inventor 袁媛李佳欣王景林徐健皓李岩伟王菁辛文文康琳高姗
Owner ACADEMY OF MILITARY MEDICAL SCI
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