Primers, kit and method for detecting methicillin-resistant Staphylococcus aureus by PSR (polymerase spiral reaction)

A technology for methicillin-resistant and detection primers, applied in the field of primers and kits for PSR detection of methicillin-resistant Staphylococcus aureus, can solve the problems of large-scale detection of samples, heavy workload, and high cost, and shorten the detection cycle , Guaranteed reliability, intuitive interpretation effect

Active Publication Date: 2019-02-19
SOUTH CHINA UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The workload of conventional detection and identification methods is relatively large, and the results of the detection are generally 4 to 5 days before the results are obtained, which takes a long time. Judging the identification results based on experimental phenomena is difficult to meet the needs of rapid identification
The gene chip detection and PCR amplification methods developed in recent years have strong specificity, rapidity and sensitivity, but the cost is relatively high
Immunological detection methods, such as ELISA, immunochromatography, etc., have high sensitivity, but the operation process is complicated and the cost is high, so it is not suitable for large-scale detection of samples
At present, the most widely used constant temperature amplification technology-LAMP also has its limitations, such as complex primer design, high false positive rate, high reagent price, and due to the protection of intellectual property rights in Japan, my country has strong limitations in transformation and application

Method used

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  • Primers, kit and method for detecting methicillin-resistant Staphylococcus aureus by PSR (polymerase spiral reaction)
  • Primers, kit and method for detecting methicillin-resistant Staphylococcus aureus by PSR (polymerase spiral reaction)
  • Primers, kit and method for detecting methicillin-resistant Staphylococcus aureus by PSR (polymerase spiral reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Polymerase helical reaction detection of methicillin-resistant Staphylococcus aureus primer screening

[0054] 1. Design primers

[0055] According to the principle of PSR amplification reaction, Primer Premier software was used to design multiple sets of primers as shown in Table 1 for femA and mecA targets respectively.

[0056] Table 1 Primer information

[0057]

[0058] 2. Establish a polymerase helical reaction detection method

[0059] (1) Reaction system

[0060] ① Combinations of primer pairs with concentrations of 50 μM in Table 1.

[0061] ②2×Reaction stock solution: Tris-HCl with a concentration of 40.0mM, ammonium sulfate 20.0mM, potassium chloride 20.0mM, magnesium sulfate 16.0mM, 0.2% (v / v) Tween 20, betaine 1.4M, dNTPs (each ) 10.0mM mixed solution composition.

[0062] ③ Bst DNA polymerase (Bst DNA Polymerase, Large Fragment, purchased from NEB Company) aqueous solution with a concentration of 8 U / μL;

[0063] (2) Detection method

...

Embodiment 2

[0070] Example 2 Microbiological method for detecting methicillin-resistant Staphylococcus aureus based on polymerase helical reaction isothermal amplification technology

[0071] 1. The method for detecting pathogenic microorganisms based on polymerase helical isothermal amplification technology, select the preferred primer combination obtained in Example 1, and this embodiment takes methicillin-resistant Staphylococcus aureus as an example, and the reagents used are as follows:

[0072] (1) Primer

[0073] The detection primers Ft aqueous solution and Bt aqueous solution with a concentration of 50 μM each, the primer sequences are as follows (5'-3'):

[0074] Target femA detection primer Ft:

[0075] 5'-AGGTATAGACTTCGATGTTTCAAAATCGCGGTCCAGTG-3' (SEQ ID NO. 1);

[0076] Target femA detection primer Bt:

[0077] 5'-TTGTAGCTTCAGATATGGAAACCAATCATTACCAGCA-3' (SEQ ID NO. 2);

[0078] Target mecA detection primer Ft

[0079] 5'-CCAATAACTGCATCATCT-GCGACTTCACATCTATTAGG-3' (SEQ I...

Embodiment 3

[0095] Example 3 polymerase helical reaction detection methicillin-resistant Staphylococcus aureus specificity test

[0096] The genomic DNA of the following bacterial strains was established according to the reaction system and conditions of Example 2 to detect the polymerase helical reaction, and the specificity test was carried out:

[0097] (1) Methicillin-resistant Staphylococcus aureus: Methicillin-resistant Staphylococcus aureus NCTC10442 [1] ; Methicillin-resistant Staphylococcus aureus N315 [1] ;Methicillin-resistant Staphylococcus aureus 85 / 2082 [1] ;Methicillin-resistant Staphylococcus aureus CA05 [1] ; methicillin-resistant Staphylococcus aureus JCSC4469 [1] .

[0098] (2) Non-methicillin-resistant Staphylococcus aureus: Salmonella ATCC29629; Salmonella ATCC19585; Salmonella ATCC14028; Salmonella ATCC13076; Salmonella ATCC29629; Listeria monocytogenes ATCC19116; Listeria ATCC15313; Listeria monocytogenes ATCC19113; Pseudomonas aeruginosa ATCC27853; Escherichia...

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Abstract

The invention discloses primers, kit and method for detecting methicillin-resistant Staphylococcus aureus by PSR (polymerase spiral reaction). The primers provided herein are shown as SEQ ID NO. 1 to4 and suitable for detecting methicillin-resistant Staphylococcus aureus by PSR; specificity, sensitivity and reliability can be detected for methicillin-resistant Staphylococcus aureus by detecting specific target sequences femA and mecA. The invention also provides a kit and method for detecting methicillin-resistant Staphylococcus aureus by PSR based on the primers; the method has the advantages of high sensitivity, good specificity, good operational convenience, high operational speed, accurate and reliable results, low detection cost and suitability to field detection; no special expensive instruments or agents are required; after developing with a fluorescent dye, the results can be judged with eyes; the kit and method are particularly suitable for detection in small- and medium-sized units and field detection and have a promising application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer, a kit and a method for PSR detection of methicillin-resistant staphylococcus aureus. Background technique [0002] Staphylococcus aureus is an important pathogen of hospital and community infections. With the widespread use of broad-spectrum antibacterial drugs in recent years, the drug resistance of Staphylococcus aureus is gradually increasing, among which methicillin-resistant Staphylococcus aureus ( The isolation rate of Meticillin-resistant staphylococcus aureus (MRSA) is increasing day by day. MRSA is not only resistant to methicillin, but also resistant to multiple antibiotics. Therefore, rapid and accurate detection of MRSA strains can not only guide clinical selection of appropriate antibiotics, but also help control the spread of MRSA. [0003] At present, the detection methods for methicillin-resistant Staphylococcus aureus mainly include the traditi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/14C12R1/445
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 徐振波苗健徐行勇刘君彦陈定强苏健裕陈玲李晓玺李冰李琳
Owner SOUTH CHINA UNIV OF TECH
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