A kind of primer, kit and method for detecting methicillin-resistant staphylococcus aureus by PSR
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
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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
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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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