LAMP primer group and kit for detecting streptococcus pneumoniae, and application of LAMP primer group
A Streptococcus pneumoniae and kit technology, applied in the field of primer sets, can solve the problems of life-threatening safety, high mortality, and low accuracy, and achieve the effects of good sensitivity and repeatability, high primer sensitivity, and reduced peak time
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Embodiment 1
[0035] The embodiment of the present invention provides a LAMP primer set and performance analysis for detecting Streptococcus pneumoniae
[0036] 1.1. Primer design
[0037] The LAMP primer set was designed using Primer Explorer V5 software, its website is http: / / primerexplorer.jp / e / , the sequence of the Streptococcus pneumoniae autolysin gene LYTA downloaded from the NCBI database was imported into the above software, and after screening Appropriate primers were obtained, and the primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd. The initial concentration of the synthesized primers was 100 μM, and the used concentrations were: the outer primers F3 and B3 were 10 μM respectively; the inner primers FIP and BIP were respectively 100 μM; 10 μM for loop primers LF and LB. Different volumes of primers were mixed and then tested for Streptococcus pneumoniae. After the above operations, the primers were obtained as follows:
[0038] Outer primer F3: TGTTCCGTCTGGT...
Embodiment 2
[0088] The embodiment of the present invention provides a kit for detecting Streptococcus pneumoniae, including a LAMP primer set, a reaction solution, betaine, a positive control substance, and a negative control substance;
[0089] Wherein, the reaction solution includes: Bst large fragment DNA polymerase, fluorescent dye Eva Green, dNTPs, Tris-Hcl, KCl, MgSO 4 , (NH 4 ) 2 SO 4 ;
[0090] LAMP primer set:
[0091] The nucleotide sequence of the outer primer F3 is shown in the sequence listing SEQ ID NO:1; the nucleotide sequence of the outer primer B3 is shown in the sequence listing SEQ ID NO:2; the nucleotide sequence of the inner primer FIP is as shown in the sequence listing SEQ Shown in ID NO:3; The nucleotide sequence of inner primer BIP is shown in sequence table SEQ ID NO:4; The nucleotide sequence of loop primer LF is shown in sequence table SEQ ID NO:5; The nucleus of loop primer LB The nucleotide sequence is shown in the sequence listing SEQ ID NO:6.
[0092...
Embodiment 3
[0096] The embodiment of the present invention provides a method for detecting Streptococcus pneumoniae, the method comprising the following steps:
[0097] A: Extract the nucleic acid genome in the sample to be tested as a template for the loop-mediated constant temperature amplification reaction system;
[0098] B: Add the reaction reagents to carry out loop-mediated constant temperature amplification;
[0099] Configure LAMP reaction system, for each 25 μL volume of LAMP reaction system: 10.5 μL of ring-mediated constant temperature reaction solution, 2 μL of betaine, 0.5 μL and 0.5 μL of outer primers F3 and B3 respectively; 0.4 μL of inner primers FIP and BIP , 0.4 μL; add 2 μL each of the loop primers LF and LB; add 2.2 μL of the DNA template extracted from the sample to be tested; ddH 2 Add 4.5 μL to make up to 25 μL;
[0100] The condition of the LAMP reaction was constant temperature reaction at 63° C. for 60 min.
[0101] Wherein, the reaction solution includes: B...
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