Detection kit and detection method for escherichia coli H serotypes
A detection kit and technology for Escherichia coli, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve problems such as labor-intensive, time-consuming, and high-technical requirements
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Embodiment 1
[0088] The design of embodiment 1 kit and the determination of amplification conditions
[0089] 1. Gene selection
[0090] Select the coding gene fliC of Escherichia coli H antigen or its homologues at the loci other than fliC, and download the gene template sequence from genebank. Primers were designed according to the conservative type of the coding gene of the same H serotype in different strains and the polymorphism of the coding gene of different H serotypes.
[0091] 2. Design and screening of amplification primers
[0092] Apply Primer3 and design according to the conventional primer design requirements. Taking the H2 serotype coding gene as an example, use Primer3 to automatically search for primers, set the Tm value range at 56-64°C, and set the product lengths to 150-300, 301-400, 401-500, 501-700, 701- 900, select 2-3 pairs of primers for each product length interval.
[0093] The coding genes of Escherichia coli H serotype H1 and H12 have a high similarity, us...
Embodiment 2
[0104] The preparation of embodiment 2 typing standards
[0105] Preparation:
[0106] ①Take 53 H serotype standard strains of Escherichia coli H1~H12, H14~H21, H23~H49, H51~H56 preserved in the Jiangsu Provincial Center for Disease Control and Prevention, resuscitate the 53 H serotype standard strains, pick a single Bacterial colonies, extraction of genomic DNA by boiling method;
[0107] ② Perform PCR amplification,
[0108] PCR reaction reagents are from TaRaKa's Premix Taq TM . The reaction system configuration is as follows: 2×PremixTaq 25μl, genomic DNA 0.1~10μl with a content of 10~100ng, corresponding primers (see Table 2 for final concentration), and use dsH 2 O to make up 50 μl.
[0109] The reaction program of amplification is: 94°C, 1 minute, 1 cycle; 94°C, 30 seconds, 59°C, 30 seconds, 72°C, 1 minute, 30 cycles; 72°C, 10 minutes, 1 cycle.
[0110] ③ The obtained 53 amplification products are the typing standards of 53 H serotypes, which are the reference mat...
Embodiment 3
[0112] The specific detection of embodiment 3 compound amplification system
[0113] The 53 H serotype standard strains of Escherichia coli H1~H12, H14~H21, H23~H49, H51~H56 preserved in the Jiangsu Provincial Center for Disease Control and Prevention were respectively amplified. Five sets of primers were used for 5 amplifications. Amplification system: 2×Premix Taq 25 μl, genomic DNA 0.1-10 μl, content 10-100 ng, primer mixture 10 μl, and dsH 2 O make up 50 μl; the reaction program of amplification is: 94°C, 1 minute, 1 cycle; 94°C, 30 seconds, the annealing temperature is 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C ℃, 62℃, 63℃, 64℃ and 65℃, 30 seconds, 72℃, 1 minute, 30 cycles; 72℃, 10 minutes, 1 cycle. Finally, the amplified products were subjected to capillary electrophoresis. The results are shown in Table 3:
[0114] table 3
[0115]
[0116]
[0117] *: + means amplified positive; - means amplified negative
[0118] The results showed that the compound amplificati...
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